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.     

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.     

Antibody Recognition Force Microscopy Shows that Outer Membrane Cytochromes OmcA and MtrC Are Expressed on the Exterior Surface of Shewanella oneidensis MR-1

Antibody recognition force microscopy showed that OmcA and MtrC are expressed on the exterior surface of living Shewanella oneidensis MR-1 cells when Fe(III), including solid-phase hematite (Fe₂O₃), was the terminal electron acceptor. OmcA was localized to the interface between the cell and mineral. MtrC displayed a more uniform distribution across the cell surface. Both cytochromes were associated with an extracellular polymeric substance.Shewanella oneidensis MR-1 is a dissimilatory metal-reducing bacterium that is well known for its ability to use a variety of anaerobic terminal electron acceptors (TEAs), including solid-phase iron oxide minerals, such as goethite and hematite (8, 10). Previous studies suggest that S. oneidensis MR-1 uses outer membrane cytochromes OmcA and MtrC to catalyze the terminal reduction of Fe(III) through direct contact with the extracellular iron oxide mineral (2, 8, 10, 15, 16, 20, 21, 23). However, it has yet to be shown whether OmcA or MtrC is actually targeted to the external surface of live S. oneidensis MR-1 cells when Fe(III) serves as the TEA.In the present study, we used atomic force microscopy (AFM) to probe the surface of live S. oneidensis MR-1 cells, using AFM tips that were functionalized with cytochrome-specific polyclonal antibodies (i.e., anti-OmcA or anti-MtrC). This technique, termed antibody recognition force microscopy (Ig-RFM), detects binding events that occur between antibodies (e.g., anti-OmcA) on an AFM tip and antigens (e.g., OmcA) that are exposed on a cell surface. While this is a relatively new technique, Ig-RFM has been used to map the nanoscale spatial location of single molecules in complex biological structures under physiological conditions (5, 9, 11, 13).Anti-MtrC or anti-OmcA molecules were covalently coupled to silicon nitride (Si₃N₄) cantilevers (Veeco or Olympus) via a flexible, heterofunctional polyethylene glycol (PEG) linker molecule. The PEG linker consists of an NHS (N-hydroxysuccinimide) group at one end and an aldehyde group at the other end (i.e., NHS-PEG-aldehyde). AFM tips were functionalized with amine groups, using ethanolamine (6, 7). The active NHS ester of the NHS-PEG-aldehyde linker molecule was then used to form a covalent linkage between PEG-aldehyde and the amine groups on the AFM tips (6, 7). Next, anti-MtrC or anti-OmcA molecules were covalently tethered to these tips via the linker molecule''s aldehyde group. This was accomplished by incubating the tips with antibody (0.2 mg/ml) and NaCNBH₃ as described previously (7). The cantilevers were purchased from Veeco and had spring constant values between 0.06 and 0.07 N/m, as determined by the thermal method of Hutter and Bechhoefer (12).Prior to conducting the Ig-RFM experiments, the specificity of each polyclonal antibody (i.e., anti-OmcA and anti-MtrC) for OmcA or MtrC was verified by Western blot analysis as described previously (24, 28). Proteins were resolved by both denaturing and nondenaturing polyacrylamide gel electrophoresis (PAGE). Briefly, 2.5 μg of purified OmcA or MtrC (23) was resolved by sodium dodecyl sulfate-PAGE or native PAGE, transferred to a polyvinylidene difluoride membrane, incubated with either anti-OmcA or anti-MtrC, and then visualized using the Amersham ECL Plus Western blotting detection kit. Anti-OmcA bound exclusively to OmcA, anti-MtrC bound exclusively to MtrC, and neither antibody showed cross-reactivity with the other cytochrome. Antibody specificities of anti-OmcA and anti-MtrC were also validated by immunoblot analysis of S. oneidensis whole-cell lysate (28).To determine if MtrC or OmcA was expressed on the external surface of live bacteria when Fe(III) served as the TEA, Ig-RFM was conducted on wild-type versus ΔomcA ΔmtrC double mutant cells. For these experiments, bacteria were cultivated anaerobically with Fe(III), in the form of Fe(III) chelated to nitrilotriacetic acid (NTA), serving as the TEA (19, 23). Growth conditions have been described elsewhere (3, 15) and were based on previous studies (3, 15, 16, 18) that suggest that S. oneidensis MR-1 targets OmcA and MtrC to the cell surface when Fe(III) serves as the TEA.An Asylum Research MFP-3D-BIO AFM or a Digital Instruments Bioscope AFM (16, 17) was used for these experiments. The z-piezoelectric scanners were calibrated as described previously (17). Cells were deposited on a hydrophobic glass coverslip and immersed in imaging buffer (i.e., phosphate-buffered saline [pH 7.4]). The hydrophobic glass coverslips were made as described previously (17) using a self-assembling silane compound called octadecyltrichlorosilane (OTS; Sigma-Aldrich). S. oneidensis MR-1 cells readily adsorbed onto OTS glass coverslips and remained attached to the coverslips during the entire experiment. No lateral cell movement was observed during the experiment, consistent with previous studies that used OTS glass to immobilize bacteria (15, 17, 18, 27).The AFM tip was brought into contact with the surface of a bacterium, and the antibody-functionalized tip was repeatedly brought into and out of contact with the sample, “fishing” for a binding reaction with cytochrome molecules that were exposed on the external cell surface. Binding events were observed upon separating anti-OmcA- or anti-MtrC-functionalized tips from wild-type S. oneidensis MR-1 cells (Fig. ​(Fig.1).1). For the wild-type cells, we observed both nonspecific and specific interactions (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Retraction force curves for anti-MtrC-functionalized tips (A) and anti-OmcA-functionalized tips (B) that are being pulled away from the surface of living ΔomcA ΔmtrC double mutant (gray dotted line) or wild-type (solid black line) S. oneidensis MR-1. These bacteria were adsorbed onto OTS glass coverslips. (C) Retraction curves exhibiting nonspecific binding, specific binding, or no binding between the AFM tip and the cell surface.The distinction between “specific” and “nonspecific” adhesion is made by observing the change in slope of the force curve during the retraction process (26). During specific binding (Fig. ​(Fig.1C),1C), the cantilever is initially relaxed as it is pulled away from the sample. Upon further retraction, the ligand-receptor complex becomes stretched and unravels, resulting in a nonlinear force profile as noted in references 26 and 16. On the other hand, nonspecific adhesion (Fig. ​(Fig.1C)1C) maintains the same slope during the retraction process because only the cantilever flexes (26).Figure ​Figure22 summarizes the frequency or probability of observing a binding event for both anti-OmcA and anti-MtrC tips. Each bar in Fig. ​Fig.22 represents one experiment in which 500 to 1,000 force curves were collected between one AFM tip and two to four live bacterial cells. This figure does not make a distinction between specific and nonspecific binding. It simply shows the frequency of observing an attractive interaction as the antibody-functionalized tip was pulled away from the surface of S. oneidensis MR-1. Binding events occurred with roughly the same frequency when wild-type S. oneidensis MR-1 cells were probed with anti-MtrC-functionalized tips as when they were probed with anti-OmcA-functionalized tips (Fig. ​(Fig.22).Open in a separate windowFIG. 