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.     

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

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

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.     

Overexpression of the groESL Operon Enhances the Heat and Salinity Stress Tolerance of the Nitrogen-Fixing Cyanobacterium Anabaena sp. Strain PCC7120

The bicistronic groESL operon, encoding the Hsp60 and Hsp10 chaperonins, was cloned into an integrative expression vector, pFPN, and incorporated at an innocuous site in the Anabaena sp. strain PCC7120 genome. In the recombinant Anabaena strain, the additional groESL operon was expressed from a strong cyanobacterial P_psbA1 promoter without hampering the stress-responsive expression of the native groESL operon. The net expression of the two groESL operons promoted better growth, supported the vital activities of nitrogen fixation and photosynthesis at ambient conditions, and enhanced the tolerance of the recombinant Anabaena strain to heat and salinity stresses.Nitrogen-fixing cyanobacteria, especially strains of Nostoc and Anabaena, are native to tropical agroclimatic conditions, such as those of Indian paddy fields, and contribute to the carbon (C) and nitrogen (N) economy of these soils (22, 30). However, their biofertilizer potential decreases during exposure to high temperature, salinity, and other such stressful environments (1). A common target for these stresses is cellular proteins, which are denatured and inactivated during stress, resulting in metabolic arrest, cessation of growth, and eventually loss of viability. Molecular chaperones play a major role in the conformational homeostasis of cellular proteins (13, 16, 24, 26) by (i) proper folding of nascent polypeptide chains; (ii) facilitating protein translocation and maturation to functional conformation, including multiprotein complex assembly; (iii) refolding of misfolded proteins; (iv) sequestering damaged proteins to aggregates; and (v) solubilizing protein aggregates for refolding or degradation. Present at basal levels under optimum growth conditions in bacteria, the expression of chaperonins is significantly enhanced during heat shock and other stresses (2, 25, 32).The most common and abundant cyanobacterial chaperones are Hsp60 proteins, and nitrogen-fixing cyanobacteria possess two or more copies of the hsp60 or groEL gene (http://genome.kazusa.or.jp/cyanobase). One occurs as a solitary gene, cpn60 (17, 21), while the other is juxtaposed to its cochaperonin encoding genes groES and constitutes a bicistronic operon groESL (7, 19, 31). The two hsp60 genes encode a 59-kDa GroEL and a 61-kDa Cpn60 protein in Anabaena (2, 20). Both the Hsp60 chaperonins are strongly expressed during heat stress, resulting in the superior thermotolerance of Anabaena, compared to the transient expression of the Hsp60 chaperonins in Escherichia coli (20). GroEL and Cpn60 stably associate with thylakoid membranes in Anabaena strain PCC7120 (14) and in Synechocystis sp. strain PCC6803 (15). In Synechocystis sp. strain PCC6803, photosynthetic inhibitors downregulate, while light and redox perturbation induce cpn60 expression (10, 25, 31), and a cpn60 mutant exhibits a light-sensitive phenotype (http://genome.kazusa.or.jp/cyanobase), indicating a possible role for Cpn60 in photosynthesis. GroEL, a lipochaperonin (12, 28), requires a cochaperonin, GroES, for its folding activity and has wider substrate selectivity. In heterotrophic nitrogen-fixing bacteria, such as Klebsiella pneumoniae and Bradyrhizobium japonicum, the GroEL protein has been implicated in nif gene expression and the assembly, stability, and activity of the nitrogenase proteins (8, 9, 11).Earlier work from our laboratory demonstrated that the Hsp60 family chaperonins are commonly induced general-stress proteins in response to heat, salinity, and osmotic stresses in Anabaena strains (2, 4). Our recent work elucidated a major role of the cpn60 gene in the protection from photosynthesis and the nitrate reductase activity of N-supplemented Anabaena cultures (21). In this study, we integrated and constitutively overexpressed an extra copy of the groESL operon in Anabaena to evaluate the importance and contribution of GroEL chaperonin to the physiology of Anabaena during optimal and stressful conditions.Anabaena sp. strain PCC7120 was photoautotrophically grown in combined nitrogen-free (BG11⁻) or 17 mM NaNO₃-supplemented (BG11⁺) BG11 medium (5) at pH 7.2 under continuous illumination (30 μE m⁻² s⁻¹) and aeration (2 liters min⁻¹) at 25°C ± 2°C. Escherichia coli DH5α cultures were grown in Luria-Bertani medium at 37°C at 150 rpm. For E. coli DH5α, kanamycin and carbenicillin were used at final concentrations of 50 μg ml⁻¹ and 100 μg ml⁻¹, respectively. Recombinant Anabaena clones were selected on BG11⁺ agar plates supplemented with 25 μg ml⁻¹ neomycin or in BG11⁻ liquid medium containing 12.5 μg ml⁻¹ neomycin. The growth of cyanobacterial cultures was estimated either by measuring the chlorophyll a content as described previously (18) or the turbidity (optical density at 750 nm). Photosynthesis was measured as light-dependent oxygen evolution at 25 ± 2°C by a Clark electrode (Oxy-lab 2/2; Hansatech Instruments, England) as described previously (21). Nitrogenase activity was estimated by acetylene reduction assays, as described previously (3). Protein denaturation and aggregation were measured in clarified cell extracts containing ∼500 μg cytosolic proteins treated with 100 μM 8-anilino-1-naphthalene sulfonate (ANS). The pellet (protein aggregate) was solubilized in 20 mM Tris-6 M urea-2% sodium dodecyl sulfate (SDS)-40 mM dithiothreitol for 10 min at 50°C. The noncovalently trapped ANS was estimated using a fluorescence spectrometer (model FP-6500; Jasco, Japan) at a λ_excitation of 380 nm and a λ_emission of 485 nm, as described previously (29).The complete bicistronic groESL operon (2.040 kb) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ608815","term_id":"222354886","term_text":"FJ608815"}}FJ608815) was PCR amplified from PCC7120 genomic DNA using specific primers (Table ​(Table1)1) and the amplicon cloned into the NdeI-BamHI restriction sites of plasmid vector pFPN, which allows integration at a defined innocuous site in the PCC7120 genome and expression from a strong cyanobacterial P_psbA1 promoter (6). The resulting construct, designated pFPNgro (Table ​(Table1),1), was electroporated into PCC7120 using an exponential-decay wave form electroporator (200 J capacitive energy at a full charging voltage of 2 kV; Pune Polytronics, Pune, India), as described previously (6). The electroporation was carried out at 6 kV cm⁻¹ for 5 ms, employing an external autoclavable electrode with a 2-mm gap. The electroporation buffer contained high concentrations of salt (10 mM HEPES, 100 mM LiCl, 50 mM CaCl₂), as have been recommended for plant cells (23) and other cell types (27). The electrotransformants, selected on BG11⁺ agar plates supplemented with 25 μg ml⁻¹ neomycin by repeated subculturing for at least 25 weeks to achieve complete segregation, were designated AnFPNgro.

