Cell entry-associated conformational changes in reovirus particles are controlled by host protease activity

Membrane penetration by reovirus requires successive formation of two cell entry intermediates, infectious subvirion particles (ISVPs) and ISVP*s. In vitro incubation of reovirus virions with high concentration of chymotrypsin (CHT) results in partial digestion of the viral outer capsid to form ISVPs. When virions are instead digested with low concentrations of chymotrypsin, the outer capsid is completely proteolyzed to form cores. We investigated the basis for the inverse relationship between CHT activity and protease susceptibility of the reovirus outer capsid. We report that core formation following low-concentration CHT digestion proceeds via formation of particles that contain a protease-sensitive form of the μ1C protein, a characteristic of ISVP*s. In addition, we found that both biochemical features and viral genetic requirements for ISVP* formation and core formation following low-concentration CHT digestion are identical, suggesting that core formation proceeds via a particle resembling ISVP*s. Furthermore, we determined that intermediates generated following low-concentration CHT digestion are distinct from ISVPs and convert to ISVP*-like particles much more readily than ISVPs. These results suggest that the activity of host proteases used to generate ISVPs can influence the efficiency with which the next step in reovirus cell entry, namely, ISVP-to-ISVP* conversion, occurs.     

Beta1 integrin mediates internalization of mammalian reovirus

Reovirus infection is initiated by interactions between the attachment protein sigma1 and cell surface carbohydrate and junctional adhesion molecule A (JAM-A). Expression of a JAM-A mutant lacking a cytoplasmic tail in nonpermissive cells conferred full susceptibility to reovirus infection, suggesting that cell surface molecules other than JAM-A mediate viral internalization following attachment. The presence of integrin-binding sequences in reovirus outer capsid protein lambda2, which serves as the structural base for sigma1, suggests that integrins mediate reovirus endocytosis. A beta1 integrin-specific antibody, but not antibodies specific for other integrin subunits, inhibited reovirus infection of HeLa cells. Expression of a beta1 integrin cDNA, along with a cDNA encoding JAM-A, in nonpermissive chicken embryo fibroblasts conferred susceptibility to reovirus infection. Infectivity of reovirus was significantly reduced in beta1-deficient mouse embryonic stem cells in comparison to isogenic cells expressing beta1. However, reovirus bound equivalently to cells that differed in levels of beta1 expression, suggesting that beta1 integrins are involved in a postattachment entry step. Concordantly, uptake of reovirus virions into beta1-deficient cells was substantially diminished in comparison to viral uptake into beta1-expressing cells. These data provide evidence that beta1 integrin facilitates reovirus internalization and suggest that viral entry occurs by interactions of reovirus virions with independent attachment and entry receptors on the cell surface.     

Localization of a C-terminal region of lambda2 protein in reovirus cores.

The 144-kDa lambda2 protein is a structural component of mammalian reovirus particles and contains the guanylyltransferase activity involved in adding 5' caps to reovirus mRNAs. After incubation of reovirus T3D core particles at 52 degrees C, the lambda2 protein became sensitive to partial protease degradation. Sequential treatments with heat and chymotrypsin caused degradation of a C-terminal portion of lambda2, leaving a 120K core-associated fragment. The four other proteins in cores--lambda1, lambda3, mu2, and sigma2--were not affected by the treatment. Purified cores with cleaved lambda2 were subjected to transmission cryoelectron microscopy and image reconstruction. Reconstruction analysis demonstrated that a distinctive outer region of lambda2 was missing from the modified cores. The degraded region of lambda2 corresponded to the one that contacts the base of the sigma1 protein fiber in reovirus virions and infectious subvirion particles, suggesting that the sigma1-binding region of lambda2 is near its C terminus. Cores with cleaved lambda2 were shown to retain all activities required to transcribe and cap reovirus mRNAs, indicating that the C-terminal region of lambda2 is dispensable for those functions.     

