Javier Benavente

Possible Involvement of the Double-Stranded RNA-Binding Core Protein ςA in the Resistance of Avian Reovirus to Interferon

ABSTRACT Treatment of primary cultures of chicken embryo fibroblasts with a recombinant chicken alpha/beta interferon (rcIFN) induces an antiviral state that causes a strong inhibition of vaccinia virus and vesicular stomatitis virus replication but has no effect on avian reovirus S1133 replication. The fact that avian reovirus polypeptides are synthesized normally in rcIFN-treated cells prompted us to investigate whether this virus expresses factors that interfere with the activation and/or the activity of the IFN-induced, double-stranded RNA (dsRNA)-dependent enzymes. Our results demonstrate that extracts of avian-reovirus-infected cells, but not those of uninfected cells, are able to relieve the translation-inhibitory activity of dsRNA in reticulocyte lysates, by blocking the activation of the dsRNA-dependent enzymes. In addition, our results show that protein ςA, an S1133 core polypeptide, binds to dsRNA in an irreversible manner and that clearing this protein from extracts of infected cells abolishes their protranslational capacity. Taken together, our results raise the interesting possibility that protein ςA antagonizes the IFN-induced cellular response against avian reovirus by blocking the intracellular activation of enzyme pathways dependent on dsRNA, as has been suggested for several other viral dsRNA-binding proteins.

Avian Reoviruses Cause Apoptosis in Cultured Cells: Viral Uncoating, but Not Viral Gene Expression, Is Required for Apoptosis Induction

ABSTRACT The cytopathic effect evidenced by cells infected with avian reovirus S1133 suggests that this virus may induce apoptosis in primary cultures of chicken embryo fibroblasts. In this report we present evidence that avian reovirus infection of cultured cells causes activation of the intracellular apoptotic program and that this activation takes place during an early stage of the viral life cycle. The ability of avian reoviruses to induce apoptosis is not restricted to a particular virus strain or to a specific cell type, since different avian reovirus isolates were able to induce apoptosis in several avian and mammalian cell lines. Apoptosis was also provoked in ribavirin-treated avian reovirus-infected cells and in cells infected with UV-irradiated reovirions, indicating that viral mRNA synthesis and subsequent steps in viral replication are not needed for apoptosis induction in avian reovirus-infected cells and that the number of inoculated virus particles, not their infectivity, is the critical factor for apoptosis induction by avian reovirus. Our finding that apoptosis is no longer induced when intracellular viral uncoating is blocked indicates that intraendosomal virion disassembly is required for apoptosis induction and that attachment and uptake of parental reovirions are not sufficient to cause apoptosis. Taken together, our results suggest that apoptosis is triggered from within the infected cell by viral products generated after intraendosomal uncoating of parental reovirions.

The Second Open Reading Frame of the Avian Reovirus S1 Gene Encodes a Transcription-Dependent and CRM1-Independent Nucleocytoplasmic Shuttling Protein

ABSTRACT It was previously shown that the second open reading frame of the avian reovirus S1 gene encodes a 146-amino-acid nonstructural protein, designated p17, which has no known function and no sequence similarity to other known proteins. The results presented in this report demonstrate that p17 accumulates in the nucleoplasm of infected and transfected cells. An examination of the deduced amino acid sequence of p17 revealed the presence of a putative monopartite nuclear localization signal (NLS) between residues 119 and 128. Mutagenesis analysis revealed both that this sequence is indeed a functional NLS and that two of its basic residues are critical for the normal nuclear distribution of p17. An interspecies heterokaryon assay further showed that p17 shuttles continuously between the nucleus and the cytoplasm and that this activity is restricted to its NLS-containing C-terminal tail. Finally, an analysis of the intracellular distribution of p17 in the presence of inhibitors of both RNA polymerase II and CRM1 further revealed that the nucleocytoplasmic distribution of p17 is coupled to transcriptional activity and that the viral protein exits the nucleus via a CRM1-independent pathway.