TABLE 1.

Plasmids, strains, and primers used in this study

Efficient Production of 2-Oxobutyrate from 2-Hydroxybutyrate by Using Whole Cells of Pseudomonas stutzeri Strain SDM

2-Oxobutyrate is an important intermediate in the chemical, drug, and food industries. Whole cells of Pseudomonas stutzeri SDM, containing NAD-independent lactate dehydrogenases, effectively converted 2-hydroxybutyrate into 2-oxobutyrate. Under optimal conditions, the biocatalytic process produced 2-oxobutyrate at a high concentration (44.4 g liter⁻¹) and a high yield (91.5%).2-Oxobutyrate (2-OBA) is used as a raw material in the synthesis of chiral 2-aminobutyric acid, isoleucine, and some kinds of medicines (1, 8). There is no suitable starting material for 2-OBA production by chemical synthesis; therefore, the development of innovative biotechnology-based techniques for 2-OBA production is desirable (12).2-Hydroxybutyrate (2-HBA) is cheaper than 2-OBA and can be substituted for 2-OBA in the production of isoleucine, as reported previously (9, 10). The results of those studies also indicated that it might be possible to produce 2-OBA from 2-HBA by a suitable biocatalytic process. In the presence of NAD, NAD-dependent 2-hydroxybutyrate dehydrogenase can catalyze the oxidation of 2-HBA to 2-OBA (4). However, due to the high cost of pyridine cofactors (11), it is preferable to use a biocatalyst that directly catalyzes the formation of 2-OBA from 2-HBA without any requirement for NAD as a cofactor.In our previous report, we confirmed that NAD-independent lactate dehydrogenases (iLDHs) in the pyruvate-producing strain Pseudomonas stutzeri SDM (China Center for Type Culture Collection no. M206010) could oxidize lactate and 2-HBA (6). Therefore, in addition to pyruvate production from lactate, P. stutzeri SDM might also have a potential application in 2-OBA production.To determine the 2-OBA production capability of P. stutzeri SDM, the strain was first cultured at 30°C in a minimal salt medium (MSM) supplemented with 5.0 g liter⁻¹ dl-lactate as the sole carbon source (5). The whole-cell catalyst was prepared by centrifuging the medium and resuspending the cell pellet, and biotransformation was then carried out under the following conditions using 2-HBA as the substrate and whole cells of P. stutzeri SDM as the biocatalyst: 2-HBA, 10 g liter⁻¹; dry cell concentration, 6 g liter⁻¹; buffer, 100 mM potassium phosphate (pH 7.0); temperature, 30°C; shaking speed, 300 rpm. After 4 h of reaction, the mixture was analyzed by high-performance liquid chromatography (HPLC; Agilent 1100 series; Hewlett-Packard) using a refractive index detector (3). The HPLC system was fitted with a Bio-Rad Aminex HPX-87 H column. The mobile phase consisted of 10 mM H₂SO₄ pumped at 0.4 ml min⁻¹ (55°C). Biotransformation resulted in the production of a compound that had a retention time of 19.57 min, which corresponded to the peak of authentic 2-OBA (see Fig. S1 in the supplemental material).After acidification and vacuum distillation, the new compound was analyzed by negative-ion mass spectroscopy. The molecular ion ([M − H]⁻, m/z 101.1) signal of the compound was consistent with the molecular weight of 2-OBA, i.e., 102.1 (see Fig. S2 in the supplemental material). These results confirmed that 2-HBA was oxidized to 2-OBA by whole cells of P. stutzeri SDM.To investigate whether iLDHs are responsible for 2-OBA production in the above-described biocatalytic process, 2-HBA oxidation activity in P. stutzeri SDM was probed by native polyacrylamide gel electrophoresis. After electrophoresis, the gels were soaked in a substrate solution [50 mM Tris-HCl buffer (pH 8.0) containing 0.1 mM phenazine methosulfate, 0.1 mM 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, and 1 mM l-lactate, dl-lactate, or dl-2-HBA] and gently shaken. As shown in Fig. ​Fig.1,1, d- and l-iLDH migrated as two bands with distinct mobilities. The activities responsible for d- and l-2-HBA oxidation were located at the same positions as the d- and l-iLDH activities, respectively. No other bands responsible for d- and l-2-HBA oxidation were detected. Moreover, the dialysis of the crude cell extract did not lead to loss of 2-HBA oxidation activity and the addition of 10 mM NAD⁺ could not stimulate the reaction (see Table S1 in the supplemental material). These results implied that in the biocatalytic system, 2-HBA was oxidized to 2-OBA by iLDHs present in P. stutzeri SDM.Open in a separate windowFIG. 