Addition of exogenous protease facilitates reovirus infection in many restrictive cells

Virion uncoating is a critical step in the life cycle of mammalian orthoreoviruses. In cell culture, and probably in extraintestinal tissues in vivo, reovirus virions undergo partial proteolysis within endosomal or/or lysosomal compartments. This process converts the virion into a form referred to as an intermediate subvirion particle (ISVP). In natural enteric reovirus infections, proteolytic uncoating takes place extracellularly within the intestinal lumen. The resultant proteolyzed particles, unlike intact virions, have the capacity to penetrate cell membranes and thereby gain access to cytoplasmic components required for viral gene expression. We hypothesized that the capacity of reovirus outer capsid proteins to be proteolyzed is a determinant of cellular host range. To investigate this hypothesis, we asked if the addition of protease to cell culture medium would expand the range of cultured mammalian cell lines that can be productively infected by reoviruses. We identified many transformed and nontransformed cell lines, as well as primary cells, that restrict viral infection. In several of these restrictive cells, virion uncoating is inefficient or blocked. Addition of proteases to the cell culture medium generates ISVP-like particles and promotes viral growth in nearly all cell lines tested. Interestingly, we found that some cell lines that restrict reovirus uncoating still express mature cathepsin L, a lysosomal protease required for virion disassembly in murine L929 cells. This finding suggests that factors in addition to cathepsin L are required for efficient intracellular proteolysis of reovirus virions. Our results demonstrate that virion uncoating is a critical determinant of reovirus cellular host range and that many cells which otherwise support productive reovirus infection cannot efficiently mediate this essential early step in the virus life cycle.     

Disulfide bonding among micro 1 trimers in mammalian reovirus outer capsid: a late and reversible step in virion morphogenesis

We examined how a particular type of intermolecular disulfide (ds) bond is formed in the capsid of a cytoplasmically replicating nonenveloped animal virus despite the normally reducing environment inside cells. The micro 1 protein, a major component of the mammalian reovirus outer capsid, has been implicated in penetration of the cellular membrane barrier during cell entry. A recent crystal structure determination supports past evidence that the basal oligomer of micro 1 is a trimer and that 200 of these trimers surround the core in the fenestrated T=13 outer capsid of virions. We found in this study that the predominant forms of micro 1 seen in gels after the nonreducing disruption of virions are ds-linked dimers. Cys679, near the carboxyl terminus of micro 1, was shown to form this ds bond with the Cys679 residue from another micro 1 subunit. The crystal structure in combination with a cryomicroscopy-derived electron density map of virions indicates that the two subunits that contribute a Cys679 residue to each ds bond must be from adjacent micro 1 trimers in the outer capsid, explaining the trimer-dimer paradox. Successful in vitro assembly of the outer capsid by a nonbonding mutant of micro 1 (Cys679 substituted by serine) confirmed the role of Cys679 and suggested that the ds bonds are not required for assembly. A correlation between micro 1-associated ds bond formation and cell death in experiments in which virions were purified from cells at different times postinfection indicated that the ds bonds form late in infection, after virions are exposed to more oxidizing conditions than those in healthy cells. The infectivity measurements of the virions with differing levels of ds-bonded micro 1 showed that these bonds are not required for infection in culture. The ds bonds in purified virions were susceptible to reduction and reformation in situ, consistent with their initial formation late in morphogenesis and suggesting that they may undergo reduction during the entry of reovirus particles into new cells.     

Monoclonal antibodies to reovirus reveal structure/function relationships between capsid proteins and genetics of susceptibility to antibody action.

