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.     

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.     

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.     

Structure of the reovirus outer capsid and dsRNA-binding protein sigma3 at 1.8 A resolution

The crystallographically determined structure of the reovirus outer capsid protein sigma3 reveals a two-lobed structure organized around a long central helix. The smaller of the two lobes includes a CCHC zinc-binding site. Residues that vary between strains and serotypes lie mainly on one surface of the protein; residues on the opposite surface are conserved. From a fit of this model to a reconstruction of the whole virion from electron cryomicroscopy, we propose that each sigma3 subunit is positioned with the small lobe anchoring it to the protein mu1 on the surface of the virion, and the large lobe, the site of initial cleavages during entry-related proteolytic disassembly, protruding outwards. The surface containing variable residues faces solvent. The crystallographic asymmetric unit contains two sigma3 subunits, tightly associated as a dimer. One broad surface of the dimer has a positively charged surface patch, which extends across the dyad. In infected cells, sigma3 binds dsRNA and inhibits the interferon response. The location and extent of the positively charged surface patch suggest that the dimer is the RNA-binding form of sigma3.     

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.     

Reovirus sigma NS protein localizes to inclusions through an association requiring the mu NS amino terminus

Cells infected with mammalian reoviruses contain phase-dense inclusions, called viral factories, in which viral replication and assembly are thought to occur. The major reovirus nonstructural protein mu NS forms morphologically similar phase-dense inclusions when expressed in the absence of other viral proteins, suggesting it is a primary determinant of factory formation. In this study we examined the localization of the other major reovirus nonstructural protein, sigma NS. Although sigma NS colocalized with mu NS in viral factories during infection, it was distributed diffusely throughout the cell when expressed in the absence of mu NS. When coexpressed with mu NS, sigma NS was redistributed and colocalized with mu NS inclusions, indicating that the two proteins associate in the absence of other viral proteins and suggesting that this association may mediate the localization of sigma NS to viral factories in infected cells. We have previously shown that mu NS residues 1 to 40 or 41 are both necessary and sufficient for mu NS association with the viral microtubule-associated protein mu 2. In the present study we found that this same region of micro NS is required for its association with sigma NS. We further dissected this region, identifying residues 1 to 13 of mu NS as necessary for association with sigma NS, but not with mu 2. Deletion of sigma NS residues 1 to 11, which we have previously shown to be required for RNA binding by that protein, resulted in diminished association of sigma NS with mu NS. Furthermore, when treated with RNase, a large portion of sigma NS was released from mu NS coimmunoprecipitates, suggesting that RNA contributes to their association. The results of this study provide further evidence that mu NS plays a key role in forming the reovirus factories and recruiting other components to them.     

Mammalian reoviruses contain a myristoylated structural protein.

The structural protein mu 1 of mammalian reoviruses was noted to have a potential N-myristoylation sequence at the amino terminus of its deduced amino acid sequence. Virions labeled with [3H]myristic acid were used to demonstrate that mu 1 is modified by an amide-linked myristoyl group. A myristoylated peptide having a relative molecular weight (Mr) of approximately 4,000 was also shown to be a structural component of virions and was concluded to represent the 4.2-kDa amino-terminal fragment of mu 1 which is generated by the same proteolytic cleavage that yields the carboxy-terminal fragment and major outer capsid protein mu 1C. The myristoylated 4,000-Mr peptide was found to be present in reovirus intermediate subviral particles but to be absent from cores, indicating that it is a component of the outer capsid. A distinct large myristoylated fragment of the intact mu 1 protein was also identified in intermediate subviral particles, but no myristoylated mu-region proteins were identified in cores, consistent with the location of mu 1 in the outer capsid. Similarities between amino-terminal regions of the reovirus mu 1 protein and the poliovirus capsid polyprotein were noted. By analogy with other viruses that contain N-myristoylated structural proteins (particularly picornaviruses), we suggest that the myristoyl group attached to mu 1 and its amino-terminal fragments has an essential role in the assembly and structure of the reovirus outer capsid and in the process of reovirus entry into 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.     