1.Activity staining of iLDHs after native polyacrylamide gel electrophoresis with lactate or 2-HBA as the substrate.Although the SDM strain could not use 2-HBA or 2-OBA for growth (see Fig. S3 in the supplemental material), 2-HBA might induce some of the enzymes responsible for 2-OBA production in the biocatalytic process. To exclude this possibility, the SDM strain was cultured in MSM containing dl-lactate or pyruvate as the sole carbon source. As shown in Fig. ​Fig.2,2, the enzyme activities that catalyzed lactate and 2-HBA oxidation were simultaneously present in the cells cultured on lactate and were absent in those cultured on pyruvate. After the lactate or pyruvate was exhausted, 5.05 g liter⁻¹ dl-2-HBA was added to the medium. It was observed that dl-2-HBA was efficiently converted to 2-OBA in the medium containing dl-lactate (Fig. ​(Fig.2a).2a). No 2-OBA production was detected in the medium containing pyruvate. Because 2-HBA addition did not induce the enzymes involved in 2-HBA oxidation (Fig. 2a and b), we concluded that the iLDHs induced by dl-lactate catalyzed 2-HBA oxidation in this biocatalytic process.Open in a separate windowFIG. 2.Time course of P. stutzeri SDM growth on media containing dl-lactate (a) and pyruvate (b). 2-HBA was added to the medium after the exhaustion of lactate or pyruvate. Symbols: ▴, lactate; ▵, pyruvate; •, 2-HBA; ○, 2-OBA; ▪, cell density; ▧, iLDHs activity with dl-lactate as the substrate; ▒, iLDHs activity with dl-2-HBA as the substrate.iLDHs could catalyze the oxidation of the substrate in a flavin-dependent manner and might use membrane quinone as the electron acceptor. Unlike the oxidases, which directly use the oxygen as the electron acceptor, this substrate oxidation mechanism could prevent the formation of H₂O₂ (see Fig. S4 in the supplemental material). The P. stutzeri SDM strain efficiently converted dl-2-HBA to 2-OBA with high yields (4.97 g liter⁻¹ 2-OBA was produced from 5.05 g liter⁻¹ dl-2-HBA); therefore, 2-OBA production by this strain can be a valuable and technically feasible process. To increase the efficiency of P. stutzeri SDM in the biotechnological production of 2-OBA, the conditions for biotransformation using whole cells of P. stutzeri SDM were first optimized. The influence of the reaction pH and 2-HBA concentration on 2-OBA production was determined in 100 mM phosphate buffer containing whole cells harvested from the medium containing dl-lactate as the sole carbon source. The reaction was initiated by adding the whole cells and 2-HBA at 37°C, followed by incubation for 10 min. After stopping the reaction by adding 1 M HCl, the 2-OBA concentration was determined by HPLC.As shown in Fig. ​Fig.3a,3a, ​,2-OBA2-OBA production was highest at pH 7.0. Under acidic or alkaline conditions, the transformation of 2-HBA to 2-OBA decreased. The optimal 2-HBA concentration was found to be 0.4 M, as shown in Fig. ​Fig.3b.3b. 2-OBA production increased as the 2-HBA concentration increased up to about 0.4 M and decreased thereafter. The concentration of the whole-cell catalyst was then optimized using 0.4 M 2-HBA as the substrate at pH 7.0. As shown in Fig. ​Fig.3c,3c, the highest 2-OBA concentration was obtained with 20 g (dry cell weight [DCW]) liter⁻¹ of P. stutzeri SDM. The 2-OBA concentration decreased with any increase beyond this cell concentration.Open in a separate windowFIG. 3.Optimization of the biocatalysis conditions. (a) Effect of pH on 2-OBA production activity. (b) Effect of 2-HBA concentrations on 2-OBA production activity. (c) Effect of the concentration of P. stutzeri SDM on biotransformation. OD, optical density.After optimizing the biocatalytic conditions, we studied the biotechnological production of 2-OBA from 2-HBA by using the whole-cell catalyst P. stutzeri SDM. As shown in Fig. ​Fig.4,4, when 20 g (DCW) liter⁻¹ P. stutzeri SDM was used as the biocatalyst, 48.5 g liter⁻¹ 2-HBA was biotransformed into 44.4 g liter⁻¹ 2-OBA in 24 h.Open in a separate windowFIG. 4.Time course of production of 2-OBA from 2-HBA under the optimum conditions. Symbols: ▪, 2-OBA; •, 2-HBA.Biocatalytic production of 2-OBA was carried out using crotonic acid, propionaldehyde, 1,2-butanediol, or threonine as the substrate (2, 7, 8, 12). Resting cells of the strain Rhodococcus erpi IF0 3730 produced 15.7 g liter⁻¹ 2-OBA from 20 g liter⁻¹ 1,2-butanediol, which is the highest reported yield of 2-OBA to date (8). By using the whole-cell catalyst P. stutzeri SDM, it was possible to produce 2-OBA at a high concentration (44.4 g liter⁻¹) and a high yield (91.5%). Due to the simple composition of the biocatalytic system (see Fig. S5 in the supplemental material), 2-HBA and 2-OBA could be easily separated on a column using a suitable resin. Separation of 2-OBA from the biocatalytic system was relatively inexpensive. The biocatalytic process presented in this report could be a promising alternative for the biotechnological production of 2-OBA.      

