Streptomyces Development in Colonies and Soils

Streptomyces development was analyzed under conditions resembling those in soil. The mycelial growth rate was much lower than that in standard laboratory cultures, and the life span of the previously named first compartmentalized mycelium was remarkably increased.Streptomycetes are gram-positive, mycelium-forming, soil bacteria that play an important role in mineralization processes in nature and are abundant producers of secondary metabolites. Since the discovery of the ability of these microorganisms to produce clinically useful antibiotics (2, 15), they have received tremendous scientific attention (12). Furthermore, its remarkably complex developmental features make Streptomyces an interesting subject to study. Our research group has extended our knowledge about the developmental cycle of streptomycetes, describing new aspects, such as the existence of young, fully compartmentalized mycelia (5-7). Laboratory culture conditions (dense inocula, rich culture media, and relatively elevated temperatures [28 to 30°C]) result in high growth rates and an orderly-death process affecting these mycelia (first death round), which is observed at early time points (5, 7).In this work, we analyzed Streptomyces development under conditions resembling those found in nature. Single colonies and soil cultures of Streptomyces antibioticus ATCC 11891 and Streptomyces coelicolor M145 were used for this analysis. For single-colony studies, suitable dilutions of spores of these species were prepared before inoculation of plates containing GYM medium (glucose, yeast extract, malt extract) (11) or GAE medium (glucose, asparagine, yeast extract) (10). Approximately 20 colonies per plate were obtained. Soil cultures were grown in petri dishes with autoclaved oak forest soil (11.5 g per plate). Plates were inoculated directly with 5 ml of a spore suspension (1.5 × 10⁷ viable spores ml⁻¹; two independent cultures for each species). Coverslips were inserted into the soil at an angle, and the plates were incubated at 30°C. To maintain a humid environment and facilitate spore germination, the cultures were irrigated with 3 ml of sterile liquid GAE medium each week.The development of S. coelicolor M145 single colonies growing on GYM medium is shown in Fig. ​Fig.1.1. Samples were collected and examined by confocal microscopy after different incubation times, as previously described (5, 6). After spore germination, a viable mycelium develops, forming clumps which progressively extend along the horizontal (Fig. 1a and b) and vertical (Fig. 1c and d) axes of a plate. This mycelium is fully compartmentalized and corresponds to the first compartmentalized hyphae previously described for confluent surface cultures (Fig. 1e, f, and j) (see below) (5); 36 h later, death occurs, affecting the compartmentalized hyphae (Fig. 1e and f) in the center of the colony (Fig. ​(Fig.1g)1g) and in the mycelial layers below the mycelial surface (Fig. 1d and k). This death causes the characteristic appearance of the variegated first mycelium, in which alternating live and dead segments are observed (Fig. 1f and j) (5). The live segments show a decrease in fluorescence, like the decrease in fluorescence that occurs in solid confluent cultures (Fig. ​(Fig.11 h and i) (5, 9). As the cycle proceeds, the intensity of the fluorescence in these segments returns, and the segments begin to enlarge asynchronously to form a new, multinucleated mycelium, consisting of islands or sectors on the colony surfaces (Fig. 1m to o). Finally, death of the deeper layers of the colony (Fig. ​(Fig.1q)1q) and sporulation (Fig. ​(Fig.1r)1r) take place. Interestingly, some of the spores formed germinate (Fig. ​(Fig.1s),1s), giving rise to a new round of mycelial growth, cell death, and sporulation. This process is repeated several times, and typical, morphologically heterogeneous Streptomyces colonies grow (not shown). The same process was observed for S. antibioticus ATCC 11891, with minor differences mainly in the developmental time (not shown).Open in a separate windowFIG. 1.Confocal laser scanning fluorescence microscopy analysis of the development-related cell death of S. coelicolor M145 in surface cultures containing single colonies. Developmental culture times (in hours) are indicated. The images in panels l and n were obtained in differential interference contrast mode and correspond to the same fields as in panels k and m, respectively. The others are culture sections stained with SYTO 9 and propidium iodide. Panels c, d, k, l, p, and q are cross sections; the other images are longitudinal sections (see the methods). Panels h and i are images of the same field taken with different laser intensities, showing low-fluorescence viable hyphae in the center of the colonies that develop into a multinucleated mycelium. The arrows in panels e and s indicate septa (e) and germinated spores (s). See the text for details.Figure ​Figure22 shows the different types of mycelia present in S. coelicolor cultures under the conditions described above, depending on the compartmentalization status. Hyphae were treated with different fluorescent stains (SYTO 9 plus propidium iodide for nucleic acids, CellMask plus FM4-64 for cell membranes, and wheat germ agglutinin [WGA] for cell walls). Samples were processed as previously described (5). The young initial mycelia are fully compartmentalized and have membranous septa (Fig. 2b to c) with little associated cell wall material that is barely visible with WGA (Fig. ​(Fig.2d).2d). In contrast, the second mycelium is a multinucleated structure with fewer membrane-cell wall septa (Fig. 2e to h). At the end of the developmental cycle, multinucleated hyphae begin to undergo the segmentation which precedes the formation of spore chains (Fig. 2i to m). Similar results were obtained for S. antibioticus (not shown), but there were some differences in the numbers of spores formed. Samples of young and late mycelia were freeze-substituted using the methodology described by Porta and Lopez-Iglesias (13) and were examined with a transmission electron microscope (Fig. 2n and o). The septal structure of the first mycelium (Fig. ​(Fig.2n)2n) lacks the complexity of the septal structure in the second mycelium, in which a membrane with a thick cell wall is clearly visible (Fig. ​(Fig.2o).2o). These data coincide with those previously described for solid confluent cultures (4).Open in a separate windowFIG. 2.Analysis of S. coelicolor hyphal compartmentalization with several fluorescent indicators (single colonies). Developmental culture times (in hours) are indicated. (a, e, and i) Mycelium stained with SYTO 9 and propidium iodide (viability). (b, f, and j) Hyphae stained with Cell Mask (a membrane stain). (c, g, and l) Hyphae stained with FM 4-64 (a membrane stain). (d, h, and m) Hyphae stained with WGA (cell wall stain). Septa in all the images in panels a to j, l, and m are indicated by arrows. (k) Image of the same field as panel j obtained in differential interference contrast mode. (n and o) Transmission electron micrographs of S. coelicolor hyphae at different developmental phases. The first-mycelium septa (n) are comprised of two membranes separated by a thin cell wall; in contrast, second-mycelium septa have thick cell walls (o). See the text for details. IP, propidium iodide.The main features of S. coelicolor growing in soils are shown in Fig. ​Fig.3.3. Under these conditions, spore germination is a very slow, nonsynchronous process that commences at about 7 days (Fig. 3c and d) and lasts for at least 21 days (Fig. 3i to l), peaking at around 14 days (Fig. 3e to h). Mycelium does not clump to form dense pellets, as it does in colonies; instead, it remains in the first-compartmentalized-mycelium phase during the time analyzed. Like the membrane septa in single colonies, the membrane septa of the hyphae are stained with FM4-64 (Fig. 3j and k), although only some of them are associated with thick cell walls (WGA staining) (Fig. ​(Fig.3l).3l). Similar results were obtained for S. antibioticus cultures (not shown).Open in a separate windowFIG. 3.Confocal laser scanning fluorescence microscopy analysis of the development-related cell death and hyphal compartmentalization of S. coelicolor M145 growing in soil. Developmental culture times (in days) are indicated. The images in panels b, f, and h were obtained in differential interference contrast mode and correspond to the same fields as the images in panels a, e, and g, respectively. The dark zone in panel h corresponds to a particle of soil containing hyphae. (a, c, d, e, g, i, j, and k) Hyphae stained with SYTO 9, propidium iodide (viability stain), and FM4-64 (membrane stain) simultaneously. (i) SYTO 9 and propidium iodide staining. (j) FM4-64 staining. The image in panel k is an overlay of the images in panels i and j and illustrates that first-mycelium membranous septa are not always apparent when they are stained with nucleic acid stains (SYTO 9 and propidium iodide). (l) Hyphae stained with WGA (cell wall stain), showing the few septa with thick cell walls present in the cells. Septa are indicated by arrows. IP, propidium iodide.In previous work (8), we have shown that the mycelium currently called the substrate mycelium corresponds to the early second multinucleated mycelium, according to our nomenclature, which still lacks the hydrophobic layers characteristic of the aerial mycelium. The aerial mycelium therefore corresponds to the late second mycelium which has acquired hydrophobic covers. This multinucleated mycelium as a whole should be considered the reproductive structure, since it is destined to sporulate (Fig. ​(Fig.4)4) (8). The time course of lysine 6-aminotransferase activity during cephamycin C biosynthesis has been analyzed by other workers using isolated colonies of Streptomyces clavuligerus and confocal microscopy with green fluorescent protein as a reporter (4). A complex medium and a temperature of 29°C were used, conditions which can be considered similar to the conditions used in our work. Interestingly, expression did not occur during the development of the early mycelium and was observed in the mycelium only after 80 h of growth. This suggests that the second mycelium is the antibiotic-producing mycelium, a hypothesis previously confirmed using submerged-growth cultures of S. coelicolor (9).Open in a separate windowFIG. 4.Cell cycle features of Streptomyces growing under natural conditions. Mycelial structures (MI, first mycelium; MII, second mycelium) and cell death are indicated. The postulated vegetative and reproductive phases are also indicated (see text).The significance of the first compartmentalized mycelium has been obscured by its short life span under typical laboratory culture conditions (5, 6, 8). In previous work (3, 7), we postulated that this structure is the vegetative phase of the bacterium, an hypothesis that has been recently corroborated by proteomic analysis (data not shown). Death in confluent cultures begins shortly after germination (4 h) and continues asynchronously for 15 h. The second multinucleated mycelium emerges after this early programmed cell death and is the predominant structure under these conditions. In contrast, as our results here show, the first mycelium lives for a long time in isolated colonies and soil cultures. As suggested in our previous work (5, 6, 8), if we assume that the compartmentalized mycelium is the Streptomyces vegetative growth phase, then this phase is the predominant phase in individual colonies (where it remains for at least 36 h), soils (21 days), and submerged cultures (around 20 h) (9). The differences in the life span of the vegetative phase could be attributable to the extremely high cell densities attained under ordinary laboratory culture conditions, which provoke massive differentiation and sporulation (5-7, 8).But just exactly what are “natural conditions”? Some authors have developed soil cultures of Streptomyces to study survival (16, 17), genetic transfer (14, 17-19), phage-bacterium interactions (3), and antibiotic production (1). Most of these studies were carried out using amended soils (supplemented with chitin and starch), conditions under which growth and sporulation were observed during the first few days (1, 17). These conditions, in fact, might resemble environments that are particularly rich in organic matter where Streptomyces could conceivably develop. However, natural growth conditions imply discontinuous growth and limited colony development (20, 21). To mimic such conditions, we chose relatively poor but more balanced carbon-nitrogen soil cultures (GAE medium-amended soil) and less dense spore inocula, conditions that allow longer mycelium growth times. Other conditions assayed, such as those obtained by irrigating the soil with water alone, did not result in spore germination and mycelial growth (not shown). We were unable to detect death, the second multinucleated mycelium described above, or sporulation, even after 1 month of incubation at 30°C. It is clear that in nature, cell death and sporulation must take place at the end of the long vegetative phase (1, 17) when the imbalance of nutrients results in bacterial differentiation.In summary, the developmental kinetics of Streptomyces under conditions resembling conditions in nature differs substantially from the developmental kinetics observed in ordinary laboratory cultures, a fact that should be born in mind when the significance of development-associated phenomena is analyzed.     