2.Histograms showing the frequency of observing a binding event for anti-MtrC-functionalized (blue) or anti-OmcA-functionalized (red) AFM tips on live wild-type S. oneidensis MR-1 (solid bars) or ΔomcA ΔmtrC double mutant (diagonally hatched bars) cells. The downward arrows designate injection of free antibody into the imaging buffer. The solid gray bars correspond to results obtained with unbaited AFM tips.A number of control experiments were performed to verify the detection of OmcA and MtrC on the surface of wild-type S. oneidensis MR-1. First, 0.1 μM of free anti-OmcA (or anti-MtrC) was added to the imaging fluid to block binding between the antibody-functionalized AFM tip and surface-exposed cytochromes (11, 16). This decreased the adhesion that was observed between the antibody-functionalized tip and the cell surface (Fig. ​(Fig.22).Second, we performed force measurements on ΔomcA ΔmtrC double mutant S. oneidensis MR-1 cells. This mutant is deficient in both OmcA and MtrC (19, 23, 24) but produces other proteins native to the outer surface of S. oneidensis MR-1. The resulting force spectra showed a noticeable reduction in binding events for the ΔomcA ΔmtrC double mutant cells (Fig. ​(Fig.2).2). The binding events that were observed for the double mutant were only nonspecific in nature (Fig. ​(Fig.1).1). This indicates that the antibodies on the tip do not participate in specific interactions with other proteins on the surface of S. oneidensis MR-1 cells.As a final control experiment, force measurements were conducted on wild-type S. oneidensis MR-1 cells, using Si₃N₄ tips conjugated with the PEG linker but not functionalized with polyclonal antibody (unbaited tips). Like the results with the double mutant, the unbaited tips were largely unreactive with the surface of the bacteria (Fig. ​(Fig.2).2). Those binding events that were observed were nonspecific in nature. Taken together, these results demonstrate that the antibody-coated tips have a specific reactivity with OmcA and MtrC molecules. Furthermore, these force measurements show that MtrC and OmcA are present on the external cell surface when Fe(III) serves as the TEA.To map the distribution of cytochromes on living cells, Ig-RFM was conducted on living S. oneidensis MR-1 cells that were growing on a hematite (α-Fe₂O₃) thin film. The conditions for these experiments were as follows. A hematite film was grown on a 10-mm by 10-mm by 1-mm oxide substrate via oxygen plasma-assisted molecular beam epitaxy (14, 16). The cells were grown anaerobically to mid-log phase with Fe(III)-NTA serving as the TEA. Cells were deposited onto the hematite thin film along with anaerobic growth medium that lacked Fe(III)-NTA. The cells were allowed to attach to the hematite surface (without drying) overnight in an anaerobic chamber. The following day, the liquid was carefully removed and immediately replaced with fresh anaerobic solution (pH 7.4). Ig-RFM was performed on the cells by raster scanning an antibody-functionalized AFM tip across the sample surface, thereby creating an affinity map (1). Force curves were collected for a 32-by-32 array. The raw pixilated force-volume data were deconvoluted using a regularized filter algorithm. The total time to acquire a complete image was approximately 20 min.As noted above, attractive interactions between an antibody tip and cell resulted in relatively short-range, nonspecific and longer-range, specific adhesive forces (Fig. ​(Fig.1C).1C). To distinguish between these two interactions, we integrated each force curve beginning at >20 nm and ending at the full retraction of the piezoelectric motor (∼1,800 nm). This integration procedure quantifies the work of binding, measured in joules, between the antibody tip and a particular position on the sample. While this integration procedure does not totally exclude nonspecific binding, it does select for those events associated primarily with specific antibody-antigen binding. Figure ​Figure33 is the antibody-cytochrome recognition images for MtrC and OmcA. The corresponding height (or topography) images of the bacterial cells are also shown in Fig. ​Fig.33.Open in a separate windowFIG. 3.Ig-RFM of live S. oneidensis MR-1 cells deposited on a hematite (α-Fe₂O₃) thin film. Height image (A) and corresponding Ig-RFM image (B) for a bare unfunctionalized Si₃N₄ tip. Height and corresponding Ig-RFM image for a tip functionalized with anti-MtrC (C and D) or anti-OmcA (E and F). Each panel contains a thin white oval showing the approximate location of the bacterium on the hematite surface. A color-coded scale bar is shown on the right (height in micrometers [μm], and the work required to separate the tip from the surface in attojoules [aJ]).OmcA molecules were concentrated at the boundary between the bacterial cell and hematite surface (Fig. 3E and F). MtrC molecules were also detected at the edge of a cell (Fig. 3C and D). Some MtrC, unlike OmcA, was observed on the cell surface distal from the point of contact with the mineral (Fig. 3C and D). Both OmcA and MtrC were also present in an extracellular polymeric substance (EPS) on the hematite surface (Fig. 3D and F), which is consistent with previous results showing MtrC and OmcA in an EPS produced by cells under anaerobic conditions (19, 24). This discovery is interesting in light of the research by Rosso et al. (22) and Bose et al. (4), who found that Shewanella can implement a nonlocal electron transfer strategy to reduce the surface of hematite at locations distant from the point of cell attachment. Rosso et al. (22) proposed that the bacteria utilize unknown extracellular factors to access the most energetically favorable regions of the Fe(III) oxide surface. The Ig-AFM results (Fig. ​(Fig.3)3) suggest the possibility that MtrC and/or OmcA are the “unknown extracellular factors” that are synthesized by Shewanella to reduce crystalline Fe(III) oxides at points distal from the cell. Additional experiments showing reductive dissolution features coinciding with the extracellular location of MtrC and/or OmcA would need to be performed to test this hypothesis.It is important to note that these affinity maps were collected on only a few cells because it so challenging to produce large numbers of quality images. Future work should be conducted on a population of cells. Until this time, these affinity maps can be used to provide a crude, lowest-order estimate of the number of cytochromes on the outer surface of living S. oneidensis MR-1. For example, there were 236 force curves collected on the bacterium shown in Fig. ​Fig.3D.3D. Thirty-eight of these curves exhibited a distinct, sawtooth-shaped, antibody-antigen binding event. In other words, MtrC molecules were detected in one out of every six force curves (16%) that were collected on the cell surface.This probability can be compared to other independent studies that estimated the density and size of MtrC and OmcA molecules from S. oneidensis MR-1. Lower et al. (16) estimated that S. oneidensis has 4 × 10¹⁵ to 7 × 10¹⁵ cytochromes per square meter by comparing AFM measurements for whole cells to force curves on purified MtrC and OmcA molecules. Wigginton et al. (25) used scanning tunneling microscopy to determine that the diameter of an individual cytochrome is 5 to 8 nm. These values can be used to create a simple, geometric, close-packing arrangement of MtrC or OmcA molecules on a surface. Using this approach, cytochromes could occupy 8 to 34% of the cell surface.This estimate is consistent with the observed number of putative MtrC molecules shown in Fig. ​Fig.3D.3D. Therefore, it appears that these affinity maps can be used as a lowest-order estimate for the number of cytochromes on S. oneidensis MR-1 even though we do not know a priori the exact configuration of the antibody tip (e.g., the concentration of antibody on the tip, the exact shape of the tip, the binding epitopes within the antibody).In summary, the data presented here show that S. oneidensis MR-1 localizes OmcA and MtrC molecules to the exterior cell surface, including an EPS, when Fe(III) is the TEA. Here, the cytochromes presumably serve as terminal reductases that catalyze the reduction of Fe(III) through direct contact with the extracellular iron-oxide mineral.     

