Journal of Molecular Biology
Avian Reovirus Morphogenesis Occurs Within Viral Factories and Begins with the Selective Recruitment of σNS and λA to μNS Inclusions
Introduction
Avian and mammalian reoviruses are members of the Reoviridae family containing a genome of ten segments of double-stranded RNA (dsRNA) encased by a double-protein capsid shell, designated the core and outer capsid.1., 2. These viruses enter the host cell by receptor-mediated endocytosis; after removal of the outer capsid within endosomes, the viral core is released to the cytoplasm to initiate viral gene expression. Reoviral genome segments are then transcribed by a core-associated RNA polymerase to produce messenger RNAs (mRNAs) with a nucleotide sequence identical with the positive strand of the dsRNA segments.3., 4. Viral mRNAs exert a dual function in the infected cell, since they program viral protein synthesis at the ribosomes and serve as templates for the generation of the dsRNA minus-strands.1
Replication of the reovirus genome begins with the assortment and packaging of one molecule of each of the ten species of viral mRNA, and is followed by one round of minus-strand synthesis to produce new dsRNA genome segments. In a coupled process, inner-capsid proteins are assembled to form core particles, which transcribe the bulk of viral mRNAs in order to amplify viral replication. Progeny core particles are thought to be subsequently coated by outer capsid proteins to generate mature reovirions.1., 5. Although the precise timing and molecular mechanisms of these processes are largely unknown, viral replication and assembly are thought to take place within phase-dense globular inclusions termed viral factories or viroplasms.6., 7., 8., 9. These inclusions, which contain both structural and non-structural proteins and which lack both membranes and cellular organelles, first appear as numerous small granules dispersed throughout the cytoplasm; as infection progresses, inclusion structures become larger, less numerous, and perinuclear in distribution.10 The molecular mechanisms that govern the genesis and evolution of reoviral inclusions, the intermolecular interactions among inclusion components, and the specific roles that inclusions play in viral RNA assortment/replication and in morphogenesis of progeny virions all remain poorly understood.
The avian reovirus genome encodes at least 12 primary translation products, eight of which are structural components of the viral particle and four of which are non-structural (NS) proteins.11., 12. Protein coding assignment of the avian reovirus strain S1133 genome has been achieved by in vitro translation of individual genome segments. Three λ polypeptides are encoded by the L genes (λA, λB and λC; molecular weights (MW) 130–150 kDa), three μ polypeptides by the M genes (μA, μB and μNS; MW 70–80 kDa) and four σ polypeptides by the S genes (σA, σB, σNS and σC; MW 30–50 kDa).13., 14. Two small non-structural proteins, p10 and p17, are also encoded by the avian reovirus genome segment S1, and a smaller version of the non-structural protein μNS, termed μNSC, is expressed by the M3 gene in both infected and transfected cells.11., 15., 16. The avian reovirus structural proteins λA, λB, μA and σA are core components, while μB, μBC, σB and σC are present in the outer reovirion shell. Finally, protein λC, which is the avian reovirus guanylyltransferase, extends from the inner core to the outer capsid.17
We have recently shown that the M3-encoded avian reovirus non-structural protein μNS forms viral-like inclusions when expressed in transfected cells, and is present within viral factories in infected cells.16 These findings suggest that μNS is the minimal viral factor required for viral factory formation. Furthermore, μNS associates with the other major non-structural viral protein σNS in both infected and transfected cells, and mediates its recruitment into factories.16 The aim of the present study was to investigate avian reovirus morphogenesis by monitoring the recruitment of avian reovirus proteins into both viral factories and viral particles, and by assessing the role that μNS plays in recruiting other viral proteins into factories. Our findings demonstrate that core assembly and core coating take place exclusively within viral factories of infected cells, in a temporally coordinated fashion, and also indicate that viral morphogenesis starts with μNS forming reovirus factories and recruiting proteins σNS and λA to these structures.
Section snippets
Cell lysis with a Triton-X-100-containing buffer allows discrimination between free and inclusion-associated viral proteins
The recruitment of individual proteins into viral factories in reovirus-infected cells cannot be directly monitored by immunochemical techniques, since antibodies do not distinguish between free and inclusion-associated proteins. In order to overcome this problem, we first assessed whether cell lysis with a Triton-X-100-containing buffer (hereinafter designated buffer A), previously used to discriminate between soluble and cytoskeleton-associated mammalian reovirus proteins,18 would also allow
Avian reovirus factories have globular morphology and are not microtubule-associated
All members of the Reoviridae family replicate and assemble within cytoplasmic inclusions called viral factories or viroplasms, which are devoid of membranes and cell organelles. The genesis and composition of viral factories, as well as their roles in the virus life cycle, are only beginning to be understood. Recent reports from different laboratories provide compelling evidence that specific non-structural proteins of various members of the Reoviridae family, including mammalian reovirus μNS,
Cells, viruses and antibodies
Primary cultures of CEFs were prepared from nine to ten-day-old chicken embryos and grown in monolayers in medium 199 supplemented with 10% (w/v) tryptose phosphate broth and 5% (v/v) calf serum. Strain S1133 of avian reovirus was grown in semiconfluent monolayers of primary CEFs and purified as described.29 Rabbit polyclonal sera against S1133 avian reovirions, S1133 avian reovirus cores and avian reovirus S1133 μNS protein were raised in our laboratory. A monoclonal antibody against the avian
Acknowledgements
We are grateful to Dr Aaron Shatkin for critical reading of the manuscript. We thank Laboratorios Intervet (Salamanca, Spain) for providing the pathogen-free embryonated eggs. We also thank Lois Hermo & Salvador Blanco for excellent technical assistance. This work was supported by grants from the Spanish Ministerio de Ciencia y Tecnologı́a (BCM2001-2839) and from the Xunta de Galicia (2002/PX001 and 2002/PX135). F.T.O. was recipient of a predoctoral fellowship from the Fundación Ramón Areces
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