Ganglioside GT1b Is a Putative Host Cell Receptor for the Merkel Cell Polyomavirus

The Merkel cell polyomavirus (MCPyV) was identified recently in human Merkel cell carcinomas, an aggressive neuroendocrine skin cancer. Here, we identify a putative host cell receptor for MCPyV. We found that recombinant MCPyV VP1 pentameric capsomeres both hemagglutinated sheep red blood cells and interacted with ganglioside GT1b in a sucrose gradient flotation assay. Structural differences between the analyzed gangliosides suggest that MCPyV VP1 likely interacts with sialic acids on both branches of the GT1b carbohydrate chain. Identification of a potential host cell receptor for MCPyV will aid in the elucidation of its entry mechanism and pathophysiology.Members of the polyomavirus (PyV) family, including simian virus 40 (SV40), murine PyV (mPyV), and BK virus (BKV), bind cell surface gangliosides to initiate infection (2, 8, 11, 15). PyV capsids are assembled from 72 pentamers (capsomeres) of the major coat protein VP1, with the internal proteins VP2 and VP3 buried within the capsids (7, 12). The VP1 pentamer makes direct contact with the carbohydrate portion of the ganglioside (10, 12, 13) and dictates the specificity of virus interaction with the cell. Gangliosides are glycolipids that contain a ceramide domain inserted into the plasma membrane and a carbohydrate domain that directly binds the virus. Specifically, SV40 binds to ganglioside GM1 (2, 10, 15), mPyV binds to gangliosides GD1a and GT1b (11, 15), and BKV binds to gangliosides GD1b and GT1b (8).Recently, a new human PyV designated Merkel cell PyV (MCPyV) was identified in Merkel cell carcinomas, a rare but aggressive skin cancer of neuroendocrine origin (3). It is as yet unclear whether MCPyV is the causative agent of Merkel cell carcinomas (17). A key to understanding the infectious and transforming properties of MCPyV is the elucidation of its cellular entry pathway. In this study, we identify a putative host cell receptor for MCPyV.Because an intact infectious MCPyV has not yet been isolated, we generated recombinant MCPyV VP1 pentamers in order to characterize cellular factors that bind to MCPyV. VP1 capsomeres have been previously shown to be equivalent to virus with respect to hemagglutination properties (4, 16), and the atomic structure of VP1 bound to sialyllactose has demonstrated that the capsomere is sufficient for this interaction (12, 13). The MCPyV VP1 protein (strain w162) was expressed and purified as described previously (1, 6). Briefly, a glutathione S-transferase-MCPyV VP1 fusion protein was expressed in Escherichia coli and purified using glutathione-Sepharose affinity chromatography. The fusion protein was eluted using glutathione and cleaved in solution with thrombin. The thrombin-cleaved sample was then rechromatographed on a second glutathione-Sepharose column to remove glutathione transferase and any uncleaved protein. The unbound VP1 was then chromatographed on a P-11 phosphocellulose column, and peak fractions eluting between 400 and 450 mM NaCl were collected. The purified protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Coomassie blue staining (Fig. ​(Fig.1A,1A, left) and immunoblotting using an antibody (I58) that generally recognizes PyV VP1 proteins (Fig. ​(Fig.1A,1A, right) (9). Transmission electron microscopy (Philips CM10) analysis confirmed that the purified recombinant MCPyV VP1 formed pentamers (capsomeres), which did not assemble further into virus-like particles (Fig. ​(Fig.1B).1B). In an initial screening of its cell binding properties, we tested whether the MCPyV VP1 pentamers hemagglutinated red blood cells (RBCs). The MCPyV VP1 pentamers were incubated with sheep RBCs and assayed as previously described (5). SV40 and mPyV recombinant VP1 pentamers served as negative and positive controls, respectively. We found that MCPyV VP1 hemagglutinated the RBCs with the same efficiency as mPyV VP1 (protein concentration/hemagglutination unit) (Fig. ​(Fig.1C,1C, compare rows B and C from wells 1 to 11), suggesting that MCPyV VP1 engages a plasma membrane receptor on the RBCs. The recombinant murine VP1 protein used for comparison was from the RA strain, a small plaque virus (4). Thus, MCPyV VP1 has the hemagglutination characteristics of a small plaque mPyV (12, 13).Open in a separate windowFIG. 1.Characterization of MCPyV VP1. Recombinant MCPyV VP1 forms pentamers and hemagglutinates sheep RBCs. (A) Coomassie blue-stained SDS-PAGE and an immunoblot of the purified recombinant MCPyV VP1 protein are shown. Molecular mass markers are indicated. (B) Electron micrograph of the purified MCPyV VP1. MCPyV VP1 (shown in panel A) was diluted to 100 μg/ml and absorbed onto Formvar/carbon-coated copper grids. Samples were washed with phosphate-buffered saline, stained with 1% uranyl acetate, and visualized by transmission electron microscopy at 80 kV. Bar = 20 nm. (C) Sheep RBCs (0.5%) were incubated with decreasing concentrations of purified recombinant SV40 VP1 (row A), mPyV VP1 (row B), and MCPyV VP1 (row C). Wells 1 to 11 contain twofold serial dilutions of protein, starting at 2 μg/ml (well 1). Well 12 contains buffer only and serves as a negative control. Well 7 (rows B and C) corresponds to 128 hemagglutination units per 2 μg/ml VP1 protein.To characterize the chemical nature of the putative receptor for MCPyV, total membranes from RBCs were purified as described previously (15). The plasma membranes (30 μg) were incubated with MCPyV VP1 (0.5 μg) and floated on a discontinuous sucrose gradient (15). After fractionation, the samples were analyzed by SDS-PAGE, followed by immunoblotting with I58. VP1 was found in the bottom of the gradient in the absence of the plasma membranes (Fig. ​(Fig.2A,2A, first panel). In the presence of plasma membranes, a fraction of the VP1 floated to the middle of the gradient (Fig. ​(Fig.2A,2A, second panel), supporting the hemagglutination results that suggested that MCPyV VP1 binds to a receptor on the plasma membrane.Open in a separate windowFIG. 2.MCPyV VP1 binds to a protease-resistant, sialic acid-containing receptor on the plasma membrane. (A) Purified recombinant MCPyV VP1 was incubated with or without the indicated plasma membranes. The samples were floated in a discontinuous sucrose gradient, and the fractions were collected from the top of the gradient, subjected to SDS-PAGE, and immunoblotted with the anti-VP1 antibody I58. (B) Control and proteinase K-treated plasma membranes were subjected to SDS-PAGE, followed by Coomassie blue staining. (C) HeLa cells treated with proteinase K (4 μg/ml) were incubated with MCPyV at 4°C, and the resulting cell lysate was probed for the presence of MCPyV VP1. (D) As described in the legend to panel C, except 293T cells were used. (E) Purified MCPyV VP1 was incubated with plasma membranes pretreated with or without α2-3,6,8 neuraminidase and analyzed as described in the legend to panel A.To determine whether the receptor is a protein or a lipid, plasma membrane preparations (30 μg) were incubated with proteinase K (Sigma), followed by analysis with SDS-PAGE and Coomassie blue staining. Under these conditions, the majority of the proteins in the plasma membranes were degraded by the protease (Fig. ​(Fig.2B,2B, compare lanes 1 and 2). Despite the lack of proteins, the proteinase K-treated plasma membranes bound MCPyV VP1 as efficiently as control plasma membranes (Fig. ​(Fig.2A,2A, compare the second and third panels), demonstrating that MCPyV VP1 interacts with a protease-resistant receptor. The absence of VP1 in the bottom fraction in Fig. ​Fig.2A2A (third panel) is consistent with the fact that the buoyant density of the membranes is lowered by proteolysis. Of note, a similar result was seen with binding of the mPyV to the plasma membrane (15). Binding of MCPyV to the cell surface of two human tissue culture cells (i.e., HeLa and 293T) was also largely unaffected by pretreatment of the cells with proteinase K (Fig. 2C and D, compare lanes 1 and 2), further indicating that a nonproteinaceous molecule on the plasma membrane engages the virus.We next determined whether the protease-resistant receptor contains a sialic acid modification. Plasma membranes (10 μg) were incubated with a neuraminidase (α2-3,6,8 neuraminidase; Calbiochem) to remove the sialic acid groups. In contrast to the control plasma membranes, the neuraminidase-treated membranes did not bind MCPyV VP1 (Fig. ​(Fig.2E,2E, compare first and second panels), indicating that the MCPyV receptor includes a sialic acid modification.Gangliosides are lipids that contain sialic acid modifications. We asked if MCPyV VP1 binds to gangliosides similar to other PyV family members. The structures of the gangliosides used in this analysis (gangliosides GM1, GD1a, GD1b, and GT1b) are depicted in Fig. ​Fig.3A.3A. To assess a possible ganglioside-VP1 interaction, we employed a liposome flotation assay established previously (15). When liposomes (consisting of phosphatidyl-choline [19 μl of 10 mg/ml], -ethanolamine [5 μl of 10 mg/ml], -serine [1 μl of 10 mg/ml], and -inositol [3 μl of 10 mg/ml]) were incubated with MCPyV VP1 and subjected to the sucrose flotation assay, the VP1 remained in the bottom fraction (Fig. ​(Fig.3B,3B, first panel), indicating that VP1 does not interact with these phospholipids. However, when liposomes containing GT1b (1 μl of 1 mM), but not GM1 (1 μl of 1 mM) or GD1a (1 μl of 1 mM), were incubated with MCPyV VP1, the vesicles bound this VP1 (Fig. ​(Fig.3B).3B). A low level of virus floated partially when incubated with liposomes containing GD1b (Fig. ​(Fig.3B),3B), perhaps reflecting a weak affinity between MCPyV and GD1b. Importantly, MCPyV binds less efficiently to neuraminidase-treated GT1b-containing liposomes than to GT1b-containing liposomes (Fig. ​(Fig.3B,3B, sixth panel), suggesting that the GT1b sialic acids are involved in virus binding. This finding is consistent with the ability of neuraminidase to block MCPyV binding to the plasma membrane (Fig. ​(Fig.2E).2E). The level of virus flotation observed in the neuraminidase-treated GT1b-containing liposomes is likely due to the inefficiency of the neuraminidase reaction with a high concentration of GT1b used to prepare the vesicles.Open in a separate windowFIG. 3.MCPyV VP1 binds to ganglioside GT1b. (A) Structures of gangliosides GM1, GD1a, GD1b, and GT1b. The nature of the glycosidic linkages is indicated. (B) Purified MCPyV VP1 protein was incubated with liposomes only or with liposomes containing the indicated gangliosides. The samples were analyzed as described in the legend to Fig. ​Fig.2A.2A. Where indicated, GT1b-containing liposomes were pretreated with α2-3,6,8 neuraminidase and analyzed subsequently for virus binding. (C to E) The indicated viruses were incubated with liposomes and analyzed as described in the legend to panel B.As controls, GM1-containing liposomes bound SV40 (Fig. ​(Fig.3C),3C), GD1a-containing liposomes bound mPyV (Fig. ​(Fig.3D),3D), and GD1b-containing liposomes bound BKV (Fig. ​(Fig.3E),3E), demonstrating that the liposomes were functionally intact. We note that, while all of the MCPyV VP1 floated when incubated with liposomes containing GT1b (Fig. ​(Fig.3B,3B, sixth panel), a significant fraction of SV40, mPyV, and BKV VP1 remained in the bottom fraction despite being incubated with liposomes containing their respective ganglioside receptors (Fig. 3C to E, second panels). This result is likely due to the fact that in contrast to MCPyV, which are assembled as pentamers (Fig. ​(Fig.1B),1B), the SV40, mPyV, and BKV used in these experiments are fully assembled particles: their larger and denser nature prevents efficient flotation. Nonetheless, we conclude that MCPyV VP1 binds to ganglioside GT1b efficiently.The observation that GD1a does not bind to MCPyV VP1 suggests that the monosialic acid modification on the right branch of GT1b (Fig. ​(Fig.3A)3A) is insufficient for binding. Similarly, the failure of GD1b to bind MCPyV VP1 suggests that the sialic acid on the left arm of GT1b is necessary for binding. Together, these observations suggest that MCPyV VP1 interacts with sialic acids on both branches of GT1b (Fig. ​(Fig.4).4). A recent structure of SV40 VP1 in complex with the sugar portion of GM1 (10) demonstrated that although SV40 VP1 binds both branches of GM1 (Fig. ​(Fig.4),4), only a single sialic acid in GM1 is involved in this interaction. In the case of mPyV, structures of mPyV VP1 in complex with different carbohydrates (12, 13) revealed that the sialic acid-galactose moiety on the left branch of GD1a (and GT1b) is sufficient for mPyV VP1 binding (Fig. ​(Fig.4).4). Although no structure of BKV in complex with the sugar portion of GD1b (or GT1b) is available, in vitro binding studies (8) have suggested that the disialic acid modification on the right branch of GD1b (and GT1b) is responsible for binding BKV VP1 (Fig. ​(Fig.4).4). Thus, it appears that the unique feature of the MCPyV VP1-GT1b interaction is that the sialic acids on both branches of this ganglioside are likely involved in capsid binding.Open in a separate windowFIG. 4.A potential model of the different VP1-ganglioside interactions (see the text for discussion).The identification of a potential cellular receptor for MCPyV will facilitate the study of its entry mechanism. An important issue for further study is to determine whether MCPyV targets Merkel cells preferentially, and if so, whether GT1b is found in higher levels in these cells to increase susceptibility.     