Molecular Chaperone BiP Interacts with Borna Disease Virus Glycoprotein at the Cell Surface

Borna disease virus (BDV) is characterized by highly neurotropic infection. BDV enters its target cells using virus surface glycoprotein (G), but the cellular molecules mediating this process remain to be elucidated. We demonstrate here that the N-terminal product of G, GP1, interacts with the 78-kDa chaperone protein BiP. BiP was found at the surface of BDV-permissive cells, and anti-BiP antibody reduced BDV infection as well as GP1 binding to the cell surface. We also reveal that BiP localizes at the synapse of neurons. These results indicate that BiP may participate in the cell surface association of BDV.Borna disease virus (BDV) belongs to the Bornaviridae family of nonsegmented, negative-strand RNA viruses and is characterized by highly neurotropic and noncytopathic infection (18, 33). BDV infects a wide variety of host species and causes central nervous system (CNS) diseases in animals, which are frequently associated with behavioral disorders (14, 19, 29, 31). BDV cell entry is mediated by endocytosis, following the attachment of viral envelope glycoprotein (G) to the cellular receptor (2, 7, 8). BDV G is translated as a precursor protein, GP, which is posttranslationally cleaved by the cellular protease furin to generate two functional subunits of the N (GP1) and C (GP2) termini (28). Recent studies revealed that GP1 is involved in virus interaction with as-yet-unidentified cell surface receptor(s) and that GP2 mediates a pH-dependent fusion event between viral and cell membranes (2, 7, 27). In addition, a previous work using a hippocampal culture system suggested that BDV G is required for viral dissemination in neurons (2); however, cellular factors involved in BDV cell entry, especially cell surface association, remain to be elucidated.To extend our understanding of the role of BDV G in the interaction with the cell plasma membrane, we transfected GP1 fused with hemagglutinin-tobacco etch virus protease cleavage site-FLAG tags (GP1-TAP) into human oligodendroglioma OL cells. GP1-TAP was purified using anti-FLAG M2 affinity gel (Sigma). To verify that GP1-TAP binds to OL cells, the cells were incubated with 4 μg/ml GP1-TAP, and binding was detected by anti-FLAG M2 antibody (Sigma). A flow cytometric analysis indicated that GP1-TAP binds to OL cells (Fig. ​(Fig.1A).1A). To further validate the binding of GP1-TAP, we tested whether GP1-TAP inhibits BDV infection. OL cells were pretreated with 4 μg/ml GP1-TAP for 30 min. Proteins purified from mock-transfected cells using an anti-FLAG M2 affinity gel served as a control. The cells were then mixed with cell-free BDV. After 1 h of absorption, the supernatants were removed and fresh medium was added. At 3 days postinfection, the viral antigens were stained with anti-nucleoprotein (N) monoclonal and anti-matrix (M) polyclonal antibodies. As shown in Fig. ​Fig.1B,1B, GP1-TAP reduced BDV infection by 40% compared to levels for mock-treated cells. This result was consistent with earlier reports showing that recombinant GP1 protein binds to the cell surface and inhibits BDV infection (6, 20).Open in a separate windowFIG. 1.BDV GP1 binds to the cell surface. (A) Binding of BDV GP1 to OL cells. OL cells were incubated with GP1-TAP (solid line), and its binding was detected using anti-FLAG M2 antibody and flow cytometry. As a control, cells incubated with proteins purified from mock-transfected cells were detected by an anti-FLAG M2 antibody (dotted line). (B) Inhibition of BDV infection by GP1. OL cells pretreated with GP1-TAP were inoculated with the BDV huP2br strain. Values are the means + standard deviations (SD) from three independent experiments. **, P < 0.01.To investigate the host factor(s) that mediates the interaction of GP1 with the cell surface, a combination of tandem affinity purification (TAP) and liquid chromatography tandem mass spectrometry analyses was designed (13). We transfected GP1-TAP into OL cells and then purified GP1 from cell homogenates using a TAP strategy. We compared the purified proteins from the whole-cell and cytosol fractions (Fig. ​(Fig.2A),2A), and the bands detected only in the whole-cell fraction were determined as GP1-binding proteins in the membrane and/or nuclear fractions. In addition to GP1 protein (Fig. ​(Fig.2A,2A, arrow), we identified a specific band around 80 kDa in the whole-cell homogenate, but not in the cytosol fraction (Fig. ​(Fig.2A,2A, arrowhead), and determined that the band corresponded to the BiP (immunoglobulin heavy chain-binding protein) molecular chaperone, also called glucose-regulated protein 78 (GRP78), by mass spectrometry analysis. We confirmed the specific interaction between endogenous BiP and BDV G in infected cells by immunoprecipitation analysis (Fig. ​(Fig.2B).2B). To map the binding domain on BiP to GP1, we constructed a series of deletion mutants of the green fluorescent protein (GFP)-tagged BiP plasmid (Fig. ​(Fig.2C).2C). We transfected the mutant plasmids into BDV-infected OL cells and then performed an immunoprecipitation assay using anti-GFP antibody (Invitrogen). As shown in Fig. ​Fig.2D,2D, BDV G was coimmunoprecipitated with truncated BiP mutants, except for BiPΔN-GFP, which lacks the ATP-binding domain of BiP (lane 3), suggesting that BiP interacts with GP1 via its N-terminal region.Open in a separate windowFIG. 2.BDV GP1 interacts with BiP molecular chaperone. (A) TAP analysis of BDV GP1. Proteins coimmunoprecipitated with GP1-TAP in OL cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by silver staining. Cyt, cytosol fraction; Wc, whole-cell homogenate. Arrow, GP1-TAP; arrowhead, BiP. (B) Coimmunoprecipitation (IP) of BDV G and endogenous BiP. BDV G was immunoprecipitated from BDV-infected OL cells by anti-BDV G polyclonal antibody. Endogenous BiP was then detected by anti-BiP monoclonal antibody (Becton Dickinson). IgG, immunoglobulin G. (C) Schematic representation of deletion mutants of recombinant BiP-GFP. The known functional regions are indicated. (D) Immunoprecipitation analysis of BiP-GFP mutants in BDV-infected OL cells. The deletion plasmids were transfected and immunoprecipitated by anti-GFP antibody. Specific binding was detected using anti-BDV G antibody. Lane 1, GFP; lane 2, BiP-GFP; lane 3, BiPΔN-GFP; lane 4, BiPΔPB-GFP; lane 5, BiPΔC-GFP.BiP is known to be resident primarily in the endoplasmic reticulum and functions as a molecular chaperone involved in the folding process of nascent proteins, mostly through interaction with its peptide-binding domain (12, 17, 21). On the other hand, BiP has been reported to serve as a coreceptor of certain viruses at the plasma membrane (15, 34). Recent studies also revealed that cell surface BiP mediates the internalization of its ligands into cells (1, 10). We first investigated whether BiP is expressed on the cell surface of BDV-permissive OL and 293T cells using an anti-BiP polyclonal antibody (H-129; Santa Cruz Biotechnology, Inc.). As shown in Fig. ​Fig.3A,3A, BiP expression is detected on the surface of both cell lines. This result is in agreement with recent observations that BiP is expressed on the surface of various types of cells (9, 10, 15, 23, 24, 34). We also investigated whether BiP is expressed on the cell surface of BDV-nonpermissive cell lines, such as HeLa and CHO cells. As shown in Fig. ​Fig.3A,3A, we detected BiP expression on the surface of HeLa, but not CHO, cells. These observations were confirmed by immunofluorescence analysis (Fig. ​(Fig.3B).3B). Note that BiP is clearly detected at the endoplasmic reticulum in the permeabilized CHO cells by the antibody (see Fig. S1 in the supplemental material), suggesting that BiP is expressed at a very low level, if at all, on the surface of CHO cells. We next examined whether cell surface BiP serves as a binding molecule of BDV GP1. To test this, we performed an inhibition assay using an anti-BiP polyclonal antibody (N-20; Santa Cruz Biotechnology, Inc.) which recognizes the N terminus of BiP. As shown in Fig. ​Fig.3C,3C, the antibody inhibited GP1 binding to the cell surface by 40%. Furthermore, BDV infection was found to decrease by 70% when cells were treated with the antibody (Fig. ​(Fig.3D3D).Open in a separate windowFIG. 