Mutations in type 3 reovirus that determine binding to sialic acid are contained in the fibrous tail domain of viral attachment protein sigma1.

The reovirus attachment protein, sigma1, determines numerous aspects of reovirus-induced disease, including viral virulence, pathways of spread, and tropism for certain types of cells in the central nervous system. The sigma1 protein projects from the virion surface and consists of two distinct morphologic domains, a virion-distal globular domain known as the head and an elongated fibrous domain, termed the tail, which is anchored into the virion capsid. To better understand structure-function relationships of sigma1 protein, we conducted experiments to identify sequences in sigma1 important for viral binding to sialic acid, a component of the receptor for type 3 reovirus. Three serotype 3 reovirus strains incapable of binding sialylated receptors were adapted to growth in murine erythroleukemia (MEL) cells, in which sialic acid is essential for reovirus infectivity. MEL-adapted (MA) mutant viruses isolated by serial passage in MEL cells acquired the capacity to bind sialic acid-containing receptors and demonstrated a dependence on sialic acid for infection of MEL cells. Analysis of reassortant viruses isolated from crosses of an MA mutant virus and a reovirus strain that does not bind sialic acid indicated that the sigma1 protein is solely responsible for efficient growth of MA mutant viruses in MEL cells. The deduced sigma1 amino acid sequences of the MA mutant viruses revealed that each strain contains a substitution within a short region of sequence in the sigma1 tail predicted to form beta-sheet. These studies identify specific sequences that determine the capacity of reovirus to bind sialylated receptors and suggest a location for a sialic acid-binding domain. Furthermore, the results support a model in which type 3 sigma1 protein contains discrete receptor binding domains, one in the head and another in the tail that binds sialic acid.     

Differences in the capacity of reovirus strains to induce apoptosis are determined by the viral attachment protein sigma 1.

Reoviruses are important models for studies of viral pathogenesis; however, the mechanisms by which these viruses produce cytopathic effects in infected cells have not been defined. In this report, we show that murine L929 (L) cells infected with prototype reovirus strains type 1 Lang (TIL) and type 3 Dearing (T3D) undergo apoptosis and that T3D induces apoptosis to a substantially greater extent than T1L. Using T1L x T3D reassortant viruses, we found that differences in the capacity of T1L and T3D to induce apoptosis are determined by the viral S1 gene segment, which encodes the viral attachment protein sigma 1 and the non-virion-associated protein sigma 1s. Apoptosis was induced by UV-inactivated, replication-incompetent reovirus virions, which do not contain sigma 1s and do not mediate its synthesis in infected cells. Additionally, T3D-induced apoptosis was inhibited by anti-reovirus monoclonal antibodies that inhibit T3D cell attachment and disassembly. These results indicate that sigma 1, rather than sigma 1s, is required for induction of apoptosis by the reovirus and suggest that interaction of virions with cell surface receptors is an essential step in this mechanism of cell killing.     

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.     

Crystal structure of reovirus attachment protein sigma1 reveals evolutionary relationship to adenovirus fiber.

Reovirus attaches to cellular receptors with the sigma1 protein, a fiber-like molecule protruding from the 12 vertices of the icosahedral virion. The crystal structure of a receptor-binding fragment of sigma1 reveals an elongated trimer with two domains: a compact head with a new beta-barrel fold and a fibrous tail containing a triple beta-spiral. Numerous structural and functional similarities between reovirus sigma1 and the adenovirus fiber suggest an evolutionary link in the receptor-binding strategies of these two viruses. A prominent loop in the sigma1 head contains a cluster of residues that are conserved among reovirus serotypes and are likely to form a binding site for junction adhesion molecule, an integral tight junction protein that serves as a reovirus receptor. The fibrous tail is mainly responsible for sigma1 trimer formation, and it contains a highly flexible region that allows for significant movement between the base of the tail and the head. The architecture of the trimer interface and the observed flexibility indicate that sigma1 is a metastable structure poised to undergo conformational changes upon viral attachment and cell entry.     

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.     

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.     

The viral protein sigma 3 participates in translation of late viral mRNA in reovirus-infected L cells.

Reovirus late (uncapped) mRNA was previously shown to be efficiently translated in vitro extracts prepared from infected cells but not from uninfected cells. We demonstrated that different fractions from infected cells can stimulate translation of late viral mRNA when added to uninfected extracts. The activity of the different fractions correlated with their relative content of the sigma 3 capsid protein; the fraction prepared by high-salt wash of the ribosomes had the highest specific activity. The activity present in this fraction was abolished by preincubation with an anti-sigma 3 serum. Purified sigma 3 protein also stimulated the translation of late viral mRNA, confirming that it was the factor involved. Altogether, these results suggest that this protein plays the role of a late-viral-mRNA-specific initiation factor. The absence of an inhibitory effect of sigma 3 on the translation of other mRNAs indicates that this protein is not directly involved in the inhibition of host and early viral mRNA translation that occurs in infected cells but that a second mechanism is probably operative.     

