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.     

Putative autocleavage of reovirus mu1 protein in concert with outer-capsid disassembly and activation for membrane permeabilization

Capsid proteins of several different families of non-enveloped animal viruses with single-stranded RNA genomes undergo autocatalytic cleavage (autocleavage) as a maturation step in assembly. Similarly, the 76 kDa major outer-capsid protein mu1 of mammalian orthoreoviruses (reoviruses), which are non-enveloped and have double-stranded RNA genomes, undergoes putative autocleavage between residues 42 and 43, yielding N-terminal N-myristoylated fragment mu1N and C-terminal fragment mu1C. Cleavage at this site allows release of mu1N, which is thought to be critical for penetration of the host-cell membrane during cell entry. Most previous studies have suggested that cleavage at the mu1N/mu1C junction precedes addition to the outer capsid during virion assembly, such that only a small number of the mu1 subunits in mature virions remain uncleaved at that site (approximately 5%). In this study, we varied the conditions for disruption of virions before running the proteins on denaturing gels and in several circumstances recovered much higher levels of uncleaved mu1 (up to approximately 60%). Elements of the disruption conditions that allowed greater recovery of uncleaved protein were increased pH, absence of reducing agent, and decreased temperature. These same elements allowed comparably higher levels of the mu1delta protein, in which cleavage at the mu1N/delta junction has not occurred, to be recovered from particle uncoating intermediates in which mu1 had been previously cleaved by chymotrypsin in a distinct protease-sensitive region near residue 580. The capacity to recover higher levels of mu1delta following disruption of these particles for electrophoresis was lost, however, in concert with a series of structural changes that activate the particles for membrane permeabilization, suggesting that the putative autocleavage is itself one of these changes.     

Monoclonal antibodies to reovirus sigma 1 and mu 1 proteins inhibit chromium release from mouse L cells.

Reovirus intermediate subviral particles (ISVPs) but not intact virions or cores have been shown to possess the capacity to permeabilize mouse L cells as determined by a 51Cr release assay. We used monoclonal antibodies (MAbs) directed against proteins exposed on the ISVP surface (sigma 1, mu 1, and lambda 2) to probe the role(s) of these proteins in membrane interaction and penetration. One sigma 1-specific MAb (MAb-G5) and two mu 1-specific MAbs (MAb-10H2 and MAb-8H6) inhibited reovirus-induced 51Cr release when added pre- or post-ISVP attachment to L cells. MAb-G5 inhibits 51Cr release by interfering with ISVP attachment (via sigma 1) to L-cell receptor sites. The mu 1-specific MAbs (MAb-10H2 and MAb-8H6) inhibit 51Cr release by interfering with an undefined post-L-cell-attachment event that involves bivalent binding of the mu 1-specific MAbs to an epitope located in a central region of the mu 1 protein.     

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.     

A single mutation in the carboxy terminus of reovirus outer-capsid protein sigma 3 confers enhanced kinetics of sigma 3 proteolysis,resistance to inhibitors of viral disassembly,and alterations in sigma 3 structure

Mammalian reoviruses undergo acid-dependent proteolytic disassembly within endosomes, resulting in formation of infectious subvirion particles (ISVPs). ISVPs are obligate intermediates in reovirus disassembly that mediate viral penetration into the cytoplasm. The initial biochemical event in the reovirus disassembly pathway is the proteolysis of viral outer-capsid protein sigma 3. Mutant reoviruses selected during persistent infection of murine L929 cells (PI viruses) demonstrate enhanced kinetics of viral disassembly and resistance to inhibitors of endocytic acidification and proteolysis. To identify sequences in sigma 3 that modulate acid-dependent and protease-dependent steps in reovirus disassembly, the sigma 3 proteins of wild-type strain type 3 Dearing; PI viruses L/C, PI 2A1, and PI 3-1; and four novel mutant sigma 3 proteins were expressed in insect cells and used to recoat ISVPs. Treatment of recoated ISVPs (rISVPs) with either of the endocytic proteases cathepsin L or cathepsin D demonstrated that an isolated tyrosine-to-histidine mutation at amino acid 354 (Y354H) enhanced sigma 3 proteolysis during viral disassembly. Yields of rISVPs containing Y354H in sigma3 were substantially greater than those of rISVPs lacking this mutation after growth in cells treated with either acidification inhibitor ammonium chloride or cysteine protease inhibitor E64. Image reconstructions of electron micrographs of virus particles containing wild-type or mutant sigma 3 proteins revealed structural alterations in sigma 3 that correlate with the Y354H mutation. These results indicate that a single mutation in sigma 3 protein alters its susceptibility to proteolysis and provide a structural framework to understand mechanisms of sigma 3 cleavage during reovirus disassembly.     