A Y526Q Mutation in the Newcastle Disease Virus HN Protein Reduces Its Functional Activities and Attenuates Virus Replication and Pathogenicity

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a multifunctional protein that plays a crucial role in virus infectivity. In this study, using the mesogenic strain Beaudette C (BC), we mutated three conserved amino acids thought to be part of the binding/catalytic active site in the HN protein. We also mutated five additional residues near the proposed active site that are nonconserved between BC and the avirulent strain LaSota. The eight recovered NDV HN mutants were assessed for effects on biological activities. While most of the mutations had surprisingly little effect, mutation at conserved residue Y526 reduced the neuraminidase, receptor binding, and fusion activities and attenuated viral virulence in eggs and young birds.Newcastle disease virus (NDV) is an avian pathogen of the genus Avulavirus in the family Paramyxoviridae (10). The envelope of NDV contains two surface glycoproteins, the fusion (F) protein and the HN (hemagglutinin-neuraminidase [NA]) protein. The F protein mediates viral penetration and requires cleavage-activation by host protease. Cleavability of the F protein is a major determinant of virulence. However, other viral proteins, including HN, also contribute to virulence (5). HN is a multifunctional glycoprotein. It recognizes sialic acid-containing receptors on cell surfaces; promotes the fusion activity of F protein, thereby allowing the virus to penetrate the cell surface; and acts as an NA that removes sialic acid from progeny virus particles to prevent viral self-aggregation (9).HN is a type II homotetrameric glycoprotein with a monomer length of 577 amino acids for most NDV strains (14). The ectodomain of the HN protein consists of a 95-amino-acid stalk region supporting a 428-amino-acid terminal globular head. Although mutations in the transmembrane and stalk regions of the HN protein can affect the structure and activities of the protein (11, 15), the antigenic, receptor recognition, and NA active sites are all localized in the globular head (12, 16). The X-ray crystal structure of the globular head of the NDV HN protein has identified residues that appear to contribute to receptor recognition, NA, and fusion activities (4). Previous studies have proposed that conserved residues R174, I175, D198, K236, R416, R498, Y526, and E547 are important in receptor recognition and NA activities and that residues R174 and E547 influence the fusion promotion activity of the HN protein (3, 4, 6). Although transfection studies using plasmids expressing HN mutants of NDV have highlighted the importance of these residues in different biological functions of the HN protein, their contribution to NDV biology and pathogenesis in the context of the complete virus was not known.In this study, we examined the roles of three of the above-named conserved residues, R416, R498, and Y526 (all located near the sialic acid binding site), in the biological activities and pathogenesis of the HN protein of NDV in the context of infectious virus. In addition, comparison of the HN protein sequence between the avirulent strain LaSota and the moderately virulent strain Beaudette C (BC) identified 12 amino acid differences in the globular head region of the HN protein (H203, T214, I219, S228, L269, A271, E293, G310, S494, E495, T502, and N568, named according to the BC amino acid assignment). We also examined five of these nonconserved residues, T214, I219, S494, E495, and N568, located in close proximity to residues identified earlier by crystal structure studies, to determine whether these might affect HN function and contribute to the difference in pathogenicity between the LaSota and BC strains (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Three-dimensional structure of the NDV HN protein showing the positions of amino acid residues that were substituted in the present study. The residues are shown in space-filling mode and represented in different colors. The MacPymol (DeLano Scientific) software was used to generate the model of the globular domain of the NDV HN monomer. The structure was derived from the crystal structure of the NDV HN protein reported by Crennell et al. (4).We used site-directed mutagenesis (2) to introduce individual amino acid substitutions into a cDNA of the HN gene of strain BC. For the conserved residues, we changed arginine at positions 416 and 498 and tyrosine at position 526 to polar glutamine. For the nonconserved residues, the assignments T214, I219, S494, E495, and N568 of strain BC were altered to the corresponding assignments of strain LaSota: S214, V219, G494, V495, and D568, respectively. Each mutagenized HN gene was then inserted into a full-length cDNA clone of the BC antigenome. These clones were transfected into HEp2 cells, and mutant viruses were recovered as previously described (8). These viruses were designated according to the substitutions introduced: T214S, I219V, R416Q, S494G, E495V, R498Q, Y526Q, and N568D. The HN genes from recovered viruses were sequenced. This confirmed the presence of each introduced mutation and the lack of adventitious mutations in the HN gene. To determine the stability of each HN mutation, the recovered viruses were passaged five times in 9-day-old embryonated chicken eggs and five times in chicken embryo fibroblast DF-1 cells. Sequence analysis of the HN gene of the mutant viruses at each passage showed that the introduced mutations were unaltered (data not shown). To rule out the possibility that change in the HN protein sequence could be compensated for by a mutation in the F protein, the F gene from each recovered virus was sequenced. No compensatory mutations in the F gene were observed (data not shown). The HN protein content of each mutant virus, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie staining, was very similar to that of the parental BC virus (pBC) (Table ​(Table1).1). The multicycle growth kinetics of the recombinant HN mutant viruses in DF-1 cells (Fig. ​(Fig.2)2) showed that the replication kinetics of all of the HN mutant viruses were similar to those of pBC, with the exception of the Y526Q mutant, which showed delayed growth and had a lower virus yield (1.5 to 2.0 log₁₀ PFU/ml) than the parental and other mutant viruses. In addition, the Y526Q mutant produced syncytia at 72 h, whereas the parental and other mutant viruses initiated syncytia at 24 h postinfection. These studies showed the importance of amino acid residue Y526 at the active site of the HN protein of NDV.Open in a separate windowFIG. 2.Multicycle growth kinetics of HN mutants of NDV in chicken embryo fibroblast (DF-1) cells. Cells were infected with the indicated parental or mutant virus at an multiplicity of infection of 0.01. Supernatant samples were collected at 8-h intervals until 64 h postinfection, and virus titers were determined at different time points by plaque assay. Values are averages from three independent experiments.

TABLE 1.