Thirteen newly isolated monoclonal antibodies (MAbs) were used to study relationships between reovirus outer capsid proteins sigma 3, mu 1c, and lambda 2 (core spike) and the cell attachment protein sigma 1. We focused on sigma 1-associated properties of serotype specificity and hemagglutination (HA). Competition between MAbs revealed two surface epitopes on mu 1c that were highly conserved between reovirus serotype 1 Lang (T1L) and serotype 3 Dearing (T3D). There were several differences between T1L and T3D sigma 3 epitope maps. Studies using T1L x T3D reassortants showed that primary sequence differences between T1L and T3D sigma 3 proteins accounted for differences in sigma 3 epitope maps. Four of 12 non-sigma 1 MAbs showed a serotype-associated pattern of binding to 25 reovirus field isolates. Thus, for reovirus field isolates, different sigma 1 proteins are associated with preferred epitopes on other outer capsid proteins. Further evidence for a close structural and functional interrelationship between sigma 3/mu 1c and sigma 1 included (i) inhibition by sigma 3 and mu 1c MAbs of sigma 1-mediated HA, (ii) enhancement of sigma 1-mediated HA by proteolytic cleavage of sigma 3 and mu 1c, and (iii) genetic studies demonstrating that sigma 1 controlled the capacity of sigma 3 MAbs to inhibit HA. These data suggest that (i) epitopes on sigma 3 and mu 1c lie in close proximity to sigma 1 and that MAbs to these epitopes can modulate sigma 1-mediated functions, (ii) these spatial relationships have functional significance, since removal of sigma 3 and/or cleavage of mu 1c to delta can enhance sigma 1 function, (iii) in nature, the sigma 1 protein places selective constraints on the epitope structure of the other capsid proteins, and (iv) viral susceptibility to antibody action can be determined by genes other than that encoding an antibody's epitope.     

Protective antibodies inhibit reovirus internalization and uncoating by intracellular proteases.

We identified in vitro correlates of in vivo protection mediated by nonneutralizing antibodies specific for reovirus capsid proteins. We defined mechanisms of antibody action by analyzing monoclonal antibody (MAb) effects at sequential steps in reovirus serotype 3 strain Dearing (T3D) infection of L cells. Two types of experiments showed that protective MAbs specific for the outer capsid proteins sigma 3 or mu 1 inhibited T3D infection independent of effects on binding. First, MAbs which had no effect on T3D binding inhibited T3D growth. Second, MAb-coated T3D attached to L cells did not replicate as efficiently as T3D without bound antibody. We therefore defined sigma 3-specific MAb effects on postbinding steps in T3D infection. T3D coated with MAb sigma 3-10G10 exhibited prolonged sensitivity to growth inhibition by ammonium chloride. Since ammonium chloride inhibits endosomal acidification and proteolytic processing of the T3D capsid, this suggested that MAbs inhibit early steps in T3D infection. This was confirmed by direct demonstration that several sigma 3-specific MAbs inhibited proteolytic uncoating of virions by fibroblasts. We identified two mechanisms for antibody-mediated inhibition of virion uncoating: (i) inhibition of internalization of T3D-MAb complexes bound to the cell surface, and (ii) inhibition of intracellular proteolysis of the T3D capsid. Studies using a cell-free system confirmed that sigma 3-specific MAbs directly block proteolytic uncoating of the T3D virion. In addition, we found that sigma 3-specific MAbs block (and therefore define) two distinct steps in proteolytic uncoating of the reovirion. We conclude that antibodies which are protective in vivo inhibit postbinding events in reovirus infection of permissive cells. Protective antibodies act by inhibiting internalization and intracellular proteolytic uncoating of the virion. Analysis of postbinding mechanisms of MAb action may identify targets for vaccine development and antiviral therapy.     

Reovirus variants selected during persistent infections of L cells contain mutations in the viral S1 and S4 genes and are altered in viral disassembly.

Reoviruses isolated from persistently infected cultures (PI viruses) can grow in the presence of ammonium chloride, a weak base that blocks acid-dependent proteolysis of viral outer-capsid proteins during viral entry into cells. We used reassortant viruses isolated from crosses of wild-type (wt) reovirus strain, type 1 Lang, and three independent PI viruses, L/C, PI 2A1, and PI 3-1, to identify viral genes that segregate with the capacity of PI viruses to grow in cells treated with ammonium chloride. Growth of reassortant viruses in ammonium chloride-treated cells segregated with the S1 gene of L/C and the S4 gene of PI 2A1 and PI 3-1. The S1 gene encodes viral attachment protein sigma1, and the S4 gene encodes outer-capsid protein sigma3. To identify mutations in sigma3 selected during persistent reovirus infection, we determined the S4 gene nucleotide sequences of L/C, PI 2A1, PI 3-1, and four additional PI viruses. The deduced amino acid sequences of sigma3 protein of six of these PI viruses contained a tyrosine-to-histidine substitution at residue 354. To determine whether mutations selected during persistent infection alter cleavage of the viral outer capsid, the fate of viral structural proteins was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after treatment of virions of wt and PI viruses with chymotrypsin in vitro. Proteolysis of PI virus outer-capsid proteins sigma3 and mu1C occurred with faster kinetics than proteolysis of wt virus outer-capsid proteins. These results demonstrate that mutations in either the S1 or S4 gene alter acid-dependent disassembly of the reovirus outer capsid and suggest that increased efficiency of proteolysis of viral outer-capsid proteins is important for maintenance of persistent reovirus infections of cultured cells.     