3.Cell surface BiP mediates cell association of BDV. (A) Flow cytometric analysis was performed with anti-BiP antibody (H-129) in BDV-permissive (OL and 293T) and -nonpermissive (HeLa and CHO) cells (solid lines). Cells stained with normal rabbit immunoglobulin G were used as a control (dotted lines). (B) Immunofluorescence analysis was performed by using anti-BiP antibody (H-129) with BDV-permissive and -nonpermissive cells. Arrows indicate BiP staining at the membrane. Scale bars, 10 μm. (C) Inhibition of GP1 binding by anti-BiP antibody (N-20). OL cells were pretreated with anti-BiP antibody, followed by labeling with GP1. GP1 binding on the cell surface was detected using flow cytometry. Values are the means + SD from three independent experiments. *, P < 0.05. (D) Inhibition of BDV infection by anti-BiP antibody. OL cells were incubated with 10 μg/ml anti-BiP antibody or normal goat immunoglobulin G and then the cells were mixed with cell-free BDV. After 1 h absorption, the supernatants were replaced with fresh medium. Virus infection was measured by immunofluorescence analysis using anti-N and -M antibodies at 3 days postinfection. Values are the means + SD from three independent experiments. *, P < 0.05. IgG, immunoglobulin G.To investigate the role of cell surface BiP in the infection of BDV, the BiP expression was inhibited by short interfering RNA (siRNA) in OL cells (see Fig. S2A in the supplemental material). We selected an siRNA (Hs_HSPA5_4; Qiagen, Inc.) which could partially downregulate the cell surface expression of BiP (see Fig. S2B in the supplemental material). However, siRNA treatment of BiP did not influence the infectivity of BDV in OL cells (see Fig. S2C in the supplemental material). This may be due to an incomplete reduction of BiP expression on the cell surface. Alternatively, while BiP mediates at least in part the cell surface association of BDV particles, this result may exhibit the presence of another, as-yet-unidentified BDV G-binding protein that is involved in the binding and subsequent cell entry of BDV.Previous studies demonstrated that BDV can be traced centripetally and transsynaptically after olfactory, ophthalmic, or intraperitoneal inoculation (3, 25). Migration of BDV to the CNS after footpad infection can be prevented by sciatic nerve transection (3). These observations suggest that BDV may disseminate primarily via neural networks. Recently, it has been demonstrated that BDV G was expressed at the termini of neurites or at contact sites of neurites (2), suggesting that local assembly of BDV may take place at the presynaptic terminals of synapses, similar to assembly of other neurotropic viruses (22, 26, 32). If BiP localizes at synapse sites, BiP may efficiently participate in the transmission of BDV particles at the synapses. To evaluate this hypothesis, we examined BiP localization in primary culture of mouse hippocampal neurons. After in vitro culture for 17 days, BiP localization was determined by an immunofluorescence assay without permeabilization. As shown in Fig. ​Fig.4A,4A, BiP signals were clearly detected at neurites, including the contact sites between dendrites and axons, as punctate staining (arrows), suggesting that BiP is expressed at the neuronal surface, most likely at the synapses. We next examined the localization of BiP with postsynaptic density 95 (PSD-95), a marker of postsynaptic density (5). Although BiP signals were detected mainly in the perinuclear area of the hippocampal neurons, punctate staining was also found at neurites colocalized with PSD-95 (Fig. ​(Fig.4B,4B, arrows). Taken together, these observations suggested that BiP is distributed at the synaptic surface, including the postsynaptic membrane, of neurons, a possible site for BDV budding and entry (2).Open in a separate windowFIG. 4.BiP localizes at the synaptic surface of hippocampus neurons. (A) Localization of BiP at synaptic surface. Hippocampal neurons were immunostained with anti-BiP antibody (N-20) without permeabilization. A differential interference contrast (DIC) image is shown. Dotted lines in the Merge panel indicate the dendrite outline. Arrows indicate BiP staining at the contact sites between axons and dendrites. (B) Colocalization between BiP and a postsynaptic protein. Hippocampal neurons were immunostained with anti-BiP (N-20) and anti-PSD-95 (Millipore) antibodies. Arrows indicate colocalized signals of BiP and PSD-95 at neurites. Scale bars, 10 μm.In summary, this study demonstrates that BiP is a GP1-binding protein at the synaptic surface. This is the first report showing the BDV G-binding factor on the cell surface. The first step of BDV entry might be mediated by the interaction of GP1 with as-yet-unidentified cell surface receptors, which may form a complex with other molecules, such as BiP. We showed that treatment with anti-BiP antibody affects BDV infection as well as GP1 binding to the cell surface (Fig. ​(Fig.3).3). Furthermore, synaptic distribution of BiP was found in hippocampal primary neurons (Fig. ​(Fig.4).4). These findings strongly suggest that BiP plays critical roles in BDV association with the neuronal surface via interaction with GP1. On the other hand, a BDV-nonpermissive cell line, HeLa, appeared to express BiP on the cell surface, suggesting that the cell surface BiP may not be necessarily involved in the infectivity of BDV. A recent study by Clemente et al. (6) revealed that following initial attachment to the cell surface, BDV is recruited to the plasma membrane lipid raft (LR) prior to internalization of the particles. The study suggested that BDV may use the LR as a platform to interact with additional host cell factor(s) required for efficient BDV internalization. Because BiP does not contain transmembrane regions, BiP needs another host protein(s) with transmembrane regions on the cell surface. It has been reported that cell surface BiP interacts with diverse proteins, such as major histocompatibility complex class I molecules (34), the voltage-dependent anion channel (9), and the DnaJ-like protein MTJ-1 (4), all of which associate with LR in the plasma membrane (16, 24, 35). Once BDV has attached to the cell surface, it might utilize such BiP-associated LR proteins for efficient cell surface attachment or internalization. Previously, it has been proposed that kainate 1 (KA-1) receptor might represent the BDV receptor within the CNS (11). Because some glutamate receptors are shown to bind to BiP (30), KA-1 receptors might interact with BiP and serve as a receptor complex for BDV. Further studies are required for a full understanding of the cell association processes, especially receptor binding, of BDV.      