Acetolactate Synthase from Bacillus subtilis Serves as a 2-Ketoisovalerate Decarboxylase for Isobutanol Biosynthesis in Escherichia coli

A pathway toward isobutanol production previously constructed in Escherichia coli involves 2-ketoacid decarboxylase (Kdc) from Lactococcus lactis that decarboxylates 2-ketoisovalerate (KIV) to isobutyraldehyde. Here, we showed that a strain lacking Kdc is still capable of producing isobutanol. We found that acetolactate synthase from Bacillus subtilis (AlsS), which originally catalyzes the condensation of two molecules of pyruvate to form 2-acetolactate, is able to catalyze the decarboxylation of KIV like Kdc both in vivo and in vitro. Mutational studies revealed that the replacement of Q487 with amino acids with small side chains (Ala, Ser, and Gly) diminished only the decarboxylase activity but maintained the synthase activity.We have previously shown that 2-keto acids generated from amino acid biosynthesis can serve as precursors for the Ehrlich degradation pathway (15) to higher alcohols (3). In order to produce isobutanol, the valine biosynthesis pathway was used to generate 2-ketoisovalerate (KIV), the precursor to valine, which was then converted to isobutanol via a decarboxylation and reduction step (Fig. ​(Fig.1A).1A). The entire pathway to isobutanol from glucose is shown in Fig. ​Fig.1A.1A. To produce isobutanol, we overexpressed five genes, alsS (Bacillus subtilis), ilvC (Escherichia coli), ilvD (E. coli), kdc (Lactococcus lactis), and ADH2 (Saccharomyces cerevisiae) (Fig. ​(Fig.1A).1A). This E. coli strain produced 6.8 g/liter isobutanol in 24 h (Fig. ​(Fig.1B)1B) and more than 20 g/liter in 112 h (3). More recently, we have found that an alcohol dehydrogenase (Adh) encoded by yqhD on the E. coli genome can convert isobutyraldehyde to isobutanol efficiently (5) (Fig. ​(Fig.1B1B).Open in a separate windowFIG. 1.Schematic representation of the pathway for isobutanol production. (A) The Kdc-dependent synthetic pathway for isobutanol production. (B) Isobutanol production with the Kdc-dependent and -independent synthetic pathways. IlvC, acetohydroxy acid isomeroreductase; IlvD, dihydroxy acid dehydratase. (C) Enzymatic reaction of Als, Ahbs, and Kdc activities.One key reaction in the production of isobutanol is the conversion of KIV to isobutyraldehyde catalyzed by 2-ketoacid decarboxylase (Kdc) (Fig. ​(Fig.1C).1C). Since E. coli does not have Kdc, kdc from L. lactis was overexpressed. Kdc is a nonoxidative thiamine PP_i (TPP)-dependent enzyme and is relatively rare in bacteria, being more frequently found in plants, yeasts, and fungi (8, 19). Several enzymes with Kdc activity have been found, including pyruvate decarboxylase, phenylpyruvate decarboxylase (18), branched-chain Kdc (8, 19), 2-ketoglutarate decarboxylase (10, 17, 20), and indole-3-pyruvate decarboxylase (13).In this work, unexpectedly, we find that Kdc is nonessential for E. coli to produce isobutanol (Fig. ​(Fig.1).1). An E. coli strain overexpressing only alsS (from B. subtilis), ilvC, and ilvD (both from E. coli) is still able to produce isobutanol. Since E. coli is not a natural producer of isobutanol, it cannot be detected from the culture media in any unmodified strain. We identify that AlsS from B. subtilis, which was introduced in E. coli for acetolactate synthesis (Als), catalyzes the decarboxylation of 2-ketoisovalerate like Kdc both in vivo and in vitro. AlsS is part of the acetoin synthesis pathway and catalyzes the aldo condensation of two molecules of pyruvate to 2-acetolactate (Als activity) (Fig. ​(Fig.1C)1C) (11). The overall reaction catalyzed by AlsS is irreversible because of CO₂ evolution. The first step in catalysis is the ionized thiazolium ring of TPP reacting with the first pyruvate, followed by decarboxylation. This intermediate then reacts with the second pyruvate. Deprotonation followed by C-C bond breakage produces 2-acetolactate. In this work, mutational approaches were used to assess the importance of Q487 in the Kdc activity of AlsS.     

Hepatitis C Virus Egress and Release Depend on Endosomal Trafficking of Core Protein