A Monoclonal Antibody Specific for Reovirus Outer-Capsid Protein ς3 Inhibits ς1-Mediated Hemagglutination by Steric Hindrance

Reovirus virions are nonenveloped icosahedral particles consisting of two concentric protein shells, termed outer capsid and core. Outer-capsid protein sigma1 is the viral attachment protein and binds carbohydrate molecules on the surface of host cells. Monoclonal antibody (MAb) 4F2, which is specific for outer-capsid protein sigma3, blocks the binding of sigma1 protein to sialic acid and inhibits reovirus-induced hemagglutination (HA). To determine whether MAb 4F2 inhibits HA by altering sigma1-sigma3 interactions or by steric hindrance, we analyzed the effect of 4F2 immunoglobulin G (IgG) and Fab fragments (Fabs) on HA induced by reovirus strain type 3 Dearing (T3D). The concentration of 4F2 IgG sufficient to inhibit T3D-induced HA was 12.5 microg per ml, whereas that of Fabs was >200 microg per ml. Dynamic light scattering analysis showed that at the concentration of IgG sufficient to inhibit HA, virion-antibody complexes were monodispersed and not aggregated. The affinity of 4F2 Fabs for T3D virions was only threefold less than that of intact IgG, which suggests that differences in HA inhibition titer exhibited by 4F2 IgG and Fabs are not attributable to differences in the affinity of these molecules for T3D virions. We used cryoelectron microscopy and three-dimensional image analysis to visualize T3D virions alone and in complex with either IgG or Fabs of MAb 4F2. IgG and Fabs bind the same site at the distal portion of sigma3, and binding of IgG and Fabs induces identical conformational changes in outer-capsid proteins sigma3 and mu1. These results suggest that MAb 4F2 inhibits reovirus binding to sialic acid by steric hindrance and provide insight into the conformational flexibility of reovirus outer-capsid proteins.     

Cathepsin S supports acid-independent infection by some reoviruses

In murine fibroblasts, efficient proteolysis of reovirus outer capsid protein sigma3 during cell entry by virions requires the acid-dependent lysosomal cysteine protease cathepsin L. The importance of cathepsin L for infection of other cell types is unknown. Here we report that the acid-independent lysosomal cysteine protease cathepsin S mediates outer capsid processing in macrophage-like P388D cells. P388D cells supported infection by virions of strain Lang, but not strain c43. Genetic studies revealed that this difference is determined by S4, the viral gene segment that encodes sigma3. c43-derived subvirion particles that lack sigma3 replicated normally in P388D cells, suggesting that the difference in infectivity of Lang and c43 virions is at the level of sigma3 processing. Infection of P388D cells with Lang virions was inhibited by the broad spectrum cysteine protease inhibitor trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane but not by NH(4)Cl, which raises the endocytic pH and thereby inhibits acid-dependent proteases such as cathepsins L and B. Outer capsid processing and infection of P388D cells with Lang virions were also inhibited by a cathepsin S-specific inhibitor. Furthermore, in the presence of NH(4)Cl, cell lines engineered to express cathepsin S supported infection by Lang, but not c43, virions. Our results thus indicate that differences in susceptibility to cathepsin S-mediated sigma3 processing are responsible for strain differences in reovirus infection of macrophage-like P388D cells and other cathepsin S-expressing cells. Additionally, our data suggest that the acid dependence of reovirus infections of most other cell types may reflect the low pH requirement for the activities of most other lysosomal proteases rather, than some other acid-dependent aspect of cell entry.     