The Carboxy-Terminal Segment of the Human Cytomegalovirus DNA Polymerase Accessory Subunit UL44 Is Crucial for Viral Replication

The amino-terminal 290 residues of UL44, the presumed processivity factor of human cytomegalovirus DNA polymerase, possess all of the established biochemical activities of the full-length protein, while the carboxy-terminal 143 residues contain a nuclear localization signal (NLS). We found that although the amino-terminal domain was sufficient for origin-dependent synthesis in a transient-transfection assay, the carboxy-terminal segment was crucial for virus replication and for the formation of DNA replication compartments in infected cells, even when this segment was replaced with a simian virus 40 NLS that ensured nuclear localization. Our results suggest a role for this segment in viral DNA synthesis.Human cytomegalovirus (HCMV) encodes a DNA polymerase which is composed of two subunits, UL54, the catalytic subunit, and UL44, an accessory protein (8, 12, 21). UL44 can be divided into two regions, a 290-residue amino (N)-terminal domain and a 143-residue carboxy (C)-terminal segment. The overall fold of the N-terminal domain is markedly similar to that of processivity factors such as herpes simplex virus type 1 (HSV-1) UL42 and eukaryotic proliferating cell nuclear antigen (6, 22, 41), which function to tether catalytic subunits to DNA to ensure long-chain DNA synthesis. In vitro, the N-terminal domain of UL44 is sufficient for all of the established biochemical activities of full-length UL44, including dimerization, binding to double-stranded DNA, interaction with UL54, and stimulation of long-chain DNA synthesis, consistent with a role as a processivity factor (4, 5, 8, 11, 23, 24, 39). In contrast, little is known about the functions of the C-terminal segment of UL44 other than its having been reported from transfection experiments to be important for downregulation of transactivation of a non-HCMV promoter (7) and to contain a nuclear localization signal (NLS) (3). Neither the importance of this NLS nor the role of the entire C-terminal segment has been investigated in HCMV-infected cells.We first examined whether the N-terminal domain is sufficient to support DNA synthesis from HCMV oriLyt in cells using a previously described cotransfection-replication assay (27, 28). A DpnI-resistant fragment, indicative of oriLyt-dependent DNA synthesis, was detected in the presence of wild-type (WT) UL44 (pSI-UL44) (34) and in the presence of the UL44 N-terminal domain (pSI-UL44ΔC290), but not in the presence of UL44-F121A (6, 34), a mutant form previously shown not to support oriLyt-dependent DNA synthesis (34) (Fig. ​(Fig.1A).1A). Thus, the N-terminal domain alone is sufficient to support oriLyt-dependent DNA synthesis in a transient-transfection assay.Open in a separate windowFIG. 1.Effects of UL44 C-terminal truncations in various assays. (A) HFF cells were cotransfected with the pSP50 plasmid (containing the oriLyt DNA replication origin), a plasmid expressing WT or mutant UL44 (as indicated at the top of the panel), and plasmids expressing all of the other essential HCMV DNA replication proteins. At 5 days posttransfection, total DNA was extracted and cleaved with DpnI to digest unreplicated DNA and a Southern blot assay was performed to detect replicated pSP50. An arrow indicates DpnI-resistant, newly synthesized pSP50 fragments. (B) FLAG-tagged constructs analyzed in panel C are cartooned as horizontal bars. The names of the constructs are above the bars. The lengths of the constructs in amino acids are indicated by the scale at the bottom of the panel. The positions of residues required but not necessarily sufficient for features of the constructs are designated by shading, as indicated at the bottom of the panel. (C) Vero cells were transfected with plasmids expressing WT UL44 (parts a to c), FLAG-UL44 (parts d to f), FLAG-UL44-290stop (parts g to i), or FLAG-UL44-290NLSstop (parts j to l). At 48 h posttransfection, cells were fixed and stained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize the nucleus (blue) (parts a, d, g, and j) and by IF with anti-UL44 (part b) or anti-FLAG (parts e, h, and k) and a secondary antibody conjugated with Alexa 488 (green). Parts c, f, i, and l are merged from images in the left and middle columns. Magnification: ×1,000. (D) Replication kinetics of rescued viruses. Rescued derivatives of UL44 mutant viruses (UL44-290stop-R and UL44-290NLSstop-R) or WT AD169 viruses were used to infect HFF cells at an MOI of 1 PFU/cell. The supernatants from infected cells were collected every 24 h, and viral titers were determined by plaque assays on HFF cells.These results were somewhat unexpected, as the C-terminal segment contains a functional NLS identified in transfection assays (3). We therefore assayed the intracellular localization of WT and mutant UL44 following transient transfection using pcDNA3-derived expression plasmids. Since the anti-UL44 antibodies that we have tested do not recognize the N-terminal domain of UL44, we constructed UL44 genes to encode N-terminally FLAG-tagged full-length UL44 (FLAG-UL44) or a FLAG-tagged N-terminal domain, the latter by inserting three in-frame tandem stop codons after codon 290 (FLAG-UL44-290stop, Fig. ​Fig.1B).1B). We also constructed a mutant form encoding a FLAG-tagged N-terminal domain, followed by the simian virus 40 (SV40) T-antigen NLS (15-17), followed by three tandem stop codons (FLAG-UL44-290NLSstop, Fig. ​Fig.1B).1B). Vero cells were transfected with each construct using Lipofectamine 2000, fixed with 4% formaldehyde at 48 h posttransfection, and assayed by indirect immunofluorescence (IF) using anti-UL44 (Virusys) or anti-FLAG antibody (Sigma). We observed mostly nuclear localization of WT UL44 or FLAG-UL44 with either diffuse or more localized intranuclear distribution (Fig. ​(Fig.1C,1C, parts a to c and d to f, respectively) and some occasional perinuclear staining, which may be due to protein overexpression. In cells expressing FLAG-UL44-290NLSstop, we observed mostly diffuse nuclear localization with little to no perinuclear staining (Fig. ​(Fig.1C,1C, parts j to l). In cells expressing FLAG-UL44-290stop, we observed mostly cytoplasmic staining, but with some cells exhibiting some nuclear staining (Fig. ​(Fig.1C,1C, parts g to i), which may explain the ability of truncated UL44 to support oriLyt-dependent DNA replication in a transient-transfection assay (Fig. ​(Fig.1A1A).We next investigated whether the C-terminal segment of UL44 is necessary for viral replication. We reasoned that we could investigate whether any requirement for this segment could be due to a requirement for an NLS by testing whether the SV40 NLS could