Biological activities of HN mutants of NDV

Murine Norovirus Infection Has No Significant Effect on Adaptive Immunity to Vaccinia Virus or Influenza A Virus

Murine norovirus (MNV) is endemic in many research mouse colonies. Although MNV infections are typically asymptomatic in immunocompetent mice, the effects of MNV infection on subsequent experimental viral infections are poorly documented. Here, we infected C57BL/6 mice with MNV and then with either vaccinia virus or influenza A virus. MNV infection had no effect on CD8⁺ T-cell or antibody responses to secondary viruses or to secondary virus-induced morbidity or mortality. While our findings suggest that MNV has little influence on host immunity in immunocompetent mice, we would urge caution regarding the potential effects of MNV on immune responses to viruses and other pathogens, which must be determined on a system-by-system basis.Human norovirus (NoV) infections cause greater than 90% of nonbacterial gastroenteritis cases (4, 5) and are an important public health concern. Murine noroviruses (MNV) were recently identified (7) as highly pathogenic agents in immunocompromised mice, and serological studies indicate that over 20% of mice in research colonies are exposed to MNV (6). As with NoV, MNV is spread through the fecal-oral route. While NoV rapidly causes gastrointestinal symptoms and fever in healthy individuals, MNV is typically asymptomatic in immunocompetent mice.MNV isolates are both genetically and biologically diverse (13). In wild-type (wt) mice, some strains of MNV are rapidly cleared, while others persist (13). Controlling MNV infections requires elements of both innate and adaptive immunity. Mice with defects in interferon (IFN) signaling pathways demonstrate increased MNV lethality (7, 9). CD4⁺ and CD8⁺ T cells and B cells are all needed for complete MNV clearance (1, 2). Natural exposure of immunocompromised mice to MNV leads to inflammation of the liver, lungs, and peritoneal and pleural cavities (14).It is well established that infection with natural mouse viruses can greatly impact immune responses to infections with other viruses. The prevalence of MNV in research mouse colonies might therefore lead to irreproducible and variable results that significantly impact research efforts. Indeed, MNV was recently reported to alter disease progression in a mouse model of bacterium-induced inflammatory bowel disease (8). Concern over the potential effects of MNV on viral immunology research prompted a dedicated workshop at the 2008 Keystone Viral Immunity meeting (http://www.keystonesymposia.org). In the present study, we examined the effect of MNV infection on adaptive immune responses in wt mice to influenza A virus (IAV) and vaccinia virus (VV).We infected C57BL/6 mice perorally with a high dose (3 × 10⁷ PFU/mouse) of a plaque-purified MNV stock derived from MNV-CR6p2 (13). The capacity of this plaque-purified virus to persist in wt mice has been confirmed by quantitative PCR analysis and a plaque assay (D. Strong, L. Thackray, and H. Virgin, unpublished observation). We confirmed that the mice were infected by measuring anti-MNV antibodies (Abs) by using an enzyme-linked immunosorbent assay (ELISA) (data not shown). For all experiments, mice were infected with MNV at Washington University and shipped 4 to 5 days later to NIAID for further study. To contain MNV, infected mice were housed in microisolator cages in a quarantine room. In some experiments, control mice were housed in the same room as MNV-infected mice. Sera collected from control mice did not contain anti-MNV Abs as determined by ELISA (data not shown), confirming that transmission of MNV between mice housed in microisolator cages can be prevented by proper cage changing and aseptic handling of samples from infected mice.Upon intraperitoneal (i.p.) infection with either VV or IAV, mice mount robust CD8⁺ T-cell responses that peak, respectively, on day 6 or 7. Anti-VV and anti-IAV CD8⁺ T-cell responses in C57BL/6 mice conform to a well-established immunodominance hierarchy (3, 10). To determine to what extent MNV infection alters the magnitude and/or immunodominance hierarchy of CD8⁺ T-cell responses, we infected C57BL/6 mice i.p. with either VV or IAV 19 days following MNV infection. As controls, naïve mice (MNV negative) were infected with either virus. Lymphocytes were isolated from mice 6 days postinfection with VV and 7 days postinfection with IAV. The fraction of antigen-specific CD8⁺ T cells present in spleen and peritoneal exudate cells (PEC) was determined by intracellular IFN-γ staining after stimulation with synthetic peptides. MNV infection had little effect on the magnitude of splenic or PEC CD8⁺ T cells responding to VV (Fig. 1A and B) or IAV (Fig. 1C and D) infection. Regardless of MNV exposure history, splenic and PEC responses were dominated by B8R- and A8R-specific CD8⁺ T cells following VV infection (Fig. 1A and B) and by PA-specific and NP-specific CD8⁺ T cells following IAV infection (Fig. 1C and D).Open in a separate windowFIG. 