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.     

G protein mutations that alter the pheromone response in Saccharomyces cerevisiae.

The GPA1 gene of Saccharomyces cerevisiae encodes a G alpha protein that couples the membrane-bound pheromone receptors to downstream elements in the mating response pathway. We have isolated seven mutant alleles of GPA1 that confer pheromone resistance: G50D (a glycine-to-aspartate change at position 50), G322E, G322R, E355K, E364K, G470D, and an E364K-G470D double mutant. All of the mutations lie within large regions that are highly conserved between Gpa1 and four other G alpha proteins; four of the changes are located in domains with proposed functions. On the basis of a gentic analysis, the pheromone-unresponsive GPA1 alleles can be divided into two classes: those that encode constitutively activated proteins and those that encode proteins unable to respond to the upstream signal. Our results support the hypothesis that the activated form of Gpa1 stimulates adaptation to pheromone.     

Inhibition of viral mRNA translation in interferon-treated L cells infected with reovirus.

Murine L cells were treated with interferon (IFN) concentrations which reduced by 75 to 80% the synthesis of viral mRNA after infection with reovirus. Protein synthesis was not inhibited in these cells up to 6 h after infection, but a large fraction of the viral mRNA was not associated with polyribosomes and sedimented at about 50S. In contrast, most of the reovirus mRNA was associated with polyribosomes in control infected cells. This mRNA was of similar size to non-polyribosomal mRNA from IFN-treated cells when analyzed by Northern blot hybridization with a cloned cDNA for the s2 reovirus mRNA, indicating that the non-polyribosomal mRNA was not appreciably degraded. Viral mRNA was labeled with [3H]uridine and the non-polyribosomal mRNA was isolated from IFN-treated cells. This mRNA could quantitatively bind to 80S initiation complexes when incubated in a rabbit reticulocyte cell-free system. These findings indicated that the non-polyribosomal RNA was translatable, but that its binding to functional initiation complexes was inhibited in IFN-treated cells by a discriminatory mechanism, which did not affect translation of cellular mRNA. Previous experiments showed that mRNA is blocked in 48S complexes when the alpha subunit of initiation factor eIF-2 is phosphorylated by the double-stranded RNA-dependent protein kinase induced by IFN. A localized activation of this kinase could explain the block of viral mRNA in 48S complexes. By labeling the phosphoproteins of IFN-treated cells with 32P, eIF-2 (alpha P) was shown to cosediment with non-polyribosomal mRNA, presumably in 48S complexes.     

A comparative molecular force spectroscopy study of homophilic JAM-A interactions and JAM-A interactions with reovirus attachment protein sigma1

JAM-A belongs to a family of immunoglobulin-like proteins called junctional adhesion molecules (JAMs) that localize at epithelial and endothelial intercellular tight junctions. JAM-A is also expressed on dendritic cells, neutrophils, and platelets. Homophilic JAM-A interactions play an important role in regulating paracellular permeability and leukocyte transmigration across epithelial monolayers and endothelial cell junctions, respectively. In addition, JAM-A is a receptor for the reovirus attachment protein, sigma1. In this study, we used single molecular force spectroscopy to compare the kinetics of JAM-A interactions with itself and sigma1. A chimeric murine JAM-A/Fc fusion protein and the purified sigma1 head domain were used to probe murine L929 cells, which express JAM-A and are susceptible to reovirus infection. The bond half-life (t(1/2)) of homophilic JAM-A interactions was found to be shorter (k(off)(o) = 0.688 +/- 0.349 s(-1)) than that of sigma1/JAM-A interactions (k(off)(o) = 0.067 +/- 0.041 s(-1)). These results are in accordance with the physiological functions of JAM-A and sigma1. A short bond lifetime imparts a highly dynamic nature to homophilic JAM-A interactions for regulating tight junction permeability while stable interactions between sigma1 and JAM-A likely anchor the virus to the cell surface and facilitate viral entry.     

Inhibition of reovirus type 3 binding to host cells by sialylated glycoproteins is mediated through the viral attachment protein.