Hepatitis C virus (HCV) assembly is known to occur in juxtaposition to lipid droplets, but the mechanisms of nascent virion transport and release remain poorly understood. Here we demonstrate that HCV core protein targets to early and late endosomes but not to mitochondria or peroxisomes. Further, by employing inhibitors of early and late endosome motility in HCV-infected cells, we demonstrate that the movement of core protein to the early and late endosomes and virus production require an endosome-based secretory pathway. We also observed that this way is independent of that of the internalization of endocytosed virus particles during virus entry.Hepatitis C virus (HCV) is a major causative agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV usually infects host cells via receptor-mediated endocytosis (6, 21), followed by the release of genomic RNA after uncoating of the nucleocapsid in the endosome. HCV core protein constitutes the viral nucleocapsid and may possess multiple functions. Intracellular HCV core protein is localized mainly in lipid droplets (LDs) (23, 29). Recent studies have indicated that core protein promotes the accumulation of LDs to facilitate virus assembly (1, 10) and recruits viral replication complexes to LD-associated membranes, where virus assembly takes place (23). However, the precise mechanisms of HCV assembly, budding, and release remain largely unclear. Most recently, HCV virion release has been shown to require the functional endosomal sorting complex required for transport III (ESCRT-III) and Vps4 (an AAA ATPase) (13), which are required for the biogenesis of the multivesicular body (MVB), a late endosomal compartment (12). Late endosomes have been implicated in the budding of several other viruses, including retroviruses (8, 17, 24, 25, 27), rhabdoviruses (14), filoviruses (18, 20), arenaviruses (26, 32), and hepatitis B virus (35). However, little is known about the roles of late endosomes in the HCV life cycle.Since LDs are associated with the endoplasmic reticulum membrane, endosomes, peroxisomes, and mitochondria (16, 37), we investigated what subcellular compartments may be involved in HCV assembly and release. We first compared the intracellular distribution of HCV core protein with that of early endosome markers Rab5a and early endosome antigen 1 (EEA1), as well as the late endosome marker CD63 in the HCV Jc1-infected Huh7.5 cells at day 10 postinfection (p.i.). In immunofluorescence studies, we demonstrated that the core protein partially colocalized with Rab5a (Fig. ​(Fig.1A,1A, left panel) or EEA1 (Fig. ​(Fig.1A,1A, right panel). This finding was confirmed by the expression of enhanced green fluorescent protein (EGFP)-tagged Rab5a (Fig. ​(Fig.1A,1A, middle panel). Similarly, core protein also showed partial colocalization with CD63 (Fig. ​(Fig.1B).1B). In particular, core protein showed numerous vesicle-like structures of homogeneous size that partially colocalized with CD63 at the cell periphery (Fig. ​(Fig.1B,1B, right panel inset and drawing). This result contrasts with that of Ai et al. (2), who observed, by confocal microscopy, that core protein did not interact with markers of early and late endosomes. Ai et al. did find, however, that multimeric core complexes cofractionated with ER/late endosomal membranes in HCV-infected cells.Open in a separate windowFIG. 1.HCV core protein colocalized with early and late endosomes but not mitochondria and peroxisomes. HCV-infected cells were costained with anti-core protein (red) and anti-Rab5a (A, left panel), -EEA1 (A, right panel), or -CD63 antibodies (green) (B) or transfected with plasmids expressing enhanced green fluorescent protein (EGFP)-Rab5a (A, middle panel), enhanced yellow fluorescent protein (EYFP) (C, left panel), EYFP-mitochondria (C, middle panel), or EYFP-peroxisome (C, right panel). Cellular DNA was labeled with DAPI (4′,6-diamidine-2-phenylindole) (blue). Images shown were collected sequentially with a confocal laser scanning microscope and merged to demonstrate colocalization (yellow merge fluorescence). Enlarged views of parts of every image are shown (insets). The cartoon in panel B illustrates the core protein-containing vesicle-like structures (depicted as red circles), which partially colocalized with CD63 at the cell periphery in HCV-infected cells. PM, plasma membrane.To demonstrate the specificity of the association of core protein with endosomes, we transfected HCV-infected cells (at day 10 p.i.) with pEYFP, pEYFP-mito, and pEYFP-peroxi (Clontech) (Fig. ​(Fig.1C),1C), which label the cytoplasm/nucleus, mitochondria, and peroxisomes, respectively. The results showed that HCV core protein did not colocalize with mitochondria or peroxisomes. Taken together, these results indicate that core protein is partially associated with early and late endosomes.To investigate the functional involvement of the endosomes in HCV release, we employed HCV-infected cells (at day 10 p.i.). In our observation, at day 10 p.i., not all the cells were infected with HCV, as revealed by immunofluorescence staining against core protein (data not shown), suggesting that these cells are a mixture of infected and noninfected cells. We examined the effects of inhibitors of endosome movement, including 10 μM nocodazole (which induces microtubule depolymerization), 100 nM wortmannin (which inhibits early endosomes), 20 nM Baf-A1 (which blocks early endosomes from fusing with late endosomes) (Sigma), and 10 μg/ml U18666A (which arrests late endosome movement) (Biomol), on the release of HCV in the HCV-infected cells. We first determined the possible cytotoxicity of these drugs. We found that within 20 h of the drug application, no significant effect on cell viability, as revealed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay, was observed (Fig. ​(Fig.2C).2C). We therefore treated the cells with the various drugs for 20 h. This protocol focuses only on virus release, not on virus entry, as the reinfection of Huh7.5 cells could not account for the effects on virus release, as one round of HCV replication requires about 24 h (11).Open in a separate windowFIG. 2.HCV virion release requires endosome motility. HCV-infected cells (at day 10 p.i.) were treated with DMSO, nocodazole (10 μM), U18666A (10 μg/ml), Baf-A1 (20 nM), or wortmannin (100 nM) for 20 h, and then the levels of extracellular (A) and intracellular (B) HCV RNA and HCV core proteins (F) in cells were analyzed by RT-qPCR and Western blotting, respectively. Results of Western blotting were quantified by PhosphorImager counting. (C) Analysis of cellular proliferation and survival by MTS assay. (D) Assay of extracellular viral infectivity. The culture supernatants from the cells treated with the various drugs as indicated were used to infect naïve Huh7.5 cells. The cells were stained with anti-core protein antibody (green) and DAPI (blue). The images were analyzed by using Metamorph, and the proportion of cells (of 5,000 counted) expressing core protein was counted (E). Results are presented as percentages and are averages and standard deviations from results of triplicate experiments. (G) In parallel, the HCV-infected cells were costained with anti-Rab5a (green) and -NS5A (red) antibodies. Cellular DNA was labeled with DAPI (blue). Enlarged views of parts of every image are shown (insets). (H) Colocalization efficiency between NS5A and early endosomes was analyzed by using Zeiss LSM Zen software. Error bars represent standard deviations of the mean result from 20 cells of two experiments. (I) Assay of intracellular HCV titers. Intracellular HCV particles were prepared from the HCV-infected cells (at day 10 p.i.) treated with U18666A at concentrations varying from 2.5 to 10 μg/ml for 20 h. The titers of intracellular HCV particles were determined by immunofluorescence staining for core-positive cell foci and are reported in focus-forming units (FFU)/ml. (J) HCV-infected cells (at day 10 p.i.) were treated with U18666A at concentrations varying from 2.5 to 20 μg/ml for 8 h, and then the levels of extracellular HCV RNA were analyzed by RT-qPCR. (K) Effects of nocodazole (10 μM), U18666A (10 μg/ml), Baf-A1 (20 nM), or wortmannin (100 nM) on HCV production in single-cycle HCV growth assays. Huh7.5 cells were infected with HCV at an MOI of 1 and then incubated with DMEM containing the various drugs. At 24 h p.i., the cells and their culture supernatants were collected and used to determine the levels of intracellular and extracellular HCV RNA, which were converted to percentages of the control levels (DMSO) as 100%. Noc, nocodazole; U18, U18666A; Baf-A1, Bafilomycin A1; Wortman, wortmannin.After treatments for 20 h, the cells and their culture supernatants were collected. Intracellular RNA was isolated from cell lysates using a High Pure RNA isolation kit (Roche), and viral RNA was isolated from cell culture supernatants using a QIAamp viral RNA kit (Qiagen). Equivalent RNA volumes were subsequently analyzed on a LightCycler 1.5 real-time PCR system (Roche) for quantitative PCR (qPCR), with a primer-probe set specific for the 5′ untranslated region (UTR) sequence of HCV Jc1 and a second set specific for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene to quantitate the RNA amount. To calculate the percentage of HCV RNA remaining after the inhibitor treatments, the mean HCV RNA levels from triplicate wells of each sample type were standardized to the mean GAPDH RNA level in the dimethyl sulfoxide (DMSO) control wells. The relative levels of HCV RNA (percentage of control) were then analyzed with LightCycler software (version 3.53) and calculated using the relative quantification method as described in Roche Applied Science, Relative Quantification, Technical Note no. LC 13/2001 (http://www.gene-quantification.de/roche-rel-quant.pdf).As indicated in Fig. ​Fig.2A,2A, the extracellular HCV RNA levels were reduced to 32.1%, 21.7%, 76.2%, and 53.8% of the DMSO control after treatments with nocodazole, U18666A, Baf-A1, and wortmannin, respectively. We further determined the intracellular levels of HCV RNA to test whether these drugs have an effect on HCV RNA replication. Intracellular HCV RNA was reduced to 64% by nocodazole treatment (Fig. ​(Fig.2B),2B), confirming previous reports that microtubules are required for HCV RNA synthesis (9, 28). Treatments with Baf-A1 and wortmannin, however, did not affect HCV RNA replication. Interestingly, the intracellular HCV RNA level was increased to 167% by U18666A treatment (Fig. ​(Fig.2B),2B), suggesting that U18666A blocks HCV particle release (Fig. ​(Fig.2A),2A), thereby causing accumulation of HCV RNA in the late endosomes. We further determined the extracellular viral infectivity after treatments with these drugs by using the culture supernatant to infect naïve Huh7.5 cells. The infectivity was checked by counting core protein-expressing cells. Raw acquired 8-bit images (Fig. ​(Fig.2D)2D) were converted to 16-bit and analyzed with the Multi Wavelength Cell Scoring application module in Metamorph (Molecular Device). All of these treatments significantly inhibited the production of infectious HCV particles, as shown by the reduced infectivity in the supernatant of the infected cells (Fig. 2D and E). Determination of the amounts of core protein in the lysates showed that the levels of core protein in cells were not significantly affected by the various drug treatments (Fig. ​(Fig.2F).2F). Taken together, these results suggest that the inhibitors of endosome movement, including U18666A, Baf-A1, and wortmannin, reduced the secretion of HCV particles but not the HCV RNA replication. On the other hand, nocodazole-induced microtubule depolymerization reduced both HCV RNA synthesis (Fig. ​(Fig.2B)2B) (9, 28) and virus release (Fig. 2A and E). Since nocodazole also blocks the movement of endosomes between pericentriolar regions and the cell periphery (3, 7), it therefore should perturb particle release. Our present results show that nocodazole had a greater effect on the reduction in extracellular HCV RNA levels (to 32%) than intracellular HCV RNA levels (to 64%) (Fig. 2A and B), and this effect may be due to blocking of endosome movement and reduced HCV RNA replication. These results can be explained on the premise that nocodazole treatments may affect both HCV RNA replication and virus release; it led us to conclude that microtubules may simultaneously play a key role in both HCV RNA replication and virus egress. Thus, both microtubules and the movement of endosomes are required for HCV particle egress.Earlier reports indicated that Rab5, an early endosomal protein, interacts with NS4B (31) and is required for HCV RNA replication. This effect was demonstrated in Rab5 small interfering RNA (siRNA)-transfected replicon cells (5, 31). However, in our current studies (Fig. ​(Fig.2B)2B) treatments with endosome inhibitors did not reduce the levels of intracellular HCV RNA. In order to investigate the discrepancy between our results and the earlier studies, we examined the effects of endosome inhibitors on the colocalization of NS5A with Rab5a in the HCV-infected cells (at day 10 p.i.) by calculating the percentage of colocalization in the cells. Colocalization scatter diagrams were generated using the colocalization function of the Zeiss LSM Zen software. The weighted colocalization coefficient, defined as the sum of intensities of colocalizing pixels for NS5A with Rab5a in comparison to the overall sum of pixel intensities (above threshold) for NS5A, was determined. Under control conditions (DMSO), NS5A was colocalized with Rab5a throughout the cytoplasm and perinuclear region (Fig. ​(Fig.2G,2G, upper left panel). The proportion of NS5A that colocalized with Rab5a was 41% (Fig. ​(Fig.2H).2H). After treatment with U18666A, the dispersed Rab5a compartments were found only in the perinuclear region (Fig. ​(Fig.2G,2G, upper right panel). Importantly, the colocalization of NS5A with Rab5a was increased to 57% by U18666A treatment. Treatments with Baf-A1 and wortmannin, however, had no significant effect. In contrast, nocodazole treatment reduced the colocalization of NS5A with Rab5a to 12% (Fig. ​(Fig.2G2G and ​and2H).2H). Previous studies have reported that the expression levels of Rab5 and its colocalization with HCV NS4B (or NS5A) play a functional role in HCV RNA replication (31). In addition, Rab5 remained in an early endosome fraction and the expression levels of Rab5 showed no significant difference between wortmannin (100 nM)-treated or untreated cells (22). More importantly, the total levels of early endosome proteins, including EEA1 (Fig. ​(Fig.3F)3F) and Rab5a (data not shown), are not altered by the endosome inhibitors. The colocalization efficiency of NS5A with Rab5a was not reduced by U18666A, Baf-A1, or wortmannin treatments, suggesting that these drugs cannot decrease HCV RNA replication. This finding is consistent with the previous results (Fig. ​(Fig.2B).2B). These findings suggest that the observed discrepancy between our results in Fig. ​Fig.2B2B and those of the other studies (5, 31) is most likely due to differences in the expression levels of Rab5a.Open in a separate windowFIG. 3.HCV particles formed are transported from early to late endosomes. HCV-infected cells (at day 10 p.i.) were treated with the various drugs and 14 h later labeled with antibodies specific for core protein (red) and CD63 (green) (A) or EEA1 (green) (D). At the right is an enlarged area from the merged image. Nuclei were stained with DAPI (blue). (B) (E) Colocalization efficiency between core protein and early or late endosomes was analyzed by using Zeiss LSM Zen software. Error bars represent standard deviations of the mean result from 20 cells of two experiments. In parallel, the cell lysates were collected and then immunoblotted with antibodies against CD63 (C) and EEA1 (F). Results were quantified by PhosphorImager counting. The HCV-infected cells (at day 10 p.i.) were fixed either for immunofluorescence microscopy (G) or for thin-section electron microscopy (H and I). Cells were costained with anti-Rab5a (green) (G, left panel), -CD63 (green) (G, right panel) and -core protein (red). Lipid droplets (LDs) and nuclei were stained with BODYPI 493/503 (blue) and DAPI (white), respectively (G). Enlarged views of parts of every image are shown (insets). (H, left panel) Early endosome (EE) (white arrow) containing particles resembling HCV adjacent to the LDs. (I, left panel) MVB/late endosome (LE) containing particles resembling HCV and internal vesicles (white arrow). High-magnification images of the early endosome (H, right panel) and MVB/late endosome (I, right panel) harboring particles resembling HCV (black arrow).To further confirm that U18666A suppresses HCV release and/or virus assembly, we determined the titer of the accumulated infectious virus particles inside the cells. The HCV-infected cells (at day 10 p.i.) were treated with U18666A at various concentrations between 2.5 and 10 μg/ml for 20 h, and then intracellular HCV particles were isolated from the cells by repeated freezing and thawing (15). The infectivity was assayed on naïve Huh7.5 cells. The results showed that the titers of infectious intracellular HCV particles were increased in a dose-dependent manner by the U18666A treatments (Fig. ​(Fig.2I).2I). These data, combined with our previous results (Fig. 2A, B, D, and E), indicate that U18666A blocks HCV particle release. Further, we determined the 50% effective dose (ED₅₀) and 50% cytotoxic concentration (CC₅₀), which are defined as the concentration of U18666A that reduced the levels of extracellular HCV RNA by 50% and the concentration of U18666A that produced 50% cytotoxicity in an MTS assay, respectively. We observed that the HCV-infected cells (at day 10 p.i.) treated with U18666A at concentrations varying from 2.5 to 20 μg/ml for 8 h showed a dose-dependent reduction in extracellular HCV RNA levels (Fig. ​(Fig.2J).2J). The ED₅₀ and CC₅₀ were calculated by polynomial regression analysis. An ED₅₀ of 8.18 μg/ml (19.2 μM) and a CC₅₀ of 40.26 μg/ml (94.9 μM) were observed for U18666A for the reduction of extracellular HCV RNA and the cytotoxicity of HCV-infected cells, respectively. These results indicate that U18666A acts as a specific inhibitor of HCV release.In our previous results in Fig. 2A and D, we used a multiple-cycle virus growth assay, which could not discriminate the role of endosome movement in infection from that in virus assembly or egress. We therefore used a single-cycle HCV growth assay to further confirm that these endosome inhibitors could suppress HCV release. Previous studies have suggested that one round of HCV replication requires about 24 h (11). Therefore, Huh7.5 cells were infected with HCV JC1 at a multiplicity of infection (MOI) of 1 for 3 h. The HCV-infected cells were washed with phosphate-buffered saline (PBS) and then incubated with Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and nocodazole (10 μM), U18666A (10 μg/ml), Baf-A1 (20 nM), or wortmannin (100 nM). Therefore, the experiment will focus on virus egress and RNA replication instead of virus entry because the cells were first inoculated with HCV and then treated with the various drugs. At 24 h p.i., the cells and their culture supernatants were collected. Intracellular RNA and viral RNA were isolated from cell lysates and cell culture supernatants, respectively. The percentage of HCV RNA remaining after the inhibitor treatments was determined in the same way as for Fig. 2A and B. As indicated in Fig. ​Fig.2K,2K, the levels of extracellular HCV RNA (or viral particles) were reduced to 61%, 48.8%, 59.7%, and 60.4% after treatments with nocodazole, U18666A, Baf-A1, and wortmannin, respectively, compared to the control DMSO treatment. We further determined the intracellular levels of HCV RNA to test whether these drugs have an effect on HCV RNA replication. Intracellular HCV RNA was reduced to 69% by nocodazole treatment (Fig. ​(Fig.2K),2K), but not by U18666A, Baf-A1, and wortmannin. Overall, these results of single-cycle HCV growth assay are similar to those of multiple-cycle virus growth assay (Fig. 2A and B), again suggesting that the endosome movement inhibitors reduced the secretion of HCV particles.To further understand the roles of the early and late endosomes in the HCV life cycle, we next examined the effects of endosome inhibitors on the colocalization of core protein with CD63 or EEA1 in the HCV-infected cells (at day 10 p.i.) by calculating the percentage of their colocalization in the cells. The percentage of colocalization of core protein with CD63 or EEA1 was determined in the same way as for Fig. ​Fig.2H.2H. Under control conditions (DMSO), core protein was colocalized with CD63 throughout the cytoplasm, perinuclear region, and cell periphery (Fig. ​(Fig.3A,3A, top row). The proportion of core protein that colocalized with CD63 was 17% (Fig. ​(Fig.3B).3B). After treatment with U18666A, a characteristic collapse of dispersed CD63 compartments to the perinuclear region of the cells was revealed (Fig. ​(Fig.3A,3A, second row). Importantly, the colocalization of core protein with CD63 increased to 30% when the movement of late endosome was arrested by U18666A, whereas it was reduced to 2% and 7% by Baf-A1 and wortmannin treatments (Fig. 3A and B), respectively, suggesting that the movement of core protein was blocked by U18666A and accumulated in the juxtanuclear region. Additionally, EEA1-labeled distinct puncta, which are dispersed throughout the cytoplasm and partially colocalized with core protein in DMSO treatment (Fig. ​(Fig.3D,3D, top row), were found clustered in the perinuclear region following treatments with U18666A and Baf-A1 (Fig. ​(Fig.3D,3D, second and third rows). Correspondingly, the colocalization of core protein and EEA1 was increased after these treatments. In contrast, very little colocalization between core protein and EEA1 was seen after treatment with wortmannin (Fig. ​(Fig.3D,3D, bottom row). The colocalization of core protein and EEA1 was 10% in the DMSO control, in contrast with 35% and 20% after treatments with U18666A and Baf-A1, respectively, and 4% after treatment with wortmannin (Fig. ​(Fig.3E).3E). These data suggest that core protein was blocked by U18666A and accumulated in the juxtanuclear region. In parallel, the levels of CD63 and EEA1 protein in the lysates were determined by Western blotting. The results indicated that the total levels of CD63 and EEA1 were not altered by the various drug treatments (Fig. 3C and F, respectively). These results collectively indicate that the colocalization of core protein with CD63 or EEA1 and the release of virus particles depend on the motility of endosomes. Thus, we suggest that HCV core and/or the viral particles formed are transported from early to late endosomes.The above results prompted us to characterize the location of the early and late endosomes in relation to the site of core-LD colocalization, where HCV assembly takes place (23). In HCV-infected cells (at day 10 p.i.), early endosomes (Fig. ​(Fig.3G,3G, left panel) were colocalized with core protein and were located in juxtaposition to LDs, whereas the late endosomes were located far away from LDs (Fig. ​(Fig.3G,3G, right panel). These data suggest that following the assembly of viral particles in juxtaposition to LDs, the HCV particles are transported through early to late endosomes. To gain further insight into the trafficking patterns of HCV particles in Huh7.5 cells, we performed electron microscopy of the HCV-infected cells (at day 10 p.i.). Particles resembling HCV were present in both the early endosomes adjacent to the LDs (Fig. ​(Fig.3H)3H) and MVBs (late endosomes) (Fig. ​(Fig.3I).3I). The morphology of these endosomal compartments is similar to that reported previously (34, 36). These results again suggest that the HCV particles formed are transported from early to late endosomes.In order to rule out the possibility that the reduction in HCV particle secretion by endosome inhibitors (Fig. ​(Fig.2)2) was caused by the inhibition of endocytosis-mediated virus entry, we determined the percentage of cells that could be infected by HCV in the presence of endosome inhibitors. Cells were first treated with inhibitors of endosome movement, followed by HCV inoculation for 3 days. This analysis revealed that the same percentage of cells was infected and produced core protein following DMSO and U18666A treatments, 52% and 54%, respectively (Fig. 4A and B), demonstrating that U18666A did not affect HCV entry, and HCV could proceed normally to RNA replication. In contrast, Baf-A1 and wortmannin treatments yielded 7% and 20% (Fig. 4A and B), respectively, of infected cells, indicating that they blocked HCV entry, as previously reported (6, 21). Overall, these findings indicate that late endosome motility is dispensable for HCV entry and subsequent RNA replication and translation but is required for viral egress.Open in a separate windowFIG. 4.HCV entry and RNA replication are not affected by inhibiting late endosome movement, and endosomal localization of core protein is not affected by inhibiting endocytosis. Huh7.5 cells were treated with the various drugs and 14 h later washed and inoculated with HCV Jc1. At 3 days p.i., cells were stained with anti-core protein antibody (green) and DAPI (blue) (A). The images were analyzed by using Metamorph and the proportion of cells (of 5,000 counted) expressing core protein was counted (B). (C) Colocalization of HCV core protein and late endosomes is not affected by DN mutants of Esp15 or Rab5a. HCV-infected cells (at day 5 p.i.) were transfected with a control plasmid pCMV-IE (C, left panel) or with dominant negative mutants of Eps15 (pEGFP-Eps15-DN) (C, middle panel) or Rab5a (pEGFP-Rab5a-DN) (C, right panel). At day 2 posttransfection, cells were labeled with antibodies specific for core protein (blue) and CD63 (red). Cells expressed EGFP-Eps15-DN or -Rab5-DN proteins (green). Nuclei were stained with DAPI (white). Enlarged views of parts of every image (insets) are shown. Colocalization of core protein and CD63 is depicted in magenta. (D) Results from colocalization analysis are shown using Zeiss LSM Zen software. Error bars represent standard deviations of the mean result from 20 cells of two experiments.Moreover, we observed an almost complete block in virus infection after expression of either an Eps15 dominant negative (DN) mutant, EGFP-Eps15D95/295 (EGFP-Eps15-DN) (4), or a Rab5a dominant negative mutant, EGFP-Rab5a-S34N (EGFP-Rab5a-DN) (30), in naïve Huh7.5 cells (data not shown). This finding is consistent with a previous report (21). We further studied the effects of the DN mutants of Eps15 and Rab5a on the colocalization of core protein and the late endosomes to confirm that this colocalization was not due to the process of virus entry. Since the DN mutants of endocytosis will block HCV infection, we first performed virus infection followed by expression of the DN mutants. This strategy has been used to demonstrate that the trafficking of HIV-1 RNA and Gag protein to late endosome is independent of the endocytosed virus (19). EGFP-Eps15-DN and EGFP-Rab5-DN were transfected into HCV-infected cells (at day 5 p.i.). As shown in Fig. ​Fig.4C,4C, the localization of core protein with CD63 in these cells was not affected by the expression of these DN mutants; core protein remained well colocalized with CD63 in both the cell periphery and juxtanuclear positions in the multiple-cycle HCV growth assays. The calculated efficiency of colocalization of core protein with CD63 (19 to ∼20%; Fig. ​Fig.4D)4D) was not altered. These results indicate that core protein localization with late endosomes is not the result of the accumulation of endocytosed viruses but rather represents the trafficking intermediates of the core protein during the late viral replication stages. Thus, we conclude that the late endosome-based secretory pathways are not involved in virus entry but rather deliver the assembled virions to the extracellular milieu.Taken together, our results indicate that HCV egress requires the motility of early to late endosomes, which is microtubule dependent, and that this pathway is independent of the one required for virus entry. Thus, we postulate that following the assembly of virus particles in juxtaposition to LDs, the HCV particles are transported through early to late endosomes to the plasma membrane, where the membrane of late endosomes is fused with plasma membrane to release virions into the extracellular milieu. This transport appears to be important for HCV egress, but it is not clear how endosomes adapt in these processes. It is possible that HCV particles are transported into endosomes after their synthesis near LD. The endosome may facilitate transport of the virus particles to the plasma membrane or even to specialized cell surface domains, such as cell junctions. Notably, the tight junction protein claudin 1 has been reported to be required for HCV cell-to-cell transmission (33), which may help viruses to sequester away from the immune system.     