Mutations in reovirus outer-capsid protein sigma3 selected during persistent infections of L cells confer resistance to protease inhibitor E64.

Mutations selected in reoviruses isolated from persistently infected cultures (PI viruses) affect viral entry into cells. Unlike wild-type (wt) viruses, PI viruses can grow in the presence of ammonium chloride, a weak base that blocks acid-dependent proteolysis of viral outer-capsid proteins in cellular endosomes during viral entry. In this study, we show that E64, an inhibitor of cysteine proteases such as those present in the endocytic compartment, blocks growth of wt reovirus by inhibiting viral disassembly. To determine whether PI viruses can grow in the presence of an inhibitor of endocytic proteases, we compared yields of wt and PI viruses in cells treated with E64. Prototype PI viruses L/C, PI 2A1, and PI 3-1 produced substantially greater yields than wt viruses type 1 Lang (T1L) and type 3 Dearing (T3D) in E64-treated cells. To identify viral genes that segregate with growth of PI viruses in the presence of E64, we tested reassortant viruses isolated from independent crosses of T1L and each of the prototype PI viruses for growth in cells treated with E64. Growth of reassortant viruses in the presence of E64 segregated exclusively with the S4 gene, which encodes viral outer-capsid protein sigma3. These results suggest that mutations in sigma3 protein selected during persistent infection alter its susceptibility to cleavage during viral disassembly. To determine the temporal relationship of acid-dependent and protease-dependent steps in reovirus disassembly, cells were infected with wt strain T1L or T3D, and medium containing either ammonium chloride or E64d, a membrane-permeable form of E64, was added at various times after adsorption. Susceptibility to inhibition by both ammonium chloride and E64 was abolished when either inhibitor was added at times greater than 60 min after adsorption. These findings indicate that acid-dependent and protease-dependent disassembly events occur with similar kinetics early in reovirus replication, which suggests that these events take place within the same compartment of the endocytic pathway.     

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.     

Temperature-sensitive mutants of reovirus type 3: evidence for aberrant mu 1 and mu 2 polypeptide species.

An analysis of reovirus-specific polypeptides in cells infected with temperature-sensitive mutants under permissive and nonpermissive conditions revealed the presence of (i) all the known viral polypeptides and (ii) aberrant migration of the mu 1 and mu 2 polypeptides in four groups of mutants.     

A sigma 1 region important for hemagglutination by serotype 3 reovirus strains.

Hemagglutination (HA) by the mammalian reoviruses is mediated by interactions between the viral sigma 1 protein and sialoglycoproteins on the erythrocyte surface. Three serotype 3 (T3) reovirus strains were identified that do not agglutinate either bovine or type O human erythrocytes (HA negative): T3 clone 43 (T3C43), T3 clone 44 (T3C44), and T3 clone 84 (T3C84). These three strains also showed a diminished capacity to bind the major erythrocyte sialoglycoprotein, glycophorin, in an enzyme-linked immunosorbent assay. To determine the molecular basis for these findings, we examined the deduced sigma 1 amino acid sequences of the three HA-negative T3 strains and four HA-positive T3 strains. The limited number of sequence differences in the sigma 1 proteins of these seven strains allowed us to identify single unique amino acid residues in each of the HA-negative strains (aspartate 198 in T3C43, leucine 204 in T3C44, and tryptophan 202 in T3C84) that cluster within a discrete region of the sigma 1 tail. The identification of sigma 1 residues important for HA and glycophorin binding suggests that tail-forming sequences are exposed on the virion surface, where they interact with carbohydrate residues on the surface of cells.     

Core protein mu2 is a second determinant of nucleoside triphosphatase activities by reovirus cores.