Incorporation of epitope-tagged viral sigma3 proteins to reovirus virions

Tagging of viral capsid proteins is a powerful tool to study viral assembly; it also raises the possibility of using viral particles to present exogenous epitopes in vaccination or gene therapy strategies. The ability of reoviruses to induce strong mucosal immune response and their large host range and low pathogenicity in humans are some of the advantages of using reoviruses in such applications. In the present study, the feasibility of introducing foreign epitopes, "tags", to the sigma3 protein, a major component of the reovirus outer capsid, was investigated. Among eight different positions, the amino-terminal end of the protein appeared as the best location to insert exogenous sequences. Additional amino acids at this position do not preclude interaction with the micro1 protein, the other major constituent of the viral outer capsid, but strongly interfere with micro1 to micro1C cleavage. Nevertheless, the tagged sigma3 protein was still incorporated to virions upon recoating of infectious subviral particles to which authentic sigma3 protein was removed by proteolysis, indicating that micro1 cleavage is not a prerequisite for outer capsid assembly. The recently published structure of the sigma3- micro1 complex suggests that the amino-terminally inserted epitope could be exposed at the outer surface of viral particles.     

Sigma 1 protein of mammalian reoviruses extends from the surfaces of viral particles.

Electron microscopy revealed structures consisting of long fibers topped with knobs extending from the surfaces of virions of mammalian reoviruses. The morphology of these structures was reminiscent of the fiber protein of adenovirus. Fibers were also seen extending from the reovirus top component and intermediate subviral particles but not from cores, suggesting that the fibers consist of either the mu 1C or sigma 1 outer capsid protein. Amino acid sequence analysis predicts that the reovirus cell attachment protein sigma 1 contains an extended fiber domain (R. Bassel-Duby, A. Jayasuriya, D. Chatterjee, N. Sonenberg, J. V. Maizell, Jr., and B. N. Fields, Nature [London] 315:421-423, 1985). When sigma 1 protein was released from viral particles with mild heat and subsequently obtained in isolation, it was found to have a morphology identical to that of the fiber structures seen extending from the viral particles. The identification of an extended form of sigma 1 has important implications for its function in cell attachment. Other evidence suggests that sigma 1 protein may occur in virions in both an extended and an unextended state.     

Reovirus variants selected for resistance to ammonium chloride have mutations in viral outer-capsid protein sigma3

Mammalian reoviruses are internalized into cells by receptor-mediated endocytosis. Within the endocytic compartment, the viral outer capsid undergoes acid-dependent proteolysis resulting in removal of the sigma3 protein and proteolytic cleavage of the mu1/mu1C protein. Ammonium chloride (AC) is a weak base that blocks disassembly of reovirus virions by inhibiting acidification of intracellular vacuoles. To identify domains in reovirus proteins that influence pH-sensitive steps in viral disassembly, we adapted strain type 3 Dearing (T3D) to growth in murine L929 cells treated with AC. In comparison to wild-type (wt) T3D, AC-adapted (ACA-D) variant viruses exhibited increased yields in AC-treated cells. AC resistance of reassortant viruses generated from a cross of wt type 1 Lang and ACA-D variant ACA-D1 segregated with the sigma3-encoding S4 gene. The deduced sigma3 amino acid sequences of six independently derived ACA-D variants contain one or two mutations each, affecting a total of six residues. Four of these mutations, I180T, A246G, I347S, and Y354H, cluster in the virion-distal lobe of sigma3. Linkage of these mutations to AC resistance was confirmed in experiments using reovirus disassembly intermediates recoated with wt or mutant sigma3 proteins. In comparison to wt virions, ACA-D viruses displayed enhanced susceptibility to proteolysis by endocytic protease cathepsin L. Image reconstructions of cryoelectron micrographs of three ACA-D viruses that each contain a single mutation in the virion-distal lobe of sigma3 demonstrated native capsid protein organization and minimal alterations in sigma3 structure. These results suggest that mutations in sigma3 that confer resistance to inhibitors of vacuolar acidification identify a specific domain that regulates proteolytic disassembly.     

