Avian Reovirus Temperature-Sensitive Mutant tsA12 Has a Lesion in Major Core Protein σA and Is Defective in Assembly

Abstract

Members of our laboratory previously generated and described a set of avian reovirus (ARV) temperaturesensitive (ts) mutants and assigned 11 of them to 7 of the 10 expected recombination groups, named A through G (M. Patrick, R. Duncan, and K. M. Coombs, Virology 284: [113][114][115][116][117][118][119][120][121][122] 2001). This report presents a more detailed analysis of two of these mutants (tsA12 and tsA146), which were previously assigned to recombination group A. The capacities of tsA12 and tsA146 to replicate at a variety of temperatures were determined. Morphological analyses indicated that cells infected with tsA12 at a nonpermissive temperature produced ϳ100-fold fewer particles than cells infected at a permissive temperature and accumulated core particles. Cells infected with tsA146 at a nonpermissive temperature also produced ϳ100-fold fewer particles, a larger proportion of which were intact virions. We crossed tsA12 with ARV strain 176 to generate reassortant clones and used them to map the temperature-sensitive lesion in tsA12 to the S2 gene. S2 encodes the major core protein A. Sequence analysis of the tsA12 S2 gene showed a single alteration, a cytosine-to-uracil transition, at nucleotide position 488. This alteration leads to a predicted amino acid change from proline to leucine at amino acid position 158 in the A protein. An analysis of the core crystal structure of the closely related mammalian reovirus suggested that the Leu 158 substitution in ARV A lies directly under the outer face of the A protein. This may cause a perturbation in A such that outer capsid proteins are incapable of condensing onto nascent cores. Thus, the ARV tsA12 mutant represents a novel assembly-defective orthoreovirus clone that may prove useful for delineating virus assembly.Avian reoviruses (ARVs) are members of the Orthoreovirus genus of the family Reoviridae. ARVs generally are considered to be similar to their mammalian counterparts in structure and molecular composition (40,43). Both ARVs and mammalian reoviruses (MRVs) have a genome consisting of 10 segments of double-stranded RNA (dsRNA) enclosed by a doubleshelled capsid made from eight different proteins. The ARV outer capsid contains three proteins (the major proteins B and B and the minor protein C), and the inner core structure is composed of five proteins (the major core proteins A and A, the core spike protein C, and the minor core proteins B and A) (25, 26). ARVs are important pathogens of poultry that may cause considerable economic losses in poultry farming (reviewed in references 22, 37, and 44). Although their pathological effects have been extensively investigated, the basic biology, structure, function, and assembly of ARVs remain poorly understood.The segmented natures of reovirus genomes, the monocistronic nature of virtually all of the gene segments, and straindependent mobility differences of the genes in polyacrylamide gels have provided intertypic reassortants for use, and these genetic tools have been extensively used to assign biologic f...

Structure of avian orthoreovirus virion by electron cryomicroscopy and image reconstruction

“…We recognized that such data may be useful to investigate possible divergence of structural features within the genus Orthoreovirus, as well as between orthoreoviruses and aquareoviruses. Given increasing attention to molecular and cellular studies of ARV in the past several years (Hsiao et al, 2002;Shmulevitz et al, 2004;Touris-Otero et al, 2004;Xu et al, 2004), we focused our efforts on one of these fusogenic isolates, ARV-138, a mildly pathogenic strain that grows to high yields in cultured cells and has been the focus of several recent analyses (Duncan and Sullivan, 1998;O'Hara et al, 2001;Patrick et al, 2001).…”

Assignment of avian reovirus temperature-sensitive mutant recombination groups E, F, and G to genome segments

“…Methods for the generation of reassortants have been described previously (Xu et al, 2004). Previous data suggests that the proportion and type of reassortant generated depends upon the multiplicity of infection of each parent (Ramig, 1983).…”

“…Of these seven groups, four prototypic members have been mapped to their respective gene segments: tsA12 mapped to S2 gene (σA), tsB31 mapped to M2 gene (μB), tsC37 mapped to S3 gene (σB), and tsD46 mapped to L2 gene (λB) (Xu et al, 2004(Xu et al, , 2005. Here, we report the mapping of prototypic mutants in the remaining three recombination groups E (tsE158), F (tsF206), and G (tsG247).…”

“…The avian reovirus assembly process has not yet been fully resolved, and it also remains less well characterized at the molecular level than for mammalian reovirus. However, ongoing work with a panel of temperature-sensitive (ts) mutants is beginning to shed light on this virus group's replication process (Patrick et al, 2001;Xu et al, 2004Xu et al, , 2005. Although the specific mechanisms of ARV assembly and replication have yet to be fully elucidated, these studies have suggested its stages of replication are similar to those deduced in MRV.…”

“…Our lab generated a panel of avian reovirus ts mutants by infecting cells with ARV138 in the presence of nitrosoguanidine (Patrick et al, 2001). A total of seven recombination groups (A to G) were clearly identified, indicating each had their ts mutations in different genes; that 7 of the 10 ARV138 dsRNA gene segments have representative ts mutations (Patrick et al, 2001;Xu et al, 2004Xu et al, , 2005.…”