substitute for the loss of the UL44 C terminus. We therefore constructed HCMV UL44 mutant viruses by introducing the UL44-290stop and UL44-290NLSstop mutations into a WT AD169 bacterial artificial chromosome (BAC) using two-step red-mediated recombination as previously described (35, 38). We also constructed the same mutants with a FLAG epitope at the N terminus of UL44 (BAC-FLAG-UL44-290stop and BAC-FLAG-UL44-290NLSstop) to monitor UL44 expression, and we constructed rescued derivatives of the mutant BACs by replacing the mutated sequences with WT UL44 sequences, as described previously (35). We introduced BACs into human foreskin fibroblast (HFF) cells using electroporation (35, 38). In several experiments using at least two independent clones for each mutant, cells electroporated with any of the mutant BACs did not exhibit any cytopathic effect (CPE) within 21 days. In contrast, within 7 to 10 days, cells electroporated with the WT AD169 BAC, a BAC expressing WT UL44 with an N-terminal FLAG tag [AD169-BACF44 (35)], or any of the rescued derivatives began displaying a CPE and yielded infectious virus. The rescued derivatives of the nontagged mutants displayed replication kinetics similar to those of the WT virus following infection at a multiplicity of infection (MOI) of 1 PFU/cell (Fig. ​(Fig.1D).1D). The rescued derivatives of the FLAG-tagged mutants also replicated to WT levels (data not shown). Thus, the replication defects of the mutants were due to the introduced mutations that result in truncated UL44 either with or without the SV40 NLS. We therefore conclude that the C-terminal segment of UL44 is required for viral replication.To investigate the stage of viral replication at which the UL44 C-terminal segment is important, we first assayed the subcellular localization of immediate-early proteins IE1 and IE2 and FLAG-UL44 in cells electroporated with BAC DNA expressing the FLAG-tagged WT or the two mutant UL44s using IF at 2 days postelectroporation. IE1/IE2 could be detected diffusely distributed in nuclei of cells electroporated with all three BACs (Fig. 2b, f, and j). In cells electroporated with AD169-BACF44 or BAC-FLAG-UL44-290NLSstop, FLAG-UL44 was localized largely within the nucleus (Fig. 2c and k, respectively). In contrast, in cells electroporated with BAC-FLAG-UL44-290stop, the FLAG epitope was mainly localized diffusely in the cytoplasm, with only a small amount diffusely distributed in the nucleus (Fig. ​(Fig.2g).2g). These data indicate that IE proteins expressed from mutant BACs are properly localized and suggest that without its C-terminal segment, which includes the NLS identified in transfection assays (3), UL44 cannot efficiently localize to the nucleus in HCMV-infected cells. However, addition of the SV40 NLS was sufficient to efficiently localize the N-terminal domain of UL44 to the nucleus. Thus, the requirement for the C-terminal segment of UL44 for viral replication is not due solely to its NLS.Open in a separate windowFIG. 2.Localization of IE1/IE2 and FLAG-UL44 proteins in electroporated cells. HFF cells were electroporated with AD169-BACF44 (panels a to d), BAC-UL44-290stop (panels e to h), or BAC-FLAG-UL44-290NLSstop (panels i to l). At 48 h posttransfection, cells were fixed and probed with anti-IE1/2 (Virusys) or anti-FLAG (Sigma). Secondary antibodies coupled to fluorophores were used for visualization of IE1/2 (anti-mouse Alexa 594; panels b, f, and j) and FLAG (anti-rabbit Alexa 488; panels c, g, and k) antibodies. DAPI was used to counterstain the nucleus (panels a, e, and i). Panels d, h, and l are merged images of the panels in the other columns. Magnification: ×1,000.We next investigated if the block in viral replication due to the loss of the C-terminal segment could be attributed to a defect in viral DNA synthesis. Cells were electroporated with AD169-BACF44 or BAC-FLAG-UL44-290NLSstop, and viral DNA accumulation was assayed by quantitative real-time PCR at various times postelectroporation (Fig. ​(Fig.3)3) as previously described (32, 35). In HFFs electroporated with AD169-BACF44, viral DNA began to accumulate above the input levels by 8 days postelectroporation and increased over time, with as much as a 350-fold increase over the input DNA level by 18 days postelectroporation. In contrast, levels of viral DNA in cells electroporated with BAC-UL44-290NLSstop did not increase above input levels, even by 18 days postelectroporation. These data are consistent with the notion that the UL44 C-terminal segment is required for viral DNA synthesis, although we caution that the assay did not detect DNA synthesis from AD169-BACF44 until day 8, when viral spread had likely occurred (see below).Open in a separate windowFIG. 3.Quantification of viral DNA accumulation in electroporated cells. HFF cells were electroporated with AD169-BACF44 or BAC-FLAG-UL44-290NLSstop, and total DNA was harvested on the days postelectroporation indicated. Viral DNA accumulation was assessed by real-time PCR by assessing levels of the UL83 gene and normalizing to levels of the cellular β-actin gene (32). The data are presented as the fold increase in normalized viral DNA levels over the amount of input DNA (day 1).We also analyzed the localization patterns of UL44 and UL57, the viral single-stranded DNA binding protein, which is a marker for viral DNA replication compartments (1, 2, 18, 26, 29). At 8 days postelectroporation with AD169-BACF44, UL57 and FLAG-UL44 largely colocalized within a single large intranuclear structure that likely represents a fully formed replication compartment, with some cells containing multiple smaller globular structures within the nucleus that likely represent earlier stages of replication compartments (1, 2, 29) (Fig. 4a to d). Neighboring cells also stained for UL57 and FLAG-UL44, indicative of viral spread. In contrast, in cells electroporated with BAC-FLAG-UL44-290NLSstop, UL57 (Fig. ​(Fig.4f)4f) was found in either punctate or small globular structures. This pattern of UL57 staining resembled that observed at very early stages of viral DNA synthesis in HCMV-infected cells, but the structures were larger and less numerous than those observed in HCMV-infected cells in the presence of a viral DNA polymerase inhibitor (2, 29). Staining for FLAG-UL44 was nuclear and largely diffuse, with some areas of more concentrated staining (Fig. ​(Fig.4g),4g), which could also be observed in some cells at day 2 postelectroporation (Fig. ​(Fig.3k).3k). This pattern of UL44 localization was generally similar to that observed in HCMV-infected cells at very early stages of infection or when HCMV DNA synthesis is blocked and also similar to the pattern in cells transfected with a UL84 null mutant BAC (2, 29, 33, 40). Importantly, little colocalization of UL57 and UL44 was observed, with areas of concentration of UL57 or UL44 occupying separate regions in the nuclei of these cells (Fig. ​(Fig.4h).4h). We are unaware of any other examples of this pattern of localization of these proteins in HCMV-infected cells and suggest that it may be a result of the loss of the UL44 C-terminal segment. These results indicate that this segment is important for efficient formation of viral DNA replication compartments, again consistent with a requirement for this portion of UL44 for viral DNA synthesis.Open in a separate windowFIG. 