Proteasomal Turnover of Hepatitis C Virus Core Protein Is Regulated by Two Distinct Mechanisms: a Ubiquitin-Dependent Mechanism and a Ubiquitin-Independent but PA28γ-Dependent Mechanism

We have previously reported on the ubiquitylation and degradation of hepatitis C virus core protein. Here we demonstrate that proteasomal degradation of the core protein is mediated by two distinct mechanisms. One leads to polyubiquitylation, in which lysine residues in the N-terminal region are preferential ubiquitylation sites. The other is independent of the presence of ubiquitin. Gain- and loss-of-function analyses using lysineless mutants substantiate the hypothesis that the proteasome activator PA28γ, a binding partner of the core, is involved in the ubiquitin-independent degradation of the core protein. Our results suggest that turnover of this multifunctional viral protein can be tightly controlled via dual ubiquitin-dependent and -independent proteasomal pathways.Hepatitis C virus (HCV) core protein, whose amino acid sequence is highly conserved among different HCV strains, not only is involved in the formation of the HCV virion but also has a number of regulatory functions, including modulation of signaling pathways, cellular and viral gene expression, cell transformation, apoptosis, and lipid metabolism (reviewed in references 9 and 15). We have previously reported that the E6AP E3 ubiquitin (Ub) ligase binds to the core protein and plays an important role in polyubiquitylation and proteasomal degradation of the core protein (22). Another study from our group identified the proteasome activator PA28γ/REG-γ as an HCV core-binding partner, demonstrating degradation of the core protein via a PA28γ-dependent pathway (16, 17). In this work, we further investigated the molecular mechanisms underlying proteasomal degradation of the core protein and found that in addition to regulation by the Ub-mediated pathway, the turnover of the core protein is also regulated by PA28γ in a Ub-independent manner.Although ubiquitylation of substrates generally requires at least one Lys residue to serve as a Ub acceptor site (5), there is no consensus as to the specificity of the Lys targeted by Ub (4, 8). To determine the sites of Ub conjugation in the core protein, we used site-directed mutagenesis to replace individual Lys residues or clusters of Lys residues with Arg residues in the N-terminal 152 amino acids (aa) of the core (C152), within which is contained all seven Lys residues (Fig. ​(Fig.1A).1A). Plasmids expressing a variety of mutated core proteins were generated by PCR and inserted into the pCAGGS (18). Each core-expressing construct was transfected into human embryonic kidney 293T cells along with the pMT107 (25) encoding a Ub moiety tagged with six His residues (His₆). Transfected cells were treated with the proteasome inhibitor MG132 for 14 h to maximize the level of Ub-conjugated core intermediates by blocking the proteasome pathway and were harvested 48 h posttransfection. His₆-tagged proteins were purified from the extracts by Ni²⁺-chelation chromatography. Eluted protein and whole lysates of transfected cells before purification were analyzed by Western blotting using anticore antibodies (Fig. ​(Fig.1B).1B). Mutations replacing one or two Lys residues with Arg in the core protein did not affect the efficiency of ubiquitylation: detection of multiple Ub-conjugated core intermediates was observed in the mutant core proteins comparable to the results seen with the wild-type core protein as previously reported (23). In contrast, a substitution of four N-terminal Lys residues (C152K6-23R) caused a significant reduction in ubiquitylation (Fig. ​(Fig.1B,1B, lane 9). Multiple Ub-conjugated core intermediates were not detected in the Lys-less mutant (C152KR), in which all seven Lys residues were replaced with Arg (Fig. ​(Fig.1B,1B, lane 11). These results suggest that there is not a particular Lys residue in the core protein to act as the Ub acceptor but that more than one Lys located in its N-terminal region can serve as the preferential ubiquitylation site. In rare cases, Ub is known to be conjugated to the N terminus of proteins; however, these results indicate that this does not occur within the core protein.Open in a separate windowFIG. 1.In vivo ubiquitylation of HCV core protein. (A) The HCV core protein (N-terminal 152 aa) is represented on the top. The positions of the amino acid residues of the core protein are indicated above the bold lines. The positions of the seven Lys residues in the core are marked by vertical ticks. Substitution of Lys with Arg (R) is schematically depicted. (B) Detection of ubiquitylated forms of the core proteins. The transfected cells with core expression plasmids and pMT107 were treated with the proteasome inhibitor MG132 and harvested 48 h after transfection. His₆-tagged proteins were purified and subsequently analyzed by Western blot analysis using anticore antibody (upper panel). Core proteins conjugated to a number of His₆-Ub are denoted with asterisks. Whole lysates of transfected cells before purification were also analyzed (lower panel). Lanes 1 to 11, C152 to C152KR, as indicated for panel A. Lane 12; empty vector.To investigate how polyubiquitylation correlates with proteasome degradation of the core protein, we performed kinetic analysis of the wild-type and mutated core proteins by use of the Ub protein reference (UPR) technique, which can compensate for data scatter of sample-to-sample variations such as levels of expression (10, 24). Fusion proteins expressed from UPR-based constructs (Fig. ​(Fig.2A)2A) were cotranslationally cleaved by deubiquitylating enzymes, thereby generating equimolar quantities of the core proteins and the reference protein, dihydrofolate reductase-hemagglutinin (DHFR-HA) tag-modified Ub, in which the Lys at aa 48 was replaced by Arg to prevent its polyubiquitylation (Ub^R48). After 24 h of transfection with UPR constructs, cells were treated with cycloheximide and the amounts of core proteins and DHFR-HA-Ub^R48 at the indicated time points were determined by Western blot analysis using anticore and anti-HA antibodies. The mature form of the core protein, aa 1 to 173 (C173) (13, 20), and C152 were degraded with first-order kinetics (Fig. 2B and D). MG132 completely blocked the degradation of C173 and C152 (Fig. ​(Fig.2B),2B), and C152K6-23R and C152KR were markedly stabilized (Fig. ​(Fig.2C).2C). The half-lives of C173 and C152 were calculated to be 5 to 6 h, whereas those of C152K6-23R and C152KR were calculated to be 22 to 24 h (Fig. ​(Fig.2D),2D), confirming that the Ub plays an important role in regulating degradation of the core protein. Nevertheless, these results also suggest possible involvement of the Ub-independent pathway in the turnover of the core protein, as C152KR is more destabilized than the reference protein (Fig. ​(Fig.2C2C and ​and2D2D).Open in a separate windowFIG. 2.Kinetic analysis of degradation of HCV core proteins. (A) The fusion constructs used in the UPR technique. Open boxes indicate the DHFR sequence, which is extended at the C terminus by a sequence containing the HA epitope (hatched boxes). Ub^R48 moieties bearing the Lys-Arg substitution at aa 48 are represented by open ellipses. Bold lines indicate the regions of the core protein. The amino acid positions of the core protein are indicated above the bold lines. The arrows indicate the sites of in vivo cleavage by deubiquitylating enzymes. (B and C) Turnover of the core proteins. After a 24-h transfection with each UPR construct, cells were treated with 50 μg of cycloheximide/ml in the presence or absence of 10 μM MG132 for the different time periods indicated. Cells were lysed at the different time points indicated, followed by evaluation via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis using antibodies against the core protein and HA. (D) Quantitation of the data shown in panels B and C. At each time point, the ratio of band intensity of the core protein relative to the reference DHFR-HA-Ub^R48 was determined by densitometry and is plotted as a percentage of the ratio at time zero.We have shown that PA28γ specifically binds to the core protein and is involved in its degradation (16, 17). Recent studies demonstrated that PA28γ is responsible for Ub-independent degradation of the steroid receptor coactivator SRC-3 and cell cycle inhibitors such as p21 (3, 11, 12). Thus, we next investigated the possibility of PA28γ involvement in the degradation of either C152KR or C152. Since C152KR carries two amino acid substitutions in the PA28γ-binding region (aa 44 to 71) (17), we tested the influence of the mutations of C152KR on the interaction with PA28γ by use of a coimmunoprecipitation assay. When Flag-tagged PA28γ (F-PA28γ) was expressed in cells along with C152 or C152KR, F-PA28γ precipitated along with both C152 and C152KR, indicating that PA28γ interacts with both core proteins (Fig. ​(Fig.3A).3A). Figure ​Figure3B3B reveals the effect of exogenous expression of F-PA28γ on the steady-state levels of C152 and C152KR. Consistent with previous data (17), the expression level of C152 was decreased to a nearly undetectable level in the presence of PA28γ (Fig. ​(Fig.3B,3B, lanes 1 and 3). Interestingly, exogenous expression of PA28γ led to a marked reduction in the amount of C152KR expressed (Fig. ​(Fig.3B,3B, lanes 5 and 7). Treatment with MG132 increased the steady-state level of the C152KR in the presence of F-PA28γ as well as the level of C152 (Fig. ​(Fig.3B,3B, lanes 4 and 8).Open in a separate windowFIG. 3.PA28γ-dependent degradation of the core protein. (A) Interaction of the core protein with PA28γ. Cells were cotransfected with the wild-type (C152) or Lys-less (C152KR) core expression plasmid in the presence of a Flag-PA28γ (F-PA28γ) expression plasmid or an empty vector. The transfected cells were treated with MG132. After 48 h, the cell lysates were immunoprecipitated with anti-Flag antibody and visualized by Western blotting with anticore antibodies. Western blot analysis of whole cell lysates was also performed. (B) Degradation of the wild-type and Lys-less core proteins via the PA28γ-dependent pathway. Cells were transfected with the UPR construct with or without F-PA28γ. In some cases, cells were treated with 10 μM MG132 for 14 h before harvesting. Western blot analysis was performed using anticore, anti-HA, and anti-Flag antibodies. (C) After 24 h of transfection with UPR-C152KR and UPR-C191KR with or without F-PA28γ (an empty vector), cells were treated with 50 μg of cycloheximide/ml for different time periods as indicated (chase time). Western blot analysis was performed using anticore and anti-HA antibodies. The precursor core protein and the core that was processed, presumably by signal peptide peptidase, are denoted by open and closed triangles, respectively.We further investigated whether PA28γ affects the turnover of Lys-less core protein through time course experiments. C152KR was rapidly destabilized and almost completely degraded in a 3-h chase experiment using cells overexpressing F-PA28γ (Fig. ​(Fig.3C,3C, left panels). A similar result was obtained using an analogous Lys-less mutant of the full-length core protein C191KR (Fig. ​(Fig.3C,3C, right panels), thus demonstrating that the Lys-less core protein undergoes proteasomal degradation in a PA28γ-dependent manner. These results suggest that PA28γ may play a role in accelerating the turnover of the HCV core protein that is independent of ubiquitylation.Finally, we examined gain- and loss-of-function of PA28γ with respect to degradation of full-length wild-type (C191) and mutated (C191KR) core proteins in human hepatoma Huh-7 cells. As expected, exogenous expression of PA28γ or E6AP caused a decrease in the C191 steady-state levels (Fig. ​(Fig.4A).4A). In contrast, the C191KR level was decreased with expression of PA28γ but not of E6AP. We further used RNA interference to inhibit expression of PA28γ or E6AP. An increase in the abundance of C191KR was observed with PA28γ small interfering RNA (siRNA) but not with E6AP siRNA (Fig. ​(Fig.4B).4B). An increase in the C191 level caused by the activity of siRNA against PA28γ or E6AP was confirmed as well.Open in a separate windowFIG. 4.Ub-dependent and Ub-independent degradation of the full-length core protein in hepatic cells. (A) Huh-7 cells were cotransfected with plasmids for the full-length core protein (C191) or its Lys-less mutant (C191KR) in the presence of F-PA28γ or HA-tagged-E6AP expression plasmid (HA-E6AP). After 48 h, cells were lysed and Western blot analysis was performed using anticore, anti-HA, anti-Flag, or anti-GAPDH. (B) Huh-7 cells were cotransfected with core expression plasmids along with siRNA against PA28γ or E6AP or with negative control siRNA. Cells were harvested 72 h after transfection and subjected to Western blot analysis.Taking these results together, we conclude that turnover of the core protein is regulated by both Ub-dependent and Ub-independent pathways and that PA28γ is possibly involved in Ub-independent proteasomal degradation of the core protein. PA28 is known to specifically bind and activate the 20S proteasome (19). Thus, PA28γ may function by facilitating the delivery of the core protein to the proteasome in a Ub-independent manner.Accumulating evidence suggests the existence of proteasome-dependent but Ub-independent pathways for protein degradation, and several important molecules, such as p53, p73, Rb, SRC-3, and the hepatitis B virus X protein, have two distinct degradation pathways that function in a Ub-dependent and Ub-independent manner (1, 2, 6, 7, 14, 21, 27). Recently, critical roles for PA28γ in the Ub-independent pathway have been demonstrated; SRC-3 and p21 can be recognized by the 20S proteasome independently of ubiquitylation through their interaction with PA28γ (3, 11, 12). It has also been reported that phosphorylation-dependent ubiquitylation mediated by GSK3 and SCF is important for SRC-3 turnover (26). Nevertheless, the precise mechanisms underlying turnover of most of the proteasome substrates that are regulated in both Ub-dependent and Ub-independent manners are not well understood. To our knowledge, the HCV core protein is the first viral protein studied that has led to identification of key cellular factors responsible for proteasomal degradation via dual distinct mechanisms. Although the question remains whether there is a physiological significance of the Ub-dependent and Ub-independent degradation of the core protein, it is reasonable to consider that tight control over cellular levels of the core protein, which is multifunctional and essential for viral replication, maturation, and pathogenesis, may play an important role in representing the potential for its functional activity.     