Subunit composition and conformational stability of the oligomeric form of the avian reovirus cell-attachment protein σC

Previous work has shown that the avian reovirus cell-attachment sigma C (sigmaC) protein is a multimer. In the first part of this study the oligomerization state of intracellularly synthesized sigmaC was analysed by different approaches, including SDS-PAGE, chemical cross-linking, sedimentation and gel filtration analysis. All these approaches indicated that protein sigmaC in its native state is a homotrimer. In the second part of the present work we investigated the effect of different factors and reagents on oligomer stability, in order to elucidate the nature of the forces that maintain the conformational stability of the homotrimer. Our results, based on the stabilizing effect conferred by reducing agents, demonstrate that the sigmaC subunits are not covalently bound via disulfide linkages. They further suggest that the formation of an intrachain disulfide bond between the two cysteine residues of the sigmaC polypeptide has a negative effect on oligomer stability. The susceptibility of the trimer to pH, temperature, ionic strength, chemical denaturants and detergents indicates that hydrophobic interactions contribute much more to oligomer stability than do ionic interactions and hydrogen bonding. Finally, our results also reveal that mammalian and avian reovirus cell attachment proteins follow different subunit dissociation pathways.

Optimal conditions for the growth, purification and storage of the avian reovirus S1133

In spite of their importance as avian pathogens causing important losses in poultry farming, the biochemistry of avian reoviruses has been little investigated. In order to facilitate the handling of these agents in the laboratory, a study was carried out to establish the best conditions both for growing the avian reovirus S1133 in primary cultures of chicken embryo fibroblasts and for purification and storage of viral suspensions. The results indicate that the conditions used currently for the manipulation of mammalian reoviruses are not always the best for handling their avian counterparts. In particular, avian reoviruses are much less stable than mammalian reoviruses, and specific conditions for the purification and storage of avian reoviruses therefore should be used. Furthermore, the instability of avian reovirions may have important implications for the life cycle and pathogenesis of the virus within the animal host.

Avian Reovirus μNS Protein Forms Homo-Oligomeric Inclusions in a Microtubule-Independent Fashion, Which Involves Specific Regions of Its C-Terminal Domain

ABSTRACT Members of the genus Orthoreovirus replicate in cytoplasmic inclusions termed viral factories. Compelling evidence suggests that the nonstructural protein μNS forms the matrix of the factories and recruits specific viral proteins to these structures. In the first part of this study, we analyzed the properties of avian reovirus factories and μNS-derived inclusions and found that they are nonaggresome cytoplasmic globular structures not associated with the cytoskeleton which do not require an intact microtubule network for formation and maturation. We next investigated the capacity of avian reovirus μNS to form inclusions in transfected and baculovirus-infected cells. Our results showed that μNS is the main component of the inclusions formed by recombinant baculovirus expression. This, and the fact that μNS is able to self-associate inside the cell, suggests that μNS monomers contain all the interacting domains required for inclusion formation. Examination of the inclusion-forming capacities of truncated μNS versions allowed us to identify the region spanning residues 448 to 635 of μNS as the smallest that was inclusion competent, although residues within the region 140 to 380 seem to be involved in inclusion maturation. Finally, we investigated the roles that four different motifs present in μNS(448-635) play in inclusion formation, and the results suggest that the C-terminal tail domain is a key determinant in dictating the initial orientation of monomer-to-monomer contacts to form basal oligomers that control inclusion shape and inclusion-forming efficiency. Our results contribute to an understanding of the generation of structured protein aggregates that escape the cellular mechanisms of protein recycling.