A glycosyl hydrolase activity of mammalian reovirus sigma1 protein can contribute to viral infection through a mucus layer

The mammalian reovirus sigma1 protein is responsible for viral attachment to host cells and hemagglutination properties of the virus. In the present study, sequence similarity between sigma1 and chicken-type lysozymes prompted us to investigate additional functions of the sigma1 protein. Expression in Pichia pastoris yeast cells showed that sigma1 can actually cleave lysozyme substrates, including complex sugars found in bacterial cell walls. Replacement by site-directed mutagenesis of acidic amino acid residues in sigma1 by their respective isosteric, uncharged, amino acid residues has allowed us to identify Glu36 and Asp54 as the catalytic pair involved in sigma1-mediated glycosidase activity. The enzyme appears inactive in virions but its activity is unmasked upon generation of infectious subviral particles (ISVPs) by partial proteolytic removal of the outer capsid proteins. Purified sigma1 protein and ISVPs can also hydrolyze mucins, heavily glycosylated glycoproteins that are a major component of the mucus layer overlaying the intestinal epithelium. Furthermore, reovirus infection of epithelial Madin Darby canine kidney cells was inhibited tenfold in cells expressing mucin at their apical surface, while this inhibition was overcome by ISVPs. Unmasking of sigma1 mucinolytic activity in the intestine, consecutive to proteolytic cleavage of virions to ISVPs, thus likely contributes to the known increase in infectivity of reovirus ISVPs compared to complete virions. This work presents the first evidence that some mammalian viruses have evolved mechanisms to facilitate their penetration through the protective barrier of the mucus layer in the intestinal tract.     

In vitro recoating of reovirus cores with baculovirus-expressed outer-capsid proteins mu1 and sigma3

Reovirus outer-capsid proteins mu1, sigma3, and sigma1 are thought to be assembled onto nascent core-like particles within infected cells, leading to the production of progeny virions. Consistent with this model, we report the in vitro assembly of baculovirus-expressed mu1 and sigma3 onto purified cores that lack mu1, sigma3, and sigma1. The resulting particles (recoated cores, or r-cores) closely resembled native virions in protein composition (except for lacking cell attachment protein sigma1), buoyant density, and particle morphology by scanning cryoelectron microscopy. Transmission cryoelectron microscopy and image reconstruction of r-cores confirmed that they closely resembled virions in the structure of the outer capsid and revealed that assembly of mu1 and sigma3 onto cores had induced rearrangement of the pentameric lambda2 turrets into a conformation approximating that in virions. r-cores, like virions, underwent proteolytic conversion to particles resembling native ISVPs (infectious subvirion particles) in protein composition, particle morphology, and capacity to permeabilize membranes in vitro. r-cores were 250- to 500-fold more infectious than cores in murine L cells and, like virions but not ISVPs or cores, were inhibited from productively infecting these cells by the presence of either NH4Cl or E-64. The latter results suggest that r-cores and virions used similar routes of entry into L cells, including processing by lysosomal cysteine proteinases, even though the former particles lacked the sigma1 protein. To examine the utility of r-cores for genetic dissections of mu1 functions in reovirus entry, we generated r-cores containing a mutant form of mu1 that had been engineered to resist cleavage at the delta:phi junction during conversion to ISVP-like particles by chymotrypsin in vitro. Despite their deficit in delta:phi cleavage, these ISVP-like particles were fully competent to permeabilize membranes in vitro and to infect L cells in the presence of NH4Cl, providing new evidence that this cleavage is dispensable for productive infection.     