Isoprenoids Are Essential for Fruiting Body Formation in Myxococcus xanthus

It was recently shown that Myxococcus xanthus harbors an alternative and reversible biosynthetic pathway to isovaleryl coenzyme A (CoA) branching from 3-hydroxy-3-methylglutaryl-CoA. Analyses of various mutants in these pathways for fatty acid profiles and fruiting body formation revealed for the first time the importance of isoprenoids for myxobacterial development.Myxobacteria are unique among the prokaryotes as (i) they can form highly complex fruiting bodies under starvation conditions, even up to microscopic tree-like structures (28); (ii) they can move on solid surfaces using different motility mechanisms (16); (iii) they produce some of the most cytotoxic secondary metabolites, with epothilone already in clinical use against cancer (2, 3); and (iv) they harbor the largest prokaryotic genomes found so far (15, 27). The large genome might be directly related to their complex life-style and the diverse secondary (3) and primary (9) metabolisms. Already in 2002 we found that myxobacteria are able to produce isovaleryl coenzyme A (IV-CoA) and compounds derived thereof via a new pathway that branches from 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), which is the central intermediate of the well-known mevalonate-dependent isoprenoid biosynthesis (Fig. ​(Fig.1)1) (22, 23). Usually IV-CoA is derived from leucine degradation via the branched-chain keto acid dehydrogenase (BKD) complex (24), which is also the preferred pathway to IV-CoA in the myxobacteria Myxococcus xanthus and Stigmatella aurantiaca (Fig. ​(Fig.2A).2A). However, in bkd mutants, where no or only residual leucine degradation is possible (30), the alternative pathway is induced (Fig. ​(Fig.2B),2B), presumably to ensure the production of iso-fatty acids (iso-FAs) (5). A possible reason for this alternative pathway is the importance of IV-CoA-derived compounds in the complex myxobacterial life cycle, which is the starvation-induced formation of fruiting bodies in which the cells differentiate into myxospores. We showed that this pathway is induced during fruiting body formation in M. xanthus when leucine is limited. Under these conditions, this pathway might be more important for protein synthesis than for lipid remodeling, as lipids are present in excess during development due to the surface reduction from vegetative rods to round myxospores as described previously (29). Examples of IV-CoA-derived compounds are the unusual iso-branched ether lipids, which are almost exclusively produced in the developing myxospores. They might serve as structural lipids and signaling compounds during fruiting body formation (26).Open in a separate windowFIG. 1.Biosynthesis of IV-CoA and compounds derived thereof and biosynthesis of isoprenoids in M. xanthus. Broken arrows indicate multistep reactions; supplementation (double-lined arrows) with MVL and IVA can be used to complement selected mutants.Open in a separate windowFIG. 2.Short representations of proposed metabolic fluxes through the IV-CoA/isoprenoid network. Broken arrows indicate no metabolic flux. (A) DK1622 (wild type); (B) DK5643 (Δbkd); (C) DK5624 (Δbkd mvaS::kan); (D) HB002 (Δbkd liuC::kan); (E) HB002 with 1 mM IVA; (F) HB002 with 1 mM MVL. Ac-CoA, acetyl-CoA; MVA, mevalonic acid.In M. xanthus, we could recently identify candidate genes involved in the alternative pathway from HMG-CoA to IV-CoA. We also described the genes required for the degradation pathway of leucine and subsequently also those involved in the transformation of IV-CoA to HMG-CoA (4). In myxobacteria leucine is an important precursor for isoprenoid biosynthesis, as was already shown elsewhere for the biosynthesis of steroids (7) and prenylated secondary metabolites like aurachin (22) or leupyrrins (6), as well as volatiles like geosmin or germacradienol in M. xanthus and S. aurantiaca (11, 13). The interconnection of iso-FAs and isoprenoid biosynthesis made it difficult to assign functions to these compound classes during fruiting body formation in M. xanthus because it cannot be excluded that reduced leucine degradation also impairs isoprenoid biosynthesis. A mutant strain of M. xanthus that was blocked in the degradation of leucine and the alternative pathway had a deletion in the bkd locus as well as a plasmid insertion in the mvaS gene encoding the HMG-CoA synthase (strain DK5624). This double mutation severely affected isoprenoid biosynthesis (5), and cultures of DK5624 must be supplemented with mevalonolactone (MVL; the cyclized form of mevalonic acid) in order to enable growth (Fig. ​(Fig.2C).2C). Since we have identified the genes involved in IV-CoA biosynthesis and the mevalonate pathway (4), we can now start to identify differences between strains that show deficiencies in iso-FAs and strains that show deficiencies in isoprenoids via simple analysis of the FA profile and analysis of the myxobacterial development of selected mutants.All mutants used in this study (HB002 [Δbkd liuC::kan], HB015 [Δbkd MXAN_4265::kan], DK5624 [Δbkd mvaS::kan], HB019 [Δbkd mvaS::kan mvaS⁺], and HB020 [Δbkd MXAN_4265::kan mvaS⁺]) have been published previously (4), and FA analysis as well as myxobacterial fruiting body formation has also been described previously (26).M. xanthus HB002 (Δbkd liuC) shows only residual amounts of iso-FAs, as both leucine degradation and the alternative pathway to IV-CoA are blocked (Fig. ​(Fig.2D)2D) and its capability to form fruiting bodies is strongly reduced (Fig. ​(Fig.3).3). The residual amount of iso-FAs results from a second BKD activity in M. xanthus that has been identified by residual leucine incorporation as well as by residual enzymatic activity in bkd mutants (23, 30). This second BKD activity might be a side activity of the pyruvate dehydrogenase or a related chemical oxidative decarboxylation, as no second bkd locus could be identified in the genome (unpublished results). Moreover, growth of HB002 is not MVL dependent because the block in the alternative pathway does not affect isoprenoid biosynthesis, as liuC encodes a dehydratase/hydratase that is involved in the conversion of HMG-CoA to 3-methylglutaconyl-CoA and vice versa (4). As expected, the FA profile (4) as well as the developmental phenotype (data not shown) can be complemented (Fig. ​(Fig.2E)2E) by the addition of isovaleric acid (IVA), the free acid of IV-CoA, indicating the importance of iso-branched compounds for development in M. xanthus. Unexpectedly, addition of MVL (Fig. ​(Fig.2F)2F) also partially restored fruiting body formation without restoring the FA profile (Fig. ​(Fig.3).3). Similarly, M. xanthus HB015 (Δbkd MXAN_4265::kan) can produce only traces of iso-FAs, as both pathways to IV-CoA are blocked. MXAN_4265 encodes a protein with similarity to a glutaconyl-CoA transferase subunit, but from our previous results, we postulated it to be involved in the alternative pathway to IV-CoA (Fig. ​(Fig.1)1) (4). The respective mutant shows a severely impaired developmental phenotype, which can be complemented not only by the addition of IVA (not shown) but also by the addition of MVL (Fig. ​(Fig.3).3). Again, no change in the FA profile was observed after the addition of MVL. However, a plasmid insertion into MXAN_4265 has a polar effect on mvaS, which is the last gene in this five-gene operon and which is crucial for HMG-CoA formation from acetoacetyl-CoA and acetyl-CoA. Therefore, we assume that both pathways to HMG-CoA are blocked in HB015: no HMG-CoA can be made from acetyl-CoA and hardly any can be made via leucine degradation. In order to prove this hypothesis, we complemented HB015 with an additional copy of mvaS under the constitutive T7A1 promoter as described previously, using the plasmid pCK4267exp (4). The resulting strain, HB020 (Δbkd MXAN_4265::kan mvaS⁺), showed a restored developmental phenotype but still produced only trace amounts of iso-FAs.Open in a separate windowFIG. 3.Fruiting body formation on TPM agar in selected mutants at 24, 48, and 72 h after starvation. Numbers refer to the relative amounts (in percentages) of the most abundant iso-FA, iso-15:0, which is indicative of iso-FAs in general. Strains were DK1622 (wild type), HB002 (Δbkd liuC::kan), HB015 (Δbkd MXAN_4265::kan), DK5624 (Δbkd mvaS::kan), HB019 (Δbkd mvaS::kan mvaS⁺), and HB020 (Δbkd MXAN_4265::kan mvaS⁺). DK5624 was grown with 0.3 mM MVL prior to starvation, and the cells were washed and plated on TPM with or without 1 mM of MVL.The data from HB002, HB015, and HB020 indicate an important function of the mevalonate-dependent isoprenoid pathway for fruiting body formation in M. xanthus. Therefore, MVL addition can at least partially complement the developmental phenotype of DK5624, which cannot form fruiting bodies without MVL (Fig. ​(Fig.3).3). However, genetic complementation with mvaS in HB019 resulted in the expected complementation of the fruiting body formation and the FA profile (Fig. ​(Fig.3,3, bottom row).Leucine is one of the most abundant proteinogenic amino acids. It is also an essential amino acid for M. xanthus (8), which has a predatory life-style (1), as it lives on other bacteria and fungi that contain a lot of leucine. Moreover, leucine is very efficiently incorporated into isoprenoids like geosmin and aurachin (10, 22). Thus, one can conclude that in fact leucine degradation is the major pathway for HMG-CoA biosynthesis instead of the usual formation via acetoacetyl-CoA and acetyl-CoA by the HMG-CoA synthase MvaS as indicated in Fig. ​Fig.2A.2A. No difference in growth was observed between culture with and culture without MVL for HB002 (Δbkd liuC::kan) and HB015 (Δbkd MXAN_4265::kan) in rich medium (data not shown), probably due to the complete MvaS activity (in HB002) or residual BKD activity (in HB002 and HB015), resulting in all precursors for the mevalonate-dependent isoprenoid biosynthesis still being present in excess under these conditions. However, under starvation conditions a small reduction in HMG-CoA biosynthesis caused by completely blocked leucine degradation (as in HB002 due to the mutation in liuC [Fig. ​[Fig.2D])2D]) or reduced leucine degradation and a mutation in mvaS (as in HB015) might each result in a reduced isoprenoid level, which can be complemented at least partially by the addition of MVL. This would also explain the difference in the developmental phenotypes of HB002 and HB015, with the phenotype being more severe in HB002 (Fig. ​(Fig.3).3). The fact that complementation with IVA is in all cases more efficient than that with MVL can be explained by the role of the already-mentioned isolipids. They can be produced only after IVA addition, which also complements the (developmental) phenotype of some of these mutants (26).As isoprenoids represent probably the most diverse class of natural products (14), it is very hard to predict which particular isoprenoids might be responsible for the observed effects. Several isoprenoids (7, 11-13), prenylated secondary metabolites (6, 22), and carotenoids (18-21) are known from myxobacteria in general, and a major volatile compound from M. xanthus is the terpenoid geosmin (13). In order to test whether geosmin might be required for fruiting body formation, we constructed a plasmid insertion mutant in MXAN_6247, which is involved in the cyclization of farnesyl diphosphate to geosmin, following published procedures (4, 5). The resulting strain, HB022, showed the expected loss in geosmin production but no developmental phenotype (data not shown).Additionally, it cannot be excluded that prenylated proteins, sugars, or quinones from the respiratory chain are important for fruiting body formation. Moreover, stigmolone has been described as a pheromone involved in fruiting body formation in S. aurantiaca (25). Although its biosynthesis has not been elucidated yet, stigmolone could be an isoprenoid as well, which is deducible from the two iso-branched residues within its chemical structure (17). Nevertheless, the importance of isoprenoids for M. xanthus is evident from the data presented, and clearly more work is needed to identify the compound(s) involved.     