Avian Reovirus SigmaA Localizes to the Nucleolus and Enters the Nucleus by a Nonclassical Energy- and Carrier-Independent Pathway

ABSTRACT Avian reovirus sigmaA is a double-stranded RNA (dsRNA)-binding protein that has been shown to stabilize viral core particles and to protect the virus against the antiviral action of interferon. To continue with the characterization of this viral protein, we have investigated its intracellular distribution in avian cells. Most sigmaA accumulates into cytoplasmic viral factories of infected cells, and yet a significant fraction was detected in the nucleolus. The protein also localizes in the nucleolus of transfected cells, suggesting that nucleolar targeting is not facilitated by the viral infection or by viral factors. Assays performed in both intact cells and digitonin-permeabilized cells demonstrate that sigmaA is able to enter the nucleus via a nucleoporin-dependent nondiffusional mechanism that does not require added cytosolic factors or energy input. These results indicate that sigmaA by itself is able to penetrate into the nucleus using a process that is mechanistically different from the classical nuclear localization signal/importin pathway. On the other hand, two sigmaA arginines that are necessary for dsRNA binding are also required for nucleolar localization, suggesting that dsRNA-binding and nucleolar targeting are intimately linked properties of the viral protein.

Avian reovirus-triggered apoptosis enhances both virus spread and the processing of the viral nonstructural muNS protein

Abstract Avian reovirus non-structural protein muNS is partially cleaved in infected chicken embryo fibroblast cells to produce a 55-kDa carboxyterminal protein, termed muNSC, and a 17-kDa aminoterminal polypeptide, designated muNSN. In this study we demonstrate that muNS processing is catalyzed by a caspase 3-like protease activated during the course of avian reovirus infection. The cleavage site was mapped by site directed mutagenesis between residues Asp-154 and Ala-155 of the muNS sequence. Although muNS and muNSC, but not muNSN, are able to form inclusions when expressed individually in transfected cells, only muNS is able to recruit specific ARV proteins to these structures. Furthermore, muNSC associates with ARV factories more weakly than muNS, sigmaNS and lambdaA. Finally, the inhibition of caspase activity in ARV-infected cells does not diminish ARV gene expression and replication, but drastically reduces muNS processing and the release and dissemination of progeny viral particles.

Crystal structure of the avian reovirus inner capsid protein sigmaA.

ABSTRACT Avian reovirus, an important avian pathogen, expresses eight structural and four nonstructural proteins. The structural σA protein is a major component of the inner capsid, clamping together λA building blocks. σA has also been implicated in the resistance of avian reovirus to the antiviral action of interferon by strongly binding double-stranded RNA in the host cell cytoplasm and thus inhibiting activation of the double-stranded RNA-dependent protein kinase. We have solved the structure of bacterially expressed σA by molecular replacement and refined it using data to 2.3-Å resolution. Twelve σA molecules are present in the P1 unit cell, arranged as two short double helical hexamers. A positively charged patch is apparent on the surface of σA on the inside of this helix and mutation of either of two key arginine residues (Arg155 and Arg273) within this patch abolishes double-stranded RNA binding. The structural data, together with gel shift assay, electron microscopy, and sedimentation velocity centrifugation results, provide evidence for cooperative binding of σA to double-stranded RNA. The minimal length of double-stranded RNA required for σA binding was observed to be 14 to 18 bp.

A Versatile Molecular Tagging Method for Targeting Proteins to Avian Reovirus muNS Inclusions. Use in Protein Immobilization and Purification

Background Avian reoviruses replicate in viral factories, which are dense cytoplasmic compartments estabilished by protein-protein interactions. The non-structural protein muNS forms the factory scaffold that attracts other viral components in a controlled fashion. To create such a three-dimensional network, muNS uses several different self-interacting domains. Methodology/Principal Findings In this study we have devised a strategy to identify muNS regions containing self-interacting domains, based on the capacity of muNS-derived inclusions to recruit muNS fragments. The results revealed that the muNS region consisting of residues 477–542 was recruited with the best efficiency, and this raised the idea of using this fragment as a molecular tag for delivering foreign proteins to muNS inclusions. By combining such tagging system with our previously established method for purifying muNS inclusions from baculovirus-infected insect cells, we have developed a novel protein purification protocol. Conclusions/Significance We show that our tagging and inclusion-targeting system can be a simple, versatile and efficient method for immobilizing and purifying active proteins expressed in baculovirus-infected cells. We also demonstrate that muNS inclusions can simultaneously recruit several tagged proteins, a finding which may be used to generate protein complexes and create multiepitope particulate material for immunization purposes.