1.MNV exposure does not alter CD8⁺ T-cell responses to VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.p. with ∼1 × 10⁶ PFU of VV (A and B) or ∼1 × 10⁷ 50% tissue culture infective dose units of IAV (C and D), and specific CD8⁺ T cells were determined by intracellular IFN-γ staining after restimulating lymphocytes with peptides. Lymphocytes isolated from the spleen (A and C) and peritoneal cavity (B and D) were tested. MNV infections were completed 19 days prior to VV or IAV infections. Means and SEM are shown in panels A and C. A two-way analysis of variance and Bonferroni statistical analysis were completed for these experiments. Cells were pooled for peritoneal lavage samples as shown in panels B and D. Four to five mice/group were used for each experiment; data are representative of two independent experiments.To examine the effect of MNV infection on antiviral Ab responses, MNV-infected and control C57BL/6 mice were infected intranasally (i.n.) with a sublethal dose of either VV or IAV. Three weeks later, levels of anti-VV and anti-IAV Abs were determined by ELISA and hemagglutination inhibition assays, respectively. MNV infection did not significantly modify the magnitude of Ab responses to VV (Fig. ​(Fig.2A)2A) or IAV (Fig. ​(Fig.2B).2B). Next, we determined the effect of MNV infection on heavy chain class switching of anti-VV or anti-IAV Ab responses. Anti-VV and anti-IAV Ab responses exhibited similar heavy chain profiles dominated by immunoglobulin G2b (IgG2b) Abs regardless of MNV status (Fig. 2C and D). Thus, the CD8⁺ T-cell and Ab response to both VV and IAV appears to be essentially unaffected by chronic MNV infection. Since IgG anti-VV or anti-IAV Ab responses are entirely dependent on CD4⁺ T-cell help (11, 12), we can also infer that MNV also does not significantly affect CD4⁺ T-cell responses to VV or IAV.Open in a separate windowFIG. 2.MNV exposure does not alter Ab responses to VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with ∼1 × 10³ PFU of VV (A and C) or ∼50 50% tissue culture infective dose units of IAV (B and D), and virus-specific Abs were determined by ELISA (A, C, and D) or hemagglutination inhibition (B). The ELISA results shown in panel A measured the total IgG, while the ELISA results shown in panels C and D measured the individual isotype indicated. MNV infections were completed 19 days prior to VV or IAV infections. Means and standard errors of the means are shown in panels A, C, and D. Means are shown as lines in panel B. A two-way analysis of variance and Bonferroni statistical analysis were completed for experiments shown in panels A, C, and D, and t tests were completed for the experiment shown in panel B. Four to five mice/group were used for each experiment. O.D., optical density; HAI, hemagglutination inhibition.T-cell and Ab responses, together with innate immune mechanisms, collaborate to control viral replication and limit pathogenesis. To examine the effect of chronic MNV infection on VV-induced or IAV-induced pathogenesis, we infected C57BL/6 mice i.n. with a lethal or sublethal dose of VV or IAV and monitored body weight over a 16-day period. MNV-CR6p2 infection had no significant effect on morbidity or mortality from either virus (Fig. ​(Fig.33 and ​and4).4). Since MNV isolates are highly diverse, we decided to examine the effects of a second strain of MNV (MNV-CW3) which is fully cleared in immunocompetent mice. Mice that cleared MNV-CW3 (19 days post-MNV infection) were infected i.n. with VV or IAV. Once again, this strain of MNV had no effect on VV-induced or IAV-induced morbidity or mortality (Fig. ​(Fig.33 and ​and4).4). Future studies should address the extent to which other MNV strains affect the generation of adaptive immune responses to secondary viral infections.Open in a separate windowFIG. 3.MNV does not increase morbidity following subsequent i.n. infection with VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with a sublethal dose of VV (∼1 × 10³ PFU) (A) or IAV (∼50 50% tissue culture infective dose units) (B), and weight loss was recorded for 16 days postinfection. MNV infections were completed 19 days prior to VV or IAV infections. A two-way analysis of variance and Bonferroni statistical analysis were completed. Four to five mice/group were used for each experiment.Open in a separate windowFIG. 4.MNV does not increase mortality following subsequent i.n. infection with VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with VV (∼1 × 10⁴ PFU) (A) or IAV (∼500 50% tissue culture infective dose units) (B), and survival was monitored for 16 days postinfection. MNV infections were completed 19 days prior to VV or IAV infections. Eight to 10 mice/group were used for each experiment.Taken together, these data demonstrate that MNV infection has no significant effects on the measured immune response to VV or IAV. Our results cannot, however, be simply extrapolated to other viruses or microorganisms. Rather, the effect of MNV infection on host immunity in mouse model disease systems needs to be established on a system-by-system basis. Without this knowledge, the possible confounding effects of MNV infection will continue to undermine the confidence in results obtained using mice in colonies in which MNV infections are endemic.     

