Tryptic peptide analysis of outer capsid polypeptides of mammalian reovirus serotypes 1, 2, and 3.

We studied the structural relationships among the outer capsid polypeptides of prototype strains of mammalian reovirus serotypes 1, 2, and 3 by tryptic peptide mapping. The micron1C polypeptide showed an extraordinary degree of conservation of its methionine-containing tryptic peptides. In contrast, the most abundant viral polypeptide, sigma 3, contained both conserved and unique methionine-containing tryptic peptides. The viral type-specific antigen, the sigma 1 polypeptide, contained both conserved and unique methionine- and tyrosine-containing tryptic peptides. These results suggested that the mammalian reovirus genome segments encoding each of the viral outer capsid polypeptides were derived from common ancestral segments which have diverged to different degrees.     

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.     

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.     

Biophysical studies of infectious pancreatic necrosis virus.

The molecular weight of infectious pancreatic necrosis virus (IPNV) has been determined by analytical ultracentrifugation and dynamic light scattering. The sedimentation coefficient of the virus was found to be 435S. The average value for molecular weight is (55 +/- 7) x 106. The virus genome consists of two segments of double-stranded RNA (molecular weights, 2.5 x 106 and 2.3 x 106), which represents 8.7% of the virion mass. The capsid protein moiety of IPNV consists of four species of polypeptides, as determined by polyacrylamide gel electrophoresis. The number of molecules of each polypeptide in the virion has been determined. There are 22 molecules of the internal polypeptide alpha (molecular weight, 90,000), 544 molecules of the outer capsid polypeptide beta (molecular weight, 57,000), and 550 and 122 molecules, respectively, of the internal polypeptides gamma1 (molecular weight, 29,000) and gamma2 (molecular weight, 27,000). IPNV top component contains only the beta polypeptide species, and its molecular weight is estimated to be 31 x 106. The hydrodynamic diameter and electron microscopic diameter (calculated by catalase crystal-calibrated electron microscopy) of IPNV was compared with those of reovirus and encephalomyocarditis virus. Due to the swelling of the outer capsid, reovirus particles were found to be much larger when hydrated (96-nm diameter) than when dehydrated (76-nm diameter), having a large water content content and low average density. In contrast, IPNV particles are more rigid, having nearly the same average diameter under hydrous (64 nm) as under anhydrous conditions (59.3 nm). Encephalomyocarditis virus has a very low water content and does not shrink at all when prepared for electron microscopy.     

Reovirus polypeptide sigma 3 and N-terminal myristoylation of polypeptide mu 1 are required for site-specific cleavage to mu 1C in transfected cells.

N-myristoylated viral polypeptide mu 1 was produced in COS cells transfected with a transient expression vector containing a DNA copy of the reovirus M2 gene. The mu 1 product was specifically cleaved to polypeptide mu 1C in cells that were cotransfected with the reovirus S4 gene and that expressed polypeptide sigma 3. Studies with site-specific mutants of the M2 gene demonstrated that conversion of mu 1 to mu 1C was dependent on myristoylation and the presence of the proteolytic cleavage sequence asparagine 42-proline 43 in mu 1, as well as on the presence of polypeptide sigma 3. The mu 1C product and polypeptide sigma 3 formed complexes that were immunoprecipitated by sigma 3-directed antibody, and a myristoylation-negative M2 double mutant, G2A-N42T, yielded mu 1 that did not undergo cleavage to mu 1C or bind sigma 3. However, the N42T single mutant did form immunoprecipitable complexes with sigma 3, indicating that binding can occur in the absence of cleavage. Polypeptide sigma 3 alternatively can bind double-stranded RNA and in COS cells stimulates translation of reporter chloramphenicol acetyltransferase mRNA translation, presumably by blocking double-stranded RNA-mediated activation of the eukaryotic initiation factor 2 alpha subunit kinase which inhibits the initiation of protein synthesis. Consistent with these observations and with the formation of mu 1C-sigma 3 complexes, coexpression of M2 with S4 DNA prevented the translational stimulatory effect of polypeptide sigma 3.     