The interaction of mammalian reoviruses with sialylated glycoproteins was studied and found to be highly serotype specific in that attachment of type 3 Dearing reovirus to murine L cell receptors could be strongly inhibited by bovine submaxillary mucin (BSM), fetuin, and alpha 1 acid glycoprotein, albeit at different efficiencies, whereas attachment of type 1 Lang reovirus was inhibited only by fetuin. We subsequently demonstrated, by using reassortants between type 3 and 1 reoviruses, that inhibition of reovirus attachment to cell receptors was specified by the viral attachment protein gene S1. Using a solid-phase binding assay, we further demonstrated that the ability of reovirus type 3 or reassortant 1HA3 and the inability of reovirus type 1 or reassortant 3HA1 to bind avidly to BSM was a property of the viral S1 genome segment and required the presence of sialic acid residues on BSM oligosaccharides. Taken together, these results demonstrated that there is a serotype-specific difference in the ability of the reovirus attachment protein, sigma 1, to interact with sialylated oligosaccharides of glycoproteins. Interaction of reovirus type 3 with sialylated oligosaccharides of BSM is dramatically affected by the degree of O-acetylation of their sialic acid residues, as indicated by the findings that chemical removal of O-acetyl groups stimulated reovirus type 3 attachment to BSM, whereas preferential removal of residues lacking or possessing reduced amounts of O-acetyl groups per sialic acid molecule with Vibrio cholerae sialidase abolished binding. We also demonstrated that BSM was 10 times more potent in inhibiting attachment of infectious reovirus to L cells than was V. cholerae-treated BSM. The results are consistent with the hypothesis that sialylated oligosaccharides on host cells or erythrocytes may act as binding sites or components of binding sites for type 3 reovirus through a specific interaction with the virus attachment protein.     

Mutations in a CCHC zinc-binding motif of the reovirus sigma 3 protein decrease its intracellular stability.

It has been demonstrated that the sigma 3 protein of reovirus harbors a zinc-binding domain in its amino-terminal portion. A putative zinc finger in the CCHH form is located in this domain and was considered to be a good candidate for the zinc-binding motif. We performed site-directed mutagenesis to substitute amino acids in this region and demonstrated that many of these mutants, although expressed in COS cells, were unstable compared with the wild-type protein. Further analysis revealed that zinc-binding capability, as measured by retention on a zinc chelate affinity adsorbent, correlates with stability. These studies also allowed us to identify a CCHC box as the most probable zinc-binding motif.     

Co-translational trimerization of the reovirus cell attachment protein.

The reovirus cell attachment protein, sigma1, is a trimer with a 'lollipop' structure. Recent findings indicate that the N-terminal fibrous tail and the C-terminal globular head each possess a distinct trimerization domain. The region responsible for N-terminal trimerization (formation of a triple alpha-helical coiled-coil) is located at the N-terminal one-third of sigma1. In this study, we investigated the temporality and ATP requirement of this trimerization event in the context of sigma1 biogenesis. In vitro co-synthesis of the full-length (FL) and a C-terminally truncated (d44) sigma1 protein revealed a preference for homotrimer over heterotrimer formation, suggesting that assembly at the N-terminus occurs co-translationally. This was corroborated by the observation that polysome-associated sigma1 chains were trimeric as well as monomeric. Truncated proteins (d234 and d294) with C-terminal deletions exceeding half the length of sigma1 were found to trimerize post-translationally. This trimerization did not require ATP since it proceeded normally in the presence of apyrase. In contrast, formation of stable FL sigma1 trimers was inhibited by apyrase treatment. Collectively, our data suggest that assembly of nascent sigma1 chains at the N-terminus is intrinsically ATP independent, and occurs co-translationally when the ribosomes have traversed past the midpoint of the mRNA.     

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.     

Synthesis in Escherichia coli of the reovirus nonstructural protein sigma NS.

The coding region of reovirus type 3 genomic segment S3, encoding the nonstructural protein sigma NS, was placed under the control of the bacteriophage lambda pL promoter in the Escherichia coli expression plasmid pRC23 (J.C. Lacal, E. Santos, V. Notario, M. Barbacid, S. Yamazaki, H.-F. Kung, C. Seamans, S. McAndrew, and R. Crowl, Proc. Natl. Acad. Sci. USA 81:5305-5309). Derepression of the pL promoter led to the synthesis of a protein of the same molecular weight as sigma NS produced in reovirus-infected L cells. The expressed protein was indistinguishable from authentic sigma NS by peptide mapping with Staphylococcus aureus V8 protease and by immunoblot analysis. Most importantly, the purified protein had nucleic acid-binding properties similar to that previously shown for sigma NS obtained from infected cells. Binding of single-stranded RNAs by recombinant sigma NS protein was inhibited by GTP.     

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.     

The reovirus nonstructural protein sigma1NS is recognized by murine cytotoxic T lymphocytes.

The cytotoxic T-lymphocyte (CTL) response in reovirus-infected C3H mice was investigated by using reovirus-vaccinia virus recombinants. Results of cytotoxicity assays indicated that the nonstructural protein sigma1NS elicited a significant CTL response. Experiments with sigma1NS-specific CTL lines showed that both strain-specific and cross-reactive epitopes exist in the sigma1NS protein.