Differential Rates of Protein Folding and Cellular Trafficking for the Hendra Virus F and G Proteins: Implications for F-G Complex Formation

Hendra virus F protein-promoted membrane fusion requires the presence of the viral attachment protein, G. However, events leading to the association of these glycoproteins remain unclear. Results presented here demonstrate that Hendra virus G undergoes slower secretory pathway trafficking than is observed for Hendra virus F. This slowed trafficking is not dependent on the G protein cytoplasmic tail, the presence of the G receptor ephrin B2, or interaction with other viral proteins. Instead, Hendra virus G was found to undergo intrinsically slow oligomerization within the endoplasmic reticulum. These results suggest that the critical F-G interactions occur only after the initial steps of synthesis and cellular transport.The Henipavirus genus of the paramyxovirus family comprises two recently emerged, zoonotic pathogens. Hendra virus, first identified in Australia in 1994, caused respiratory illness in and the subsequent death of over one dozen horses and two of the three humans infected (12, 21, 25). Nipah virus led to an outbreak of respiratory and encephalitic illnesses in Malaysia in 1999, affecting both swine and humans and leading to fatality in 105 of the 265 human cases (11). Additional periodic outbreaks of infections with these viruses have occurred (11), and evidence indicates human-to-human transmission of Nipah virus in at least one outbreak (16). Henipavirus, like many paramyxoviruses, requires the presence of two surface glycoproteins for virus-cell and cell-cell fusion (8, 9, 36): the fusion protein, F, which mediates the membrane fusion event, and the attachment protein, G, which binds cellular receptors ephrin B2 (6, 22) and ephrin B3 (23) and which is required for F-mediated membrane fusion. Interactions between the fusion and attachment proteins of a number of paramyxoviruses have been observed previously (30, 33, 35), and interactions between the henipavirus F and G proteins have been demonstrated by coimmunoprecipitation (1-5, 18). However, important questions remain concerning the timing of these interactions and the mechanism by which the attachment protein regulates F-mediated fusion. Results from studies of measles virus (30), Newcastle disease virus (33), and human parainfluenza virus (35) have suggested that the initial interaction between the two glycoproteins occurs within the endoplasmic reticulum (ER) at the time of synthesis, potentially allowing the attachment protein to hold the F protein in its prefusion conformation. In contrast, the retention of the parainfluenza virus type 5 (PIV5) F protein in the ER does not lead to the retention of the PIV5 attachment protein (29), suggesting that interaction between these glycoproteins does not occur soon after synthesis. Recently, henipavirus F proteins have been shown to undergo processing through a complex intracellular trafficking pathway, with expression on the cell surface in a nonfusogenic precursor form (F₀), subsequent endocytosis, cleavage by cathepsin L into the fusogenic (F₁+F₂) form, and retrafficking to the plasma membrane (10, 19, 26-28). While the half-life (t_1/2) of F protein uncleaved by cathepsin L is approximately 2 h (28), results from initial studies of Hendra virus G trafficking indicate much slower trafficking of this protein through the secretory pathway (37), a result inconsistent with the formation of an F-G complex in the ER.To more closely examine the trafficking of the henipavirus glycoproteins, endoglycosidase H (endo H) analysis was used as a marker for trafficking time to the medial-Golgi compartment. We previously reported that wild-type (wt) Hendra virus G protein becomes endo H resistant, with a t_1/2 of between 2 and 3 h (37), suggesting slow trafficking through the secretory pathway. Porotto et al. (31) described a mutant G protein lacking the first 32 residues of the cytoplasmic tail (G-Δ32), which showed enhanced fusion promotion. This Hendra virus G-Δ32 mutant also exhibited higher overall expression than the wt Hendra virus G (S. D. Whitman and R. E. Dutch, unpublished results). To determine if this mutant exhibited altered trafficking kinetics, Vero cells transfected with pCAGGS-Hendra G-Δ32 were examined by pulse-chase analysis followed by endo H treatment as described previously (37). Trafficking kinetics similar to those of wt G were observed, as G-Δ32 became endo H resistant, with a t_1/2 of approximately 2 to 3 h (Fig. ​(Fig.1A).1A). In contrast, endo H analysis of Hendra virus F revealed a resistant population of F₀ within 30 min (Fig. ​(Fig.1B),1B), with the majority of F converted to either an F₀ endo H-resistant form or to the F₁ cleaved form by 2 h. The cleavage of F₀ was observed concomitantly with the appearance of two partially endo H-resistant F₁ bands, suggestive of differential complex sugar additions within the Golgi compartment. These data confirm that Hendra virus F and G traffic through the secretory pathway at different rates, with Hendra virus G trafficking unaffected by the 32-amino-acid deletion. Endo H analysis of the Nipah virus G dimeric form (Fig. ​(Fig.1C),1C), for which the mobility shift after endo H treatment was most apparent, mirrors that of wt Hendra virus G, suggesting that slow trafficking through the secretory pathway is a property of both henipavirus G proteins.Open in a separate windowFIG. 1.Endo H digestion indicates differential rates of trafficking for the henipavirus F and G proteins. (A) Vero cells were transfected with pCAGGS-Hendra G-Δ32, and 24 h posttransfection, the cells were labeled for 30 min and chased for various times and Hendra virus G was immunoprecipitated using an antibody directed to a soluble form of G (7). Endo H digestion was performed as described previously (37). Proteins were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized using the Typhoon imaging system. (B) Vero cells were transfected with pCAGGS-Hendra F. Twenty-four hours later, the cells were labeled for 30 min. Hendra virus F was immunoprecipitated with a Hendra virus F-specific antibody (28) and subjected to endo H treatment. (C) Pulse-chase analysis of Vero cells transfected with pCAGGS-Nipah G was performed. Immunoprecipitation with antibody to a soluble form of G (7) and endo H analysis were performed as described previously. R and S denote the endo H-resistant and -sensitive species, respectively. +, present; −, absent.Slow trafficking of Hendra virus G through the secretory pathway may be caused by interactions with other viral proteins or with its receptor ephrin B2. Chinese hamster ovary (CHO) cells do not express ephrin B2 (39) and thus were utilized to examine Hendra virus G trafficking in the absence of its cognate receptor. When Hendra virus G-Δ32 was expressed in CHO cells, an endo H-resistant population appeared at 2 h (Fig. ​(Fig.2A)2A) and was found to have increased at subsequent time points, consistent with results obtained using Vero cells. These data suggest that the low trafficking rate of Hendra virus G is not due to association with ephrin B2 and is not cell type specific. Coexpression of either the Hendra virus matrix (M) protein or the F protein with G-Δ32 did not alter trafficking kinetics, indicating that an interaction with either M or F is not responsible for the low rate of G-Δ32 trafficking (Whitman and Dutch, unpublished).Open in a separate windowFIG. 