Comparison of the Structure and Polypeptide Composition of Three Double-Stranded Ribonucleic Acid-Containing Viruses (Diplornaviruses): Cytoplasmic Polyhedrosis Virus, Wound Tumor Virus, and Reovirus

Iodination of reovirus, cytoplasmic polyhedrosis virus (CPV), and wound tumor virus (WTV), and their respective subviral forms, followed by analysis of the labeled polypeptides by using polyacrylamide gel electrophoresis, has been used to compare the protein contents of these three diplornaviruses. This approach, when combined with electron microscopy and buoyant density determinations, appears capable of localizing individual polypeptides in some of the viral and subviral forms. CPV (p = 1.435 g/cm(3)) seems to resemble reovirus cores (p = 1.440 g/cm(3)) in both ultrastructure and polypeptide composition. CPV is composed of five polypeptides with molecular weights of about 151,000, 142,000, 130,000, 67,000, and 33,000. The polyhedral matrix, which in nature encapsulates the virions, is, in turn, composed mainly of two polypeptide species with molecular weights of about 30,000 and 20,000, and several minor proteins. The proteins of WTV consist mainly of four species of polypeptide with molecular weights of about 156,000, 122,000, 63,000, and 44,000, and several minor components. These molecular weight determinations are consistent with the hypothesis that, as has been suggested for reovirus, the viral proteins of CPV and WTV seem to be coded for by monocistronic mes senger RNA molecules transcribed from distinct segments of the double-stranded RNA viral genomes.     

Association of reovirus outer capsid proteins sigma 3 and mu 1 causes a conformational change that renders sigma 3 protease sensitive.

Association of the reovirus proteins sigma 3 and mu 1 influences viral entry, initiation of outer capsid assembly, and modulation of the effect of sigma 3 on cellular translation. In this study, we have addressed whether structural changes occur in sigma 3 as a result of its interaction with mu 1. Using differences in protease sensitivity to detect conformationally distinct forms of sigma 3, we showed that association of sigma 3 with mu 1 caused a conformational change in sigma 3 that converted it from a protease-resistant to a protease-sensitive structure and occurred posttranslationally. The effect of mu 1 on the structure of sigma 3 was stoichiometric. Our results are consistent with a model in which sigma 3's association with mu 1 shifts its function from translational control to assembly of an outer capsid in which sigma 3 is folded into the protease-sensitive conformation that is required for its cleavage during the next round of infection.     

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.     

Genetic diversity in natural populations of mammalian reoviruses: tryptic peptide analysis of outer capsid polypeptides of murine, bovine, and human type 1 and 3 reovirus strains.

We have studied the structural relationships between the outer capsid polypeptides of eight murine, bovine, and human isolates of type 1 and 3 mammalian reoviruses. Our results show that the outer capsid polypeptides of reoviruses isolated from different mammalian species, in different years and different geographical areas, have both conserved and unique methionine-containing tryptic peptides. We found that tryptic peptides from mu 1C polypeptides of two human, one murine, and two bovine type 3 isolates and one human and two bovine type 1 reoviruses are highly conserved. Our data show that only one tryptic peptide pattern of the mu 1C polypeptide (encoded by the M2 gene) was present in reoviruses isolated from the three different mammalian species. The mu 1C polypeptide of the type 3 Dearing strain contained one tryptic peptide not found in any other reovirus isolate examined. In marked contrast to the mu 1C polypeptides, the sigma 3 polypeptides (encoded by the S4 gene) of three type 1 and three type 3 isolates were divided into two patterns based on significant differences in their tryptic peptides. In addition, at least seven tryptic peptides were conserved among the sigma 3 polypeptides of all virus strains examined. The sigma 3 polypeptide of the type 3 Dearing strain was distinguishable from the sigma 3 polypeptides of all other strains examined. The one mu 1C and two sigma 3 tryptic peptide patterns were found to occur interchangeably in isolates of type 1 or type 3. About 1/3 of the tyrosine-containing tryptic peptides of sigma 1 polypeptides of four type 3 isolates examined were conserved. Comparison of peptide differences in sigma 1 polypeptides of these isolates showed that each had one or more unique tryptic peptides, suggesting that the S1 genes coding for these polypeptides had undergone genetic drift or, alternatively, that there are at least two tryptic peptide patterns present among the sigma 1 polypeptides of these isolates. Our results suggest that genetic drift and reassortment are the most likely explanation for the extensive genetic diversity found in natural populations of mammalian reoviruses.     