NTPase activities in mammalian reovirus cores were examined under various conditions that permitted several new differences to be identified between strains type 1 Lang (T1L) and type 3 Dearing (T3D). One difference concerned the ratio (at pH 8.5) of ATP hydrolysis at 50 degrees C to that at 35 degrees C. A genetic analysis using T1L x T3D reassortant viruses implicated the L3 and M1 gene segments in this difference, with M1 influencing ATPase activity most strongly at high temperatures. L3 and M1 encode the core proteins lambda1 and mu2, respectively. Another difference concerned the absolute levels of GTP hydrolysis by cores at 45 degrees C and pH 6.5. A genetic analysis using T1L x T3D reassortants implicated the M1 gene as the sole determinant of this difference. The results of these experiments, coupled with previous findings (S. Noble and M. L. Nibert, J. Virol. 71:2182-2191, 1997), suggest either that a single type of NTPase in cores is strongly influenced by two different core proteins--lambda1 and mu2--or that cores contain two different types of NTPase influenced by the two proteins. The findings appear relevant for understanding the complex functions of reovirus cores in RNA synthesis and capping.     

Evidence that the sigma 1 protein of reovirus serotype 3 is a multimer.

In this report, we study the reovirus serotype 3 (strain Dearing) sigma 1 protein obtained from various sources: from Escherichia coli expressing sigma 1 protein, from reovirus-infected mouse L cells, and from purified reovirions. We demonstrate that the sigma 1 protein is a multimer in its undisrupted form and present biochemical evidence suggesting that the multimer is made up of four sigma 1 subunits.     

A molecular dynamics study of reovirus attachment protein sigma1 reveals conformational changes in sigma1 structure

Molecular dynamics simulations were performed using the recently determined crystal structure of the reovirus attachment protein, sigma1. These studies were conducted to improve an understanding of two unique features of sigma1 structure: the protonation state of Asp(345), which is buried in the sigma1 trimer interface, and the flexibility of the protein at a defined region below the receptor-binding head domain. Three copies of aspartic acids Asp(345) and Asp(346) cluster in a solvent-inaccessible and hydrophobic region at the sigma1 trimer interface. These residues are hypothesized to mediate conformational changes in sigma1 during viral attachment or cell entry. Our results indicate that protonation of Asp(345) is essential to the integrity of the trimeric structure seen by x-ray crystallography, whereas deprotonation induces structural changes that destabilize the trimer interface. This finding was confirmed by electrostatic calculations using the finite difference Poisson-Boltzmann method. Earlier studies show that sigma1 can exist in retracted and extended conformations on the viral surface. Since protonated Asp(345) is necessary to form a stable, extended trimer, our results suggest that protonation of Asp(345) may allow for a structural transition from a partially detrimerized molecule to the fully formed trimer seen in the crystal structure. Additional studies were conducted to quantify the previously observed flexibility of sigma1 at a defined region below the receptor-binding head domain. Increased mobility was observed for three polar residues (Ser(291), Thr(292), and Ser(293)) located within an insertion between the second and third beta-spiral repeats of the crystallized portion of the sigma1 tail. These amino acids interact with water molecules of the solvent bulk and are responsible for oscillating movement of the head of approximately 50 degrees during 5 ns of simulations. This flexibility may facilitate viral attachment and also function in cell entry and disassembly. These findings provide new insights about the conformational dynamics of sigma1 that likely underlie the initiation of the reovirus infectious cycle.     

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.     

Characterization of an ATPase activity in reovirus cores and its genetic association with core-shell protein lambda1.

A previously identified nucleoside triphosphatase activity in mammalian reovirus cores was further characterized by comparing two reovirus strains whose cores differ in their efficiencies of ATP hydrolysis. In assays using a panel of reassortant viruses derived from these strains, the difference in ATPase activity at standard conditions was genetically associated with viral genome segment L3, encoding protein lambda1, a major constituent of the core shell that possesses sequence motifs characteristic of other ATPases. The ATPase activity of cores was affected by several other reaction components, including temperature, pH, nature and concentration of monovalent and divalent cations, and nature and concentration of anions. A strain difference in the response of core ATPase activity to monovalent acetate salts was also mapped to L3/lambda1 by using reassortant viruses. Experiments with different nucleoside triphosphates demonstrated that ATP is the preferred ribonucleotide substrate for cores of both strains. Other experiments suggested that the ATPase is latent in reovirus virions and infectious subviral particles but undergoes activation during production of cores in close association with the protease-mediated degradation of outer-capsid protein mu1 and its cleavage products, suggesting that mu1 may play a role in regulating the ATPase.     

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.