2.Endo H analysis indicates that slow trafficking through the secretory pathway is not dependent on ephrin B2 or on sequences present in the Hendra virus G cytoplasmic tail. (A) CHO cells expressing Hendra virus G-Δ32 were metabolically labeled with ³⁵S for 30 min and chased for the times indicated. Following immunoprecipitation and endo H analysis, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized via the Typhoon imaging system. (B) Pulse-chase analysis of Vero cells expressing the Hendra virus G-Δ41 tail was performed, followed by immunoprecipitation and endo H analysis. R and S denote the endo H-resistant and -sensitive species, respectively. +, present; −, absent.Lysine-rich motifs (KKXX or KXKXX) have been shown previously to be involved in the retention of type I glycoproteins in the ER (17), while motifs containing multiple arginines have been implicated in the retention of type II integral membrane proteins (20, 32). The type II Hendra and Nipah virus G proteins contain no and one arginine residue in their cytoplasmic domains, respectively, but do have several lysine-rich motifs. To determine if these motifs facilitated ER retention of Hendra virus G, thus resulting in a lowered trafficking rate, an additional mutant (Hendra virus G-Δ41) with the removal of the first 41 amino acids of the cytoplasmic tail, deleting both KKXX and KXKXX motifs and the majority of the cytoplasmic tail, was constructed. Consistent with the results from endo H analyses of wt Hendra virus G and Hendra virus G-Δ32, an endo H-resistant form of Hendra virus G-Δ41 appeared after only 2 h (Fig. ​(Fig.2B),2B), suggesting that Hendra virus G is not retained in the ER by the putative ER retention motifs KKXX and KXKXX or by any other sequence in the cytoplasmic tail.Endo H data strongly indicated differential rates of trafficking through the secretory pathway for Hendra virus G and Hendra virus F, suggesting that interactions between these two proteins likely occur subsequent to transport to the cell surface. To verify the different trafficking kinetics of these two proteins, the rates at which Hendra virus F and G appear on the cell surface were determined by using surface biotinylation, with the more highly expressed mutant Hendra virus G-Δ32 utilized in these experiments to facilitate visualization of the surface population. Hendra virus F was present on the cell surface as F₀ at the completion of the 30-min labeling (Fig. ​(Fig.3A)3A) (time zero), with F₁+F₂ appearing on the surface at approximately 2 h (Fig. ​(Fig.3A),3A), consistent with the complex trafficking pathway observed for F. In contrast, the majority of Hendra virus G-Δ32 arrived on the cell surface after the 2-h time point (Fig. ​(Fig.3B).3B). Similar cell surface profiles were observed when the proteins were coexpressed (Fig. ​(Fig.3C).3C). These data confirm the differential trafficking rates and suggest that Hendra virus F and G do not interact within the secretory pathway but traffic independently to the cell surface.Open in a separate windowFIG. 3.Analysis of cell surface populations confirms the differential trafficking rates of the Hendra virus F and G proteins. (A) Vero cells were transfected with pCAGGS-Hendra F. Twenty-four hours later, the cells were metabolically labeled with ³⁵S for 30 min and chased for the indicated times. Analysis of biotinylated surface proteins was performed as described previously (37). (B) Surface biotinylation of Vero cells expressing Hendra virus G-Δ32 was performed, and samples were analyzed as described previously (37). (C) Biotinylation analyses of Vero cells expressing both Hendra virus F and Hendra virus G-Δ32 were performed as described previously.Exit out of the ER is a carefully controlled process, allowing only properly folded proteins to be transported to the Golgi compartment (15). Thus, slow folding kinetics for Hendra virus G may explain the delayed appearance of an endo H-resistant population. Unlike Hendra virus F, which folds as a trimer (13, 14), Hendra virus G is a tetramer composed of two disulfide-linked dimers (7). To examine folding kinetics, cross-linking analysis (13) was performed at various time points post-metabolic labeling. By the end of the 30-min labeling, the majority of Hendra virus F was folded into a trimeric state which could be stabilized by the addition of a cross-linker (Fig. ​(Fig.4A),4A), and little of the F protein was present in the monomeric form after the addition of the cross-linker. No further increases in trimers were subsequently observed, suggesting very rapid oligomerization of Hendra virus F. Rapid oligomerization of paramyxovirus fusion proteins is consistent with the prefusion structure (38), in which monomers are tightly folded together to form the trimeric unit. The presence of a dimeric species with the addition of a cross-linker is due likely to incomplete cross-linking and does not represent an intermediate conformational population. The folding of Hendra virus G into a tetramer, however, occurs at a lower rate than that of F. In the absence of a cross-linker, monomeric G is present until 2 h postlabeling, suggesting that the formation of the disulfide-linked dimer is intrinsically slow (Fig. ​(Fig.4B).4B). The formation of the disulfide-linked dimer occurs at a rate similar to that of tetramer formation, as the majority of G can be cross-linked to a tetramer by the 2-h time point. Tetramerization of other paramyxovirus attachment proteins, such as PIV5 hemagglutinin-neuraminidase, occurs more rapidly, with t_1/2s for these proteins of 25 to 30 min (24), contrasting greatly with the t_1/2 of 1 to 2 h observed for Hendra virus G. These results suggest that, unlike other paramyxovirus attachment proteins, Hendra virus G undergoes very slow tetramerization and that the slow trafficking of this protein through the secretory pathway is likely a direct reflection of the low intrinsic rate of folding and oligomerization.Open in a separate windowFIG. 4.Cross-linking analysis of Hendra virus glycoproteins indicates slow tetramerization of the Hendra virus G protein. (A) Vero cells expressing Hendra virus F were metabolically labeled for 30 min and chased for the indicated times. Cross-linking with DTSSP [3,3′-dithiobis(sulfosuccinimidyl propionate)] was performed as described previously (14). (B) Vero cells expressing Hendra virus G-Δ32 were labeled for 30 min and chased for the indicated times, and cross-linking analysis with DTSSP was performed as described previously (14). +, present; −, absent.While the majority of paramyxovirus F proteins require their homotypic attachment protein for fusogenic activity, the role of the attachment protein in controlling F protein function remains unclear. The attachment protein has been proposed to hold the F protein in its prefusion conformation until receptor binding occurs (34), and research from several systems has suggested that this F protein-attachment protein interaction occurs in the ER during initial protein folding (30, 33, 35). Data from experiments presented here demonstrating differential rates of oligomerization and secretory pathway transport for Hendra virus F and G strongly indicate that the association of the newly synthesized proteins does not occur in the ER but that they instead traffic independently through the secretory pathway. Thus, at least for the henipaviruses, F-G interactions are unlikely to play a role in preventing premature triggering of the newly synthesized F protein. An alternative model for paramyxovirus fusion has suggested that attachment protein-fusion protein interactions occur only after the attachment protein binds the receptor. However, analysis of henipavirus G and F mutants suggests that F-G avidity inversely correlates with fusion (2, 3, 5), and Hendra virus G protein mutants deficient in receptor binding also lose the ability to coimmunoprecipitate Hendra virus F (4). These data support a model in which F-G interactions occur prior to receptor binding, with the subsequent G-ephrin B2 interaction leading to the release of the F-G interaction and the triggering of fusion. Taken together, these results support a model in which the henipavirus F and G associate only after trafficking to the cell surface. The mechanisms by which this interaction is promoted and/or regulated represent an exciting area of future research.