Adaptation of reovirus to growth in the presence of protease inhibitor E64 segregates with a mutation in the carboxy terminus of viral outer-capsid protein sigma3

Reovirus virions are internalized into cells by receptor-mediated endocytosis. Within the endocytic compartment, the viral outer capsid undergoes acid-dependent proteolysis leading to degradation of sigma3 protein and proteolytic cleavage of micro1/micro1C protein. E64 is a specific inhibitor of cysteine-containing proteases that blocks disassembly of reovirus virions. To identify domains in reovirus proteins that influence susceptibility to E64-mediated inhibition of disassembly, we selected variant viruses by serial passage of strain type 3 Dearing (T3D) in murine L929 cells treated with E64. E64-adapted variant viruses (D-EA viruses) produced 7- to 17-fold-greater yields than T3D did after infection of cells treated with 100 microM E64. Viral genes that segregate with growth of D-EA viruses in the presence of E64 were identified by using reassortant viruses isolated from independent crosses of E64-sensitive strain type 1 Lang and two prototype D-EA viruses. Growth of reassortant viruses in the presence of E64 segregated with the S4 gene, which encodes outer-capsid protein sigma3. Sequence analysis of S4 genes of three D-EA viruses isolated from independent passage series revealed a common tyrosine-to-histidine mutation at amino acid 354 in the deduced amino acid sequence of sigma3. Proteolysis of D-EA virions by endocytic protease cathepsin L occurred with faster kinetics than proteolysis of wild-type T3D virions. Treatment of D-EA virions, but not T3D virions, with cathepsin D resulted in proteolysis of sigma3, a property that also was found to segregate with the D-EA S4 gene. These results indicate that a region in sigma3 protein containing amino acid 354 influences susceptibility of sigma3 to proteolysis during reovirus disassembly.     

Protective anti-reovirus monoclonal antibodies and their effects on viral pathogenesis.

We used a recently isolated and characterized panel of monoclonal antibodies (MAbs) specific for cross-reactive determinants on reovirus outer capsid proteins to define mechanisms of antibody-mediated protection in vivo. We studied the capacities of MAbs to protect against lethal infection with reoviruses which differ in site of primary replication, route of spread, and central nervous system tropism. We found the following. (i) MAbs specific for each of the viral outer capsid proteins (sigma 1, sigma 3, and mu 1) and the core spike protein (lambda 2) were protective under certain circumstances. (ii) In vitro properties of MAbs, including isotype, neutralization of viral infectivity, inhibition of virus-induced hemagglutination, and avidity of binding, were poorly predictive of the capacities of MAbs to protect in vivo. (iii) MAbs did not act at a single stage during pathogenesis to mediate protection; instead, protective MAbs were capable of altering a variety of stages in reovirus pathogenesis. (iv) MAbs protective against one reovirus also protected against other reoviruses that utilized different pathogenetic strategies, suggesting that the viral epitope bound by an antibody rather than the pathogenetic strategy employed by the virus is a critical determinant of antibody-mediated protection in vivo. (v) A prominent mechanism of protective MAb action is inhibition of viral spread through nerves from a site of primary replication (e.g., the intestine or muscle tissue) to the central nervous system.     

Cryoelectron microscopy map of Atadenovirus reveals cross-genus structural differences from human adenovirus

A three-dimensional (3D) cryoelectron microscopy reconstruction of the prototype Atadenovirus (OAdV [an ovine adenovirus isolate]) showing information at a 10.6-A resolution (0.5 Fourier shell correlation) was derived by single-particle analysis. This is the first 3D structure solved for any adenovirus that is not a Mastadenovirus, allowing cross-genus comparisons between structures and the assignment of genus-specific capsid proteins. Viable OAdV mutants that lacked the genus-specific LH3 and p32k proteins in purified virions were also generated. Negatively stained 3D reconstructions of these mutants were used to identify the location of protein LH3 and infer that of p32k within the capsid. The key finding was that LH3 is a critical protein that holds the outer capsid of the virus together. In its absence, the outer viral capsid is unstable. LH3 is located in the same position among the hexon subunits as its protein IX equivalent from mastadenoviruses but sits on top of the hexon trimers, forming prominent "knobs" on the virion surface that visually distinguish OAdV from other known AdVs. Electron density was also assigned to hexon and penton subunits and to proteins IIIa and VIII. There was good correspondence between OAdV density and human AdV hexon structures, which also validated the significant differences that were observed between the penton base protein structures.