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.     

Regulated, stable expression and nuclear presence of retrovirus double-stranded RNA-binding protein sigma3 in HeLa cells.

Reovirus genome segment S4 codes for polypeptide sigma3, a major outer capsid component of virions and a double-stranded RNA (dsRNA)-binding protein implicated in viral cytopathogenesis. We have constructed a stable HeLa cell line (S4tTA) that produces functional sigma3 under tetracycline transactivator control. In the absence of tetracycline, S4tTA cells synthesized stable dsRNA-binding sigma3 that accumulated in the nucleus as well as in the cytoplasm. However, in induced S4tTA cells also expressing reovirus outer shell polypeptide mu1/mu1C, migration of sigma3 into the nucleus was blocked, probably as a result of formation of a complex with mu1/mu1C which was exclusively in the cytoplasm. Mutant analyses indicated a correlation between dsRNA-binding activity and nuclear entry of sigma3, suggesting an additional role(s) for this capsid protein in virus-cell interactions.     

Reovirus serotypes 1 and 3 differ in their in vitro association with microtubules.

Utilizing negative-stain electron microscopy in which similar concentrations of reovirus types 1 and 3 are incubated with a carbon support film containing chick brain, rabbit brain, or HeLa cell microtubules, 81% of the type 1 and 56% of type 3 exhibited an association with the apparent "edge" of the microtubule. This implies that there is a high level of specific affinity for type 1 but not for type 3 to microtubules, since it has previously been determined that only 50% of randomly associated particles would be associated with the edge. The high edge binding of reovirus type 1 is virtually independent of the origin of microtubule, or of whether microtubules or virus has been initially adhered to the support film. On the other hand, reovirus type 1-specific antiserum reduced the edge binding or reovirus type 1 to 45%, whereas type 3 specific antiserum caused no less (within the variability of the assay) of the edge binding of reovirus type 1 to microtubules (76% edge bound). High edge binding of reovirus type 1 to microtubules is correlated with the presence of type 1 or sigma 1 polypeptide. This minor outer capsid polypeptide is encoded in the S1 double-stranded RNA segment and is the viral hemagglutinin and neutralization antigen. Recombinant reovirus clones containing the S1 double-stranded RNA segment of type 1 (80 and 802) show about 85% edge binding, as compared to a value of 42% for clones and the S1 gene of type 3 (204. Electron microscopy of purified reovirus types 1 and 3 by negative staining reveals that type 1 and 802 capsomers are distinctly visualized, whereas those of type 3 and 204 appear diffuse. Thus, the greater in vitro binding of type 1 to microtubules may reflect an increased accessibility of certain of its outer capsomers, and thereby, sigma 1 polypeptides to microtubules. Examination of its outer sections of reovirus type 1- and 3-infected cells at 24 to 48 h postinfection at 31 degrees C showed that about eight times as many viral factoris in type 1-infected cells exhibited an extensive association of virus particles with microtubules, as compared to viral factories of type 3-infected cells. Thus, both in vivo and in vitro there appears to be a greater specificity for the association of reovirus type 1 particles with microtubules, as compared to reovirus type 3 particles.     

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.     

Intracellular posttranslational modifications of S1133 avian reovirus proteins.

Avian reovirus S1133 specifies at least 10 primary translation products, eight of which are present in the viral particle and two of which are nonstructural proteins. In the work presented here, we studied the covalent modifications undergone by these translation products in the infected cell. The structural polypeptide mu2 was shown to be intracellularly modified by both myristoylation and proteolysis. The site-specific cleavage of mu2 yielded a large carboxy-terminal fragment and a myristoylated approximately 5,500-Mr peptide corresponding to the amino terminus. Both mu2 and its cleavage products were found to be structural components of the reovirion. Most avian reovirus proteins were found to be glycosylated and to have a blocking group at the amino terminus. In contrast to the mammalian reovirus system, none of the avian reovirus polypeptides was found to incorporate phosphorus during infection. Our results add to current understanding of the similarities and differences between avian and mammalian reoviruses.