Structural Study of the Serratia entomophila Antifeeding Prophage: Three-Dimensional Structure of the Helical Sheath

The sheath of the Serratia entomophila antifeeding prophage, which is pathogenic to the New Zealand grass grub Costelytra zealandica, is a 3-fold helix formed by a 4-fold symmetric repeating motif disposed around a helical inner tube. This structure, determined by electron microscopy and image processing, is distinct from that of the other known morphologically similar bacteriophage sheaths.The antifeeding prophage (Afp) of Serratia entomophila and Serratia proteamaculans is a naturally occurring virus tail-like structure which delivers a putative toxin molecule that leads to starvation of the New Zealand grass grub Costelytra zealandica (5). Afp is composed of 18 different gene products (molecular masses of 6.5 to 263 kDa). The first 16 open reading frames have orthologues (Photorhabdus virulence cassettes [PVC]) in the insecticidal bacterium Photorhabdus luminescens TTO1 genome (5). Afp and PVCs morphologically resemble a typical R-type bacteriocin (6, 12, 16) However, Afp is the only known phage tail-like protein complex that is not a bacteriocin-protein complex of considerable medical relevance that targets the same or closely related bacterial strains (1, 3, 8, 12). The major component of Afp is a contractile cylindrical outer sheath encasing an inner tube speculated to house the toxin molecule (6). A dome-shaped “head” defines one extremity of the tube, while the other end is attached to a “bell-shaped” structure with a base morphologically similar to the base plate of the T4 bacteriophage tail (9).Transmission electron micrographs of two-dimensional (2D) projections of negatively stained (Fig. ​(Fig.11 A) or frozen-hydrated and vitrified (Fig. ​(Fig.1B)1B) recombinant Afp particles (see Fig. S1 in the supplemental material) were used for computational image analysis. A globally averaged image of the Afp particle in the major configuration (called E here) (Fig. ​(Fig.1C),1C), generated using negatively stained specimens, clearly distinguished the morphologies of the various constituent structural parts. Thus, the cylindrical sheath appears to be formed by a periodic structure harboring a distinctive, inverted-V-shaped feature. A minor population of Afp particles displays an alternate configuration (called C here) where, concomitant with contraction of the sheath (averaged axial compression of ∼52% [see Table S1 in the supplemental material]), the inner tube, shorn off the bell-shaped structure, is revealed (Fig. ​(Fig.1A)1A) (6). Several other bacteriocins undergo such a high degree of compression, which has been characterized in detail for the tail sheath of bacteriophage T4 (9). We also generated individual global averages for the periodic sheath structure, for the bell-shaped structure, and for the inner tube (Fig. ​(Fig.1C)1C) which provide more accurate dimensions of these different sections (see Table S1 in the supplemental material) than those reported earlier (6).Open in a separate windowFIG. 1.(A) Electron micrograph of a negatively stained preparation of partially purified recombinant Afp particles. The gray, white, and black arrows point to an Afp particle in the extended (E) configuration, to an Afp particle with the sheath contracted, exposing the inner tube in the contracted (C) configuration, and to an inner tube with the bell-shaped structure attached at one end, respectively. (B) Cryoelectron micrograph recorded from a preparation similar to that shown in panel A. (C) Globally averaged images of Afp particles (3,026 images) in the E state and the three distinguishable parts visualized by negative staining. Bars, 200 Å. (D) Averaged power spectrum of the sheath of vitrified Afp particles. The black and white arrows indicate reflections delineating the axial rise (1/78.5 Å) and the helical pitch (1/118 Å), respectively. In panels A and C, lighter regions represent protein and the contrast is reversed in panel B. Global averages were created using classalign2 of the EMAN suite (10) and visualized in bshow (4).For a better insight, we determined the 3D structure of the central periodic section of the Afp particle in the E state. A global power spectrum derived from the cryoimages established the structure to be helical with a clear first meridional reflection at 1/78.5 Å and the first, strongest nonmeridional reflection at 1/118 Å (Fig. ​(Fig.1D).1D). These reflections correspond to the axial rise (Δx) and the pitch of the helix, respectively, and reflect a turn angle (Δψ) of about ±240° (3₂ helix) for the repeating motif. The correct sign, i.e., the hand, of the helix remains to be determined. Computationally excised overlapping segments of this helical section from images of vitrified and negatively stained Afp particles were subjected to 3D reconstruction using the iterative helical real-space reconstruction (IHRSR) algorithm (2) using the determined helical parameters (see Fig. S2 in the supplemental material). After a few iterations, the presence of an in-plane 4-fold symmetry (C4) was apparent in the density map (see Fig. S2 in the supplemental material), which was then imposed in the subsequent reconstruction exercises. However, no stable solution was forthcoming, even after many (e.g., 30) iterative cycles. This is generally indicative of the presence of heterogeneity in the form of variations in helix translation and/or twist angle (15) in the structure. As a first step, we focused our attention on the pitch value, and following classification (see Fig. S3 in the supplemental material), we found that the majority of the image segments correspond to a pitch of 120 Å. These segments were then selected out of the full data set and led to a stable and refined 3D reconstruction. We also obtained very comparable results for the helical section when images of negatively stained Afp sheath sections were used, thus supporting our computational approach (see Fig. S4 in the supplemental material) and general conclusions about the E state described below.Figure ​Figure22 A is an isosurface representation of the density map of the helical Afp sheath in the E state calculated at ∼21.5-Å resolution (see Fig. S5 in the supplemental material). To the best of our knowledge, a 4-fold rotational symmetry has not been seen for any other contractile T4 bacteriophage taillike structure, which points to the unique architecture of the Afp sheath. A power spectrum generated using the 2D projection from the final density map, compared to the experimental global power spectrum (Fig. ​(Fig.2D),2D), showed strong agreement, confirming the fidelity of the computational image analysis. The density map displays protein layers, ∼80 Å thick, that are stacked on each other in a periodic fashion. The uneven outer surface of the sheath is perforated and decorated with ∼35-Å protrusions. When rendered with a raised threshold, a characteristic feature of the map is a contiguous, high-electron-density region having an inverted-Y-shaped structure (Fig. 2C and E; see Fig. S4 in the supplemental material). At the modest ∼21.5-Å resolution, the boundary of the repeating subunit cannot be defined. A 25 ± 3-Å-wide central lumen is seen clearly when viewed along the helix axis (Fig. ​(Fig.2B)2B) and likely represents the pore of the inner tube (see also below). Using scanning transmission electron microscopy (STEM) (see Fig. S6 in the supplemental material), we estimated the averaged molecular mass of the central helical section of an Afp particle to be 9.8 ± 0.4 kDa/Å (Fig. ​(Fig.3)3) (14) and that of only the inner tube to be ∼2.5 kDa/Å, based on a relatively small pool of such images. These values translate to a mass contribution of approximately 145 kDa of the subunit whose periodic arrangement forms the outer component of the sheath (i.e., excluding the inner tube) (see Fig. S6 in the supplemental material). This value is not very different from the cumulative mass of the different proteins, i.e., homologous afp2, afp3, and afp4, thought to be involved in Afp sheath formation (5) (see Fig. S7 in the supplemental material).Open in a separate windowFIG. 2.Orthogonal isosurface rendering, at 1 σ (standard deviation) of the computed ∼21.5-Å density map of the helical sheath of the vitrified Afp particle viewed normal (A) and parallel (B) to the helix. The images were generated using the software package CHIMERA (13). The arrow indicates a surface perforation. (C) The Afp density map rendered at 3.5 σ to highlight the largest contiguous high-electron-density regions; one circumscribed by a black ellipse is computationally extracted and shown in panel E. (D) Comparison of the experimental, averaged power spectrum of the helical sheath of Afp (left) with that computed (right) from the 2D projection of the calculated density map. (F) Global average of the inner tube of the Afp particle and a plot of the surface density variation (scaled from 0 to 1) along the helical (y) axis. The dimension along the tube is plotted on the x axis.Open in a separate windowFIG. 3.(A) Dark-field micrograph of a freeze-dried, unstained preparation of Afp particles used in STEM measurements. An Afp particle in the E state, an Afp inner tube with the attached bell-shaped structure, and a tobacco mosaic virus particle, used as a calibration standard, are marked by the arrowhead and the gray and white arrows, respectively. (B) Histogram plot of the measured distributions of mass per unit length corresponding to the uniform periodic section of the Afp particles overlaid with a fitted Gaussian curve produced by using the ORIGIN6 software package.A paucity of images of the C state (∼5% of the complete data set) precluded a full, refined 3D reconstruction, but based on the available 2,774 overlapping image segments of the isolated inner tube, a global average was calculated. A plot of contrast variation (Fig. ​(Fig.2F)2F) indicates that the surface is characterized by ∼40-Å spaced elevated crests and invaginated grooves, in agreement with the calculated axial rise of ∼39 Å for the subunit (see Fig. S8 in the supplemental material) comprising the tube. Based on these preliminary results, it appears that the helical symmetry of the inner tube is markedly different from that of the sheath.Our observation that the pitch of the helix in the E state can vary by as much as ∼50 Å attests to the flexible nature of the sheath, which is required for compressibility and may be facilitated by the somewhat porous nature of the sheath (Fig. ​(Fig.2).2). Preliminary deductions (data not shown) based on a small pool of images of the C state suggest profound rearrangement of the elements of the sheath. How that translates to extrusion of the toxin remains to be revealed.      

Computational Analysis of Human Adenovirus Type 22 Provides Evidence for Recombination among Species D Human Adenoviruses in the Penton Base Gene