4.Localization of UL57 and FLAG-UL44 proteins in electroporated cells. HFF cells were electroporated with AD169-BACF44 (panels a to d) or BAC-FLAG-UL44-290NLSstop (panels e to h). At 8 days posttransfection, cells were fixed and then stained with antibodies specific for UL57 (Virusys) or FLAG (Sigma), followed by a secondary antibody coupled to fluorophores to detect UL57 (anti-mouse Alexa 594; panels b and f) and FLAG (anti-rabbit Alexa 488; panels c and g) antibodies. DAPI stain was used to counterstain the nucleus (panels a and e). Panels d and h are merged images of the panels in the other columns. White arrows identify punctate UL57 staining. Yellow arrows identify areas of concentration of FLAG-UL44 staining. Magnification: ×1,000.Our results, taken together, argue for a role for the C-terminal segment of UL44 in HCMV-infected cells in efficient nuclear localization of UL44 and a role in viral DNA synthesis beyond its role in nuclear localization. It is possible that this segment interacts with host or viral proteins involved in DNA replication. Of the various proteins reported to interact with UL44 (10, 19, 30, 31, 35-37), interesting candidates include the host protein nucleolin, which has been shown to associate with UL44 and be important for viral DNA synthesis (35), and the viral UL112-113 proteins, which in transfection assays were shown to recruit UL44 to early sites of DNA replication (2, 29, 33). After this paper was submitted, Kim and Ahn reported that the C-terminal segment of UL44 is necessary for interaction with a UL112-113 protein and, similar to our findings, crucial for viral replication (19). However, contrary to our findings, they reported that this segment was not necessary for efficient nuclear localization of UL44 (19). It may well be that the C-terminal segment of UL44 also has some other role later in viral replication, perhaps in gene expression, as has been suggested (7, 13, 14).A virus with a deletion of the C-terminal 150 amino acids of the HSV-1 polymerase accessory subunit UL42 displays no obvious defect in replication (9). Thus, it appears that HSV-1 and HCMV exhibit different requirements for the C-terminal segments of their respective accessory proteins. This and many other differences between these functionally and structurally orthologous proteins (5, 6, 20, 24, 25) suggest considerable selection for different features during evolution.     

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.     

High-Resolution Cryo-Electron Microscopy Structures of Murine Norovirus 1 and Rabbit Hemorrhagic Disease Virus Reveal Marked Flexibility in the Receptor Binding Domains

Our previous structural studies on intact, infectious murine norovirus 1 (MNV-1) virions demonstrated that the receptor binding protruding (P) domains are lifted off the inner shell of the virus. Here, the three-dimensional (3D) reconstructions of recombinant rabbit hemorrhagic disease virus (rRHDV) virus-like particles (VLPs) and intact MNV-1 were determined to ∼8-Å resolution. rRHDV also has a raised P domain, and therefore, this conformation is independent of infectivity and genus. The atomic structure of the MNV-1 P domain was used to interpret the MNV-1 reconstruction. Connections between the P and shell domains and between the floating P domains were modeled. This observed P-domain flexibility likely facilitates virus-host receptor interactions.Murine norovirus 1 (MNV-1) (3, 14, 15) and rabbit hemorrhagic disease virus (RHDV) are members of the genera Norovirus and Lagovirus of the family Caliciviridae that offer a comparison to recombinant human norovirus (rNV) virus-like particles (VLPs) for assessing the structures and roles of domains within the capsid proteins of this family of viruses. Calicivirus particles contain 180 copies of the 56- to 76-kDa major capsid protein (Orf2), which is comprised of the internal/buried N terminus (N), shell (S), and protruding (P) domains (9, 10). The S domain, an eight-stranded β-barrel, forms an ∼300-Å contiguous shell around the RNA genome. A flexible hinge connects the shell to a “protruding” (P) domain at the C-terminal half of the capsid protein, which can be further divided into a globular head region (P2) and a stem region (P1) that connects the shell domain to P2. The accompanying article (13) describes the determination of the structure of the P domain of MNV-1 to a resolution of 2.0 Å.We recently determined the cryo-transmission electron microscopy (TEM) structure of MNV-1 to ∼12-Å resolution (4) and found that, compared to rNV VLPs (10) and San Miguel sea lion virus (SMSV) (1, 2), the protruding domains are rotated by ∼40° in a clockwise fashion and lifted up by ∼16 Å. To better understand the unusual conformation of MNV-1 and whether it is unique to this particular member of the calicivirus family, the ∼8-Å cryo-TEM structures of infectious MNV-1 and the VLPs of RHDV were determined.MNV-1 was produced as previously described (4). Three liters of cell culture yielded 0.5 to 1.0 mg of purified virus with a particle/PFU ratio of less than 100. Baculovirus expression and purification of recombinant RHDV (rRHDV) VLPs were performed as previously described (8). Cryo-electron microscopy (EM) data were collected at the National Resource for Automated Molecular Microscopy (NRAMM) facility in San Diego, CA (4). Images were collected at a nominal magnification of ×50,000 at a pixel size of 0.1547 nm at the specimen level using Leginon software (12) and processed with Appion software (5). The contrast transfer function for each set of particles from each image was estimated and corrected using ACE2 (a variation of ACE [7]). Particle images were automatically selected (11). The final stacks of particle images contained 20,425 MNV virions and 7,856 rRHDV VLPs, and EMAN 3D (6) was used for the reconstructions. Resolutions were estimated by Fourier shell correlations (FSC) of the three-dimensional (3D) reconstructions and application of a cutoff of 0.5. An amplitude correction of the final electron density was performed using GroEL small-angle X-ray scattering (SAXS) data.3D reconstructions of MNV-1 and rRHDV were calculated to resolutions of 8 Å and 8.1 Å, respectively (Fig. ​(Fig.1).1). The P domains of rNV VLPs rest directly on top of the shell domain (10) (Fig. ​(Fig.1A).1A). In contrast, the P domains of MNV-1 are lifted and rotated above the shell of the capsid (4) (Fig. ​(Fig.1B).1B). At this higher resolution, there was a clear connection between the P1 domain and the shell domain in all three capsid subunits (Fig. ​(Fig.1B,1B, arrow A). Unlike the smooth protruding domains of rNV, MNV-1 has two clear “horns” (arrow B), not dissimilar to those observed for the sapoviruses (1, 2). There also are islands of density in the interior of the shell, directly beneath the 5-fold axes, that may represent ordered regions of RNA.Open in a separate windowFIG. 