Sites and determinants of early cleavages in the proteolytic processing pathway of reovirus surface protein sigma3

Entry of mammalian reovirus virions into target cells requires proteolytic processing of surface protein sigma3. In the virion, sigma3 mostly covers the membrane-penetration protein mu1, appearing to keep it in an inactive form and to prevent it from interacting with the cellular membrane until the proper time in infection. The molecular mechanism by which sigma3 maintains mu1 in this inactive state and the structural changes that accompany sigma3 processing and mu1 activation, however, are not well understood. In this study we characterized the early steps in sigma3 processing and determined their effects on mu1 function and particle infectivity. We identified two regions of high protease sensitivity, "hypersensitive" regions located at residues 208 to 214 and 238 to 244, within which all proteases tested selectively cleaved sigma3 as an early step in processing. Further processing of sigma3 was required for infection, consistent with the fact that the fragments resulting from these early cleavages remained bound to the particles. Reovirus type 1 Lang (T1L), type 3 Dearing (T3D), and T1L x T3D reassortant virions differed in the sites of early sigma3 cleavage, with T1L sigma3 being cleaved mainly at residues 238 to 244 and T3D sigma3 being cleaved mainly at residues 208 to 214. These virions also differed in the rates at which the early cleavages occurred, with cleavage of T1L sigma3 occurring faster than cleavage of T3D sigma3. Analyses using chimeric and site-directed mutants of recombinant sigma3 identified carboxy-proximal residues 344, 347, and 353 as the primary determinants of these strain differences. The spatial relationships between these more carboxy-proximal residues and the hypersensitive regions were discerned from the sigma3 crystal structure. The results indicate that proteolytic processing of sigma3 during reovirus disassembly is a multistep pathway with a number of molecular determinants.     

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.     

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.     

Comparative sequence analysis of the reovirus S4 genes from 13 serotype 1 and serotype 3 field isolates.

The reovirus sigma 3 protein is a major outer capsid protein that may function to regulate translation within infected cells. To facilitate the understanding of sigma 3 structure and functions and the evolution of mammalian reoviruses, we sequenced cDNA copies of the S4 genes from 10 serotype 3 and 3 serotype 1 reovirus field isolates and compared these sequences with sequences of prototypic strains of the three reovirus serotypes. We found that the sigma 3 proteins are highly conserved: the two longest conserved regions contain motifs proposed to function in binding zinc and double-stranded RNA. We used the 16 viral isolates to investigate the hypothesis that structural interactions between sigma 3 and the cell attachment protein, sigma 1, constrain their evolution and to identify a determinant within sigma 3 that is in close proximity to the sigma 1 hemagglutination site.     

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.     

Reovirus sigma NS and mu NS proteins form cytoplasmic inclusion structures in the absence of viral infection

Reovirus replication occurs in the cytoplasm of infected cells and culminates in the formation of crystalline arrays of progeny virions within viral inclusions. Two viral nonstructural proteins, sigma NS and micro NS, and structural protein sigma 3 form protein-RNA complexes early in reovirus infection. To better understand the minimal requirements of viral inclusion formation, we expressed sigma NS, mu NS, and sigma 3 alone and in combination in the absence of viral infection. In contrast to its concentration in inclusion structures during reovirus replication, sigma NS expressed in cells in the absence of infection is distributed diffusely throughout the cytoplasm and does not form structures that resemble viral inclusions. Expressed sigma NS is functional as it complements the defect in temperature-sensitive, sigma NS-mutant virus tsE320. In both transfected and infected cells, mu NS is found in punctate cytoplasmic structures and sigma 3 is distributed diffusely in the cytoplasm and the nucleus. The subcellular localization of mu NS and sigma 3 is not altered when the proteins are expressed together or with sigma NS. However, when expressed with micro NS, sigma NS colocalizes with mu NS to punctate structures similar in morphology to inclusion structures observed early in viral replication. During reovirus infection, both sigma NS and mu NS are detectable 4 h after adsorption and colocalize to punctate structures throughout the viral life cycle. In concordance with these results, sigma NS interacts with mu NS in a yeast two-hybrid assay and by coimmunoprecipitation analysis. These data suggest that sigma NS and mu NS are the minimal viral components required to form inclusions, which then recruit other reovirus proteins and RNA to initiate viral genome replication.     

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.