Recombination in human adenoviruses (HAdV) may confer virulence upon an otherwise nonvirulent strain. The genome sequence of species D HAdV type 22 (HAdV-D22) revealed evidence for recombination with HAdV-D19 and HAdV-D37 within the capsid penton base gene. Bootscan analysis demonstrated that recombination sites within the penton base gene frame the coding sequences for the two external hypervariable loops in the protein. A similar pattern of recombination was evident within other HAdV-D types but not other HAdV species. Further study of recombination among HAdVs is needed to better predict possible recombination events among wild-type viruses and adenoviral gene therapy vectors.Adenoviruses were first isolated in 1953 (18, 31) and currently cause an array of human diseases. These include respiratory, gastrointestinal, and ocular surface infections, opportunistic infections in immune-deficient individuals, and possibly, obesity (9, 13, 14, 20, 40). Adenoviruses have also recently been used as gene therapy vectors (19, 35). Thus, while adenoviruses continue to cause significant morbidity and mortality in the human population, their existence also provides a potential benefit for the treatment of patients with an even broader range of ailments.Since the characterization of the first human adenovirus (HAdV), 53 types have been identified and subsequently classified into seven species (A to G) on the basis of serology, restriction endonuclease digestion patterns, and to a lesser degree, genotyping. Recently, high-throughput sequencing technology has made whole-genome sequencing both rapid and affordable (27). However, the genomic sequences of 23 out of 53 HAdV types remain to be determined, with most of those within species D.Species D HAdV type 22 (HAdV-D22) was originally isolated from a child in 1956 (3). While it is not clear what role HAdV-D22 plays in human disease, one report revealed a possible tropism for the eye (32). Recently, recombination with HAdV-D22 has been identified as the source of a novel HAdV, HAdV-D53, causing an outbreak of keratoconjunctivitis in Germany (37). HAdV-D22 recombination was also identified in a possible variant of HAdV-D53 that was isolated from a patient in Japan (2). Therefore, HAdV-D22 has shown the propensity to recombine with other viruses, with clinically important consequences. The emergence of new pathogenic HAdV genotypes, along with continued interest in HAdVs as vectors for human gene therapy, make adenovirus recombination a critically important issue.HAdV-D22 was acquired from the American Type Culture Collection (Manassas, VA). The complete genome of the prototype strain AV-2711 (ATCC VR-1100) was sequenced on an Applied Biosystems (Foster City, CA) 3730 XL DNA sequencer in the Laboratory for Genomics and Bioinformatics at the University of Oklahoma Health Sciences Center using a previously described protocol (29). The sequence was validated by sequencing on an ABI SOLiD DNA sequencer. Sequences from both methodologies were 100% identical and provided 7,727-fold coverage for the genome.The mVISTA Limited Area Global Alignment of Nucleotides (LAGAN) tool (http://genome.lbl.gov/vista/index.shtml) was used to align and compare the whole HAdV genomes (6) of HAdV-D22 and each of the other nine completely sequenced HAdV-Ds. Analysis revealed sequence diversity in the penton base, hexon, E3, and fiber open reading frames (Fig. ​(Fig.1).1). Surprisingly, comparison of HAdV-D22 to HAdV-D19 strain C (30) and HAdV-D37 (29) revealed considerable sequence conservation in the penton base gene (Fig. ​(Fig.11 and 2A and B).Open in a separate windowFIG. 1.Global pairwise sequence alignment of HAdV-D22 with nine other HAdV-D types. The percent sequence conservation is reflected in the height of each data point along the y axis. A conserved sequence in the penton base open reading frame is designated by an asterisk. GenBank accession numbers are as follows: HAdV-D8, AB448768; HAdV-D9, AJ854486; HAdV-D19, EF121005; HAdV-D26, EF153474; HAdV-D37, DQ900900; HAdV-D46, AY875648; HAdV-D48, EF153473; HAdV-D49, DQ393829; and HAdV-D53, FJ169625.Open in a separate windowFIG. 2.Multisequence alignment of HAdV-D penton base genes. Nucleotide alignment of the bp 397 to 594 (A) and bp 694 to 1089 (B) regions of the penton base gene.Based on the sequence conservation seen in the penton base, Simplot 3.5.1 (http://sray.med.som.jhmi.edu/SCRoftware/simplot/) and Recombination Detection Program (RDP) version 3.34 (http://darwin.uvigo.es/rdp/rdp.html) were used to identify possible recombination sites (21, 24). Bootscan analysis identified two possible recombination loci in the HAdV-D22 penton base, the first in HAdV-D37, encompassing nucleotides 400 to 600, and the second in HAdV-D19 at nucleotides 750 to 1350 (Fig. ​(Fig.3A).3A). In silico amino acid analysis showed that these two probable recombination areas code for the two variable loops in the penton base protein (Fig. ​(Fig.3A),3A), located on the exterior of the viral capsid (16, 44).Open in a separate windowFIG. 3.Bootscan analysis of HAdV-D penton base genes. Comparison of HAdV-D22 (A) with completely sequenced HAdV types. Similar comparisons of the same region were performed with HAdV-D9 (B), HAdV-D49 (C), HAdV-A12 (D), HAdV-B3 (E), HAdV-C2 (F), or HAdV-F41 (G) as the reference type. HAdV-D53 was left out of the analysis due to the 100% identity of its penton base to that of HAdV-D37. The axes of all panels are as labeled in panel A. GenBank accession numbers are as follows: HAdV-A12, NC_001460; HAdV-B3, DQ086466; HAdV-C2, AC_000007; and HAdV-F41, DQ315364.We extended our investigation to determine if these recombination sites were common to penton base genes of other HAdV genomes. We found evidence for recombination between HAdV-D9 and HAdV-D26 in both the nucleotide 400 to 600 and 750 to 1350 regions (Fig. ​(Fig.3B),3B), between HAdV-D49 and HAdV-D48 in the nucleotide 400 to 600 region, and between HAdV-D49 and HAdV-D8 in the nucleotide 750 to 1350 region (Fig. ​(Fig.3C).3C). A similar pattern was observed in one or both of these nucleotide regions for HAdV-D8, -17, -19, -26, -37, and -48 (data not shown). Remarkably, this pattern of recombination in the penton base gene was unique to HAdV species D (Fig. 3D, E, F, and G).Bootstrap-confirmed neighbor-joining phylogenetic trees of HAdV-D penton base genes were constructed with Molecular Evolutionary Genetics Analysis (MEGA) 4.0.2 (http://www.megasoftware.net/index.html) to examine viral evolution in HAdV-D (34). Analysis of the entire penton gene revealed a close relationship of HAdV-D22 and HAdV-D19 strain C (Fig. ​(Fig.4A)4A) (30). Additional phylogenetic trees were constructed to encompass the two proposed recombination sites within this gene. Phylogenetic analysis of the nucleotide 400 to 600 region revealed a close identity among HAdV-D22, HAdV-D37, and HAdV-D53 (Fig. ​(Fig.4B).4B). Analysis of the nucleotide 750 to 1350 region of the penton base gene revealed a close identity of HAdV-D22 and HAdV-D19 (Fig. ​(Fig.4C4C).Open in a separate windowFIG. 4.Phylogenetic analysis of HAdV-D penton base genes. Bootstrap neighbor-joining tree designed using MEGA 4.0.2. The Gonnet protein weight matrix in ClustalX alignment was used, along with complete deletion options. Percent bootstrap confidence levels (1,000 replicates) are shown on the relevant branches. (A) Analysis of sequenced HAdV-D penton base genes. (B) Analysis of the nucleotide 400 to 600 region of sequenced HAdV-D penton base genes. (C) Analysis of the nucleotide 750 to 1350 region of sequenced HAdV-D penton base genes.In summary, comparison of the HAdV-D22 genome to other sequenced HAdV-D genomes identified substantial sequence divergence in the penton, hexon, E3, and fiber open reading frames. Differences between HAdV-D genomes in these areas have previously been described by us in reports on HAdV-D19 strain C and HAdV-D37 genomes (29, 30). However, the sequence conservation among HAdV-D22, HAdV-D19, and HAdV-D37 in the penton base gene was unexpected and suggests recombination among these viruses. Bootscan analysis identified recombination events at two regions within the penton base gene, encompassing nucleotides 400 to 600 and 750 to 1350. Further analysis of other HAdV-D penton genes suggests that these areas represent hot spots for recombination.The propensity for recombination among different HAdV-Ds in the penton base gene can be understood in the context of penton base function. The adenovirus penton base acts as the ligand for a secondary attachment event that is critical to host cell internalization (38) and thus is critical to infection. The penton base protein contains two hypervariable loops located on the surface of the viral capsid (16, 44). One loop contains the host cell integrin binding Arg-Gly-Asp (RGD) motif mediating attachment to host cell integrins (8, 38). The RGD motif is conserved in almost every HAdV penton base protein, with the exception of HAdV-F40 and HAdV-F41, which do not use host cell integrins for internalization (1, 11). The second exterior loop, known as the variable loop (HVL1), has no known function. Both of these regions are highly variable among different HAdV types. We identified recombination around both of these loops for multiple viruses within HAdV-D (Fig. ​(Fig.3A3A).In recent epidemiological studies, patients were identified with coincident clinical infections with two or more adenovirus types (17, 36). Simultaneous infection by more than a single HAdV type is possible because of conserved host receptor affinity, common tissue tropisms, and an absence of immunity across serotypes. Our data, along with evidence from previous studies (22, 23, 37, 43), identified recombination events among viruses with similar tissue tropisms, providing evidence that the restriction of tissue tropism might determine in part the observed recombination within adenoviruses of the same species. Recently, a novel HAdV, HAdV-D53, was isolated from an outbreak of keratoconjunctivitis. Subsequent analysis revealed recombination among HAdV-D22, HAdV-D37, HAdV-D8, and a previously unknown adenoviral sequence, suggesting the potential for the emergence of new pathogens, with important ramifications for human disease.Previous work provides evidence for recombination in HAdVs (10, 12, 22, 25, 26, 37, 39, 41-43), but the mechanisms of recombination have yet to be identified. Recombination may result from selective pressure from the host immune system relative to surface capsid proteins, a nucleotide motif that directs cellular recombination machinery to the local sites on the viral DNA, or a combination of both. Two eukaryotic recombinases, RAD51 and Dmc1, both homologues of the bacterial recombinase RecA, act in host cell DNA recombination (5, 33). RAD51 mediates recombination during mitosis, while Dmc1 acts during meiosis. RAD51 is of potential interest because it colocalizes with promyelocytic leukemia nuclear bodies in the nucleus (4). Adenoviral proteins E1A and E4 Orf3 have been shown to interact with promyelocytic leukemia nuclear bodies, which play an important role in adenoviral replication (7, 15). An interaction between RAD51 and adenoviral DNA has not been studied. A proposed motif consisting of CCNCCNTNNCCNC was recently identified as being associated with loci of recombination and genome instability in humans (28). Although not present in the viruses we studied, sequencing of other viruses within HAdV-D may yet reveal a consensus site for recombination. The elucidation of recombination mechanisms for HAdVs should allow a better understanding of adenoviral evolution.In conclusion, our analysis of the penton base gene of HAdV-D identified a potential paradigm for adenovirus recombination and the emergence of pathogenic strains. An in depth understanding of adenovirus recombination and evolution is critical to ensure the safety of adenoviral gene therapy.     

Mutations in the Ectodomain of Newcastle Disease Virus Fusion Protein Confer a Hemagglutinin-Neuraminidase-Independent Phenotype

The entry of enveloped viruses into host cells is preceded by membrane fusion, which in paramyxoviruses is triggered by the fusion (F) protein. Refolding of the F protein from a metastable conformation to a highly stable postfusion form is critical for the promotion of fusion, although the mechanism is still not well understood. Here we examined the effects of mutations of individual residues of the F protein of Newcastle disease virus, located at critical regions of the protein, such as the C terminus of the N-terminal heptad repeat (HRA) and the N terminus of the C-terminal heptad repeat (HRB). Seven of the mutants were expressed at the cell surface, showing differences in antibody reactivity in comparison with the F wild type. The N211A, L461A, I463A, and I463F mutants showed a hyperfusogenic phenotype both in syncytium and in dye transfer assays. The four mutants promoted fusion more efficiently at lower temperatures than the wild type did, meaning they probably had lower energy requirements for activation. Moreover, the N211A, I463A, and I463F mutants exhibited hemagglutinin-neuraminidase (HN)-independent activity when influenza virus hemagglutinin (HA) was coexpressed as an attachment protein. The data are discussed in terms of alterations of the refolding pathway and/or the stability of the prefusion and fusion conformations.Newcastle disease virus (NDV) is an avian enveloped virus belonging to the family Paramyxoviridae. Two viral membrane-associated proteins are responsible for the entry of the virus into the host cell: they are hemagglutinin-neuraminidase (HN), a receptor-binding protein that interacts with sialoglycoconjugates at the cell surface, and F, a trimeric class I fusion protein that, upon activation, triggers the fusion of the viral and target membranes. F protein is activated after the attachment of its homotypic HN protein to the proper receptor; however, how HN activates F is not well understood. F protein is synthesized as an inactive precursor, F₀, that is activated by proteolytic cleavage to the disulfide-linked F₁-F₂ fusion-competent form (Fig. ​(Fig.1)1) (10). The crystal structures of several paramyxoviral fusion proteins, in both the prefusion and postfusion conformations (3, 26, 27), have revealed that these proteins undergo major conformational changes, from a metastable conformation to a highly stable, postfusion form. Several regions in the ectodomain of class I viral fusion proteins are involved in these conformational conversions, including a hydrophobic fusion peptide at the N terminus of the F1 protein and two hydrophobic heptad repeat motifs, HRA and HRB, located at its N and C termini, respectively (Fig. ​(Fig.1).1). In the prefusion form, HRB shows a triple-stranded coiled-coil conformation forming the stalk of the mushroom-like protein (3, 19, 27). Its globular head contains three domains, DI to DIII (Fig. ​(Fig.1),1), with the base of the head being formed by the DI and DII domains, with residues predominantly located between HRA and HRB. The top of the head is formed by DIII, consisting mainly of HRA and the fusion peptide, located on the side of the head sequestered between adjacent subunits. In this prefusion state, HRA is folded as two antiparallel β-strands and four (h1 to h4) helices (27) (see Fig. ​Fig.6).6). The DIII domain undergoes major structural changes from the prefusion to the final postfusion conformation. HRA refolds as an α-helix, propelling the fusion peptide into the target membrane and generating a prehairpin intermediate (see Fig. ​Fig.6).6). The final, stable conformation consists of a six-helical bundle (6HB), comprising a dimer of trimers in which the trimeric HRA coiled coil forms the core, packed along the outside by three antiparallel HRB α-helices (1, 3, 19, 27).Open in a separate windowFIG. 1.Schematic representation of the structure of the NDV fusion protein. (A) Domain structure of F protein (27). (B) Locations of the fusion peptide, HR regions, and sequences studied. Mutated residues are indicated in bold.Open in a separate windowFIG. 6.Scheme of conformational changes in HRA from prefusion to postfusion state. (A) Ribbon model of PIV5 F protein in its metastable prefusion conformation (PDB accession number 2b9b) (27), showing some residues (named in white) from the A subunit and the corresponding residues in the NDV F protein (named in yellow). Subunits B and C are depicted in gray for clarity. (B) In the metastable, prefusion conformation, HRA is folded as a spring-loaded mixture of α-helices, turns, and β-strands, comprising 11 segments in the DIII head domain of the trimer (27). (C) After fusion, HRA is presented as a single long helix that allows the fusion peptide to be buried in the target membrane. The approximate positions of HRC and the core β-sheet are shown as dashed lines for both conformations.The refolding mechanism that triggers F protein activation is still not well understood. Mutational analysis of the HRA and HRB domains of paramyxovirus F proteins (3, 13, 18, 19, 22, 23), as well as the use of HRA- and HRB-derived peptides (6, 17), has led to the proposal of a series of discrete refolding intermediates of the F protein, from the metastable native conformation, through the prehairpin intermediate, and to the final postfusion hairpin structure (6HB) (17, 19, 27). To gain further insight into the individual residues critical for this mechanism, in this work we mutated several residues of the head and stalk of the NDV F protein (Fig. ​(Fig.1).1). The mutations disrupted F protein antibody reactivity, fusogenicity, and HN dependence in different ways. Interestingly, a mutant of the C-terminal h4 α-helix of HRA (N211A mutant) and two mutants of a residue located at the most N-terminal position of HRB (I463A and I463F mutants) exhibited a hyperfusogenic phenotype and HN-independent activity when influenza virus hemagglutinin (HA) was coexpressed as an attachment protein. The data are discussed in terms of alterations of the refolding pathway and/or the stability of the prefusion and fusion conformations.