1.Stereo diagrams (left) and thin sections (right), with radius coloring, of rNV (A), MNV-1 (B), and an rRHDV VLP (C). For rNV, the atomic coordinates (10) were used. In MNV, arrow A indicates the thin connector between the P1 and S domains. Arrow B denotes the horns found at the tips of the P2 domains. Arrow C denotes the large gap between the P1 and S domains in the rRHDV VLP. Arrow D denotes the false connectivity in rRHDV VLPs between the P1 domain and the S domain near the 5-fold axes.As with MNV-1, there is a marked gap between the P and S domains in the rRHDV VLP (Fig. ​(Fig.1C,1C, arrow C). This gap is not as pronounced as in MNV-1 because the P domains are not rotated as in MNV-1. In this electron density map, the A/B dimers appear to be touching the shell domain near the 5-fold axes. This contact difference between the A/B dimers and the C/C dimers could be the reason why the tops of the C/C dimers appear to be markedly disordered compared to the A/B dimers in rRHDV and the C/C dimers in MNV-1.Shown in Fig. ​Fig.22 is the fitting of the atomic structures of the MNV-1 P domain (13) and the rNV S domains into the MNV-1 3D reconstruction electron density. The horns (arrow A, loops A′-B′ and E′-F′) observed at the tips of the P domain match exceedingly well with the electron density. As discussed in the accompanying publication (13), the A′-B′ and E′-F′ loops displayed two discrete conformations, a closed structure, where the two loops were tightly associated, and an open structure, where the loops were splayed apart. The horns of the closed conformation fit better into the reconstruction, as the E′-F′ loop in the open form jutted out of the density at the base of the horns. The unmodified density in the lower panel of Fig. ​Fig.22 shows fine features in the shell domain and a very clear connection between the shell and P1 domains. The connections between the P1 and S domains were of sufficient quality to build a basic backbone model by uncoiling the linker region (arrow B). The P domain in the unfiltered 3D reconstruction was far less ordered than the S domain (Fig. ​(Fig.2).2). This was likely due to movement of the entire P domain with respect to the shell.Open in a separate windowFIG. 2.Fitting of the MNV-1 P domain and the rNV shell domain into the MNV-1 electron density. A, B, and C subunits are represented by blue, green, and red, respectively. The electron density is shown in transparent gray. The top panel is the 8.0-Å-resolution 3D reconstruction modified using a low-pass filter. The bottom panel is the reconstruction without modification. The horns on the tops of the P domains are denoted by arrow A. Arrow B denotes the connection between the S and P domains.Using the structure of rNV VLP P domains for modeling, the rRHDV P domains are lifted off the surface of the shell, but not rotated as with MNV-1. This places the bottom edge of the A subunit P1 domain near the S domain at the 5-fold axes. The P-domain dimers of rNV and rRHDV have a more “arch-like” shape than MNV-1. Unlike in MNV-1, the electron densities of the C/C dimers in rRHDV are far more diffuse than those of the A/B dimers (Fig. ​(Fig.3B)3B) and the connector between the S and P1 domains is not clear. During fitting, the connector region was not as extended as with MNV-1. This may afford greater flexibility, leading to more diffuse electron density.Open in a separate windowFIG. 3.Fitting of the rNV atomic structure into the rRHDV VLP electron density. The upper stereo image shows the 8.1-Å-resolution 3D reconstruction after modification by a low-pass filter. Below is the same reconstruction prior to density modification.When the atomic models for the MNV-1 P domains (13) were placed into the cryo-TEM electron density (Fig. ​(Fig.4),4), the C termini extended deep into the cores of adjacent P domains. Possible connections not accounted for by the P-domain structures were also observed in the electron density between the P domains. A bulge between the P1 and P2 domains in the 3D reconstruction indicated a possible interaction between the C termini and the adjacent P domains. These same interactions were observed in the crystal lattice. This highly mobile C terminus may be a flexible tether between the P domains in the intact virion.Open in a separate windowFIG. 4.Possible carboxyl-terminus interactions between the P domains of MNV-1. (A) Stereo image of MNV-1 calculated to 12-Å resolution with (red) and without (yellow) the last 10 residues of the P domain. (B) The calculated MNV-1 density with the carboxyl terminus removed (yellow) overlaid onto the 3D reconstruction of MNV-1 (blue). Note the strands of difference density that roughly correspond to the C terminus in panel A. (C) The C-terminus interactions observed in the structure of the MNV-1 P domains. Shown in blue and green are ribbon diagrams of an A/B P-domain dimer. In mauve is a surface rendering of the C terminus from a crystallographically related dimer. (D) Surface rendering of the final MNV-1 model with possible interactions between the P domains in MNV-1. The carboxyl termini of the A subunits (blue) interact with the counterclockwise-related B subunits around the 5-fold axes (white arrows). Around the 3-fold (quasi-6-fold) axes, the C subunits interact with the A subunits and the B subunits interact with the C subunits (orange arrows).It is absolutely clear that the hinge region between the S and P domains affords a remarkable degree of flexibility in the P domains that is not genus specific or related to differences between rVLPs and authentic virions. The simplest explanation for the role of this transition is that it gives the P domains flexibility that may be used to optimize interactions with cell receptors during attachment and entry. In this way, the P domains can increase their avidity for the cell surface by being more facile in adapting to the presentation of cellular recognition motifs.     

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.