Persistent reovirus infections of L cells select mutations in viral attachment protein sigma1 that alter oligomer stability

Abstract

During maintenance of L-cell cultures persistently infected with reovirus, mutations are selected in viruses and cells. Cells cured of persistent infection support growth of viruses isolated from persistently infected cultures (PI viruses) significantly better than that of wild-type (wt) viruses. In a previous study, the capacity of PI virus strain L/C to grow better than wt strain type 1 Lang (T1L) in cured cells was mapped genetically to the S1 gene (R. S. Kauffman, R. Ahmed, and B. N. Fields, Virology 131:79-87, 1983), which encodes viral attachment protein 1. To investigate mechanisms by which mutations in S1 confer growth of PI viruses in cured cells, we determined the S1 gene nucleotide sequences of L/C virus and six additional PI viruses isolated from independent persistently infected L-cell cultures. The S1 sequences of these viruses contained from one to three mutations, and with the exception of PI 2A1, mutations in each S1 gene resulted in changes in the deduced amino acid sequence of 1 protein. Using electrophoresis conditions that favor migration of 1 oligomers, we found that 1 proteins of L/C, PI 1A1, PI 3-1, and PI 5-1 migrated as monomers, whereas 1 proteins of wt reovirus and PI 2A1 migrated as oligomers. These findings suggest that mutations in 1 protein affecting stability of 1 oligomers are important for the capacity of PI viruses to infect mutant cells selected during persistent infection. Since no mutation was found in the deduced amino acid sequence of PI 2A1 1 protein, we used T1L ؋ PI 2A1 reassortant viruses to identify viral genes associated with the capacity of this PI virus to grow better than wt in cured cells. The capacity of PI 2A1 to grow better than T1L in cured cells was mapped to the S4 gene, which encodes outer-capsid protein 3. This finding suggests that in some cases, mutations in 3 protein in the absence of 1 mutations confer growth of PI viruses in mutant cells. To confirm the importance of the S1 gene in PI virus growth in cured cells, we used T1L ؋ PI 3-1 reassortant viruses to genetically map the capacity of this PI virus to grow better than wt in cured cells. In contrast to our results using PI 2A1, we found that growth of PI 3-1 in cured cells was determined by the 1-encoding S1 gene. Given that the 1 and 3 proteins play important roles in reovirus disassembly, findings made in this study suggest that stability of the viral outer capsid is an important determinant of the capacity of reoviruses to adapt to host cells during persistent infection.

A Plasmid-Based Reverse Genetics System for Animal Double-Stranded RNA Viruses

“…Following 0-5 days of incubation, recombinant virus was isolated from transfected cells by plaque puri-fication on monolayers of L cells (Virgin et al, 1988). Electrophoretic analysis of viral dsRNA was performed as described (Wilson et al, 1996). Confirmation of mutations in the S1, S4, and L1 genes of recombinant viruses was performed using the Onestep RT-PCR kit (Qiagen), gene-specific primer sets, and viral dsRNA extracted from purified virions as template.…”

Cleavage Susceptibility of Reovirus Attachment Protein ς1 during Proteolytic Disassembly of Virions Is Determined by a Sequence Polymorphism in the ς1 Neck

“…Another function of the 1 neck may be to promote intraendosomal virion disassembly by undergoing conformational adjustments that alter interactions between 1 and neighboring outer-capsid components, such as the 3 protein (34,38,63). Indeed, mutations in both 1 and 3 proteins confer the ability of virions to bypass blocks to disassembly within the endosome (66,69), suggesting a cooperative interaction of 1 with 3 in the dissociation of outer-capsid proteins. Additional sites in the 1 tail, near the middle and near the virion-proximal amino terminus, also show evidence of flexibility (24), and concerted adjustments at these positions and the neck region may facilitate the dramatic conformational change in 1 observed when the outer capsid is degraded by protease to produce an ISVP (25).…”

Reovirus Growth in Cell Culture Does Not Require the Full Complement of Viral Proteins: Identification of a ς1s-Null Mutant

“…3 and 4). The fact that the 1s ORF is conserved in every S1 gene sequence reported to date, representing 28 independent reovirus isolates (1,2,7,9,12,14,29,30,32,55), suggests that 1s confers some selective advantage to reovirus replication and plays an important biological role. Examination of the deduced amino acid sequences of nonstructural proteins of other members of the Reoviridae family does not reveal a homologue for the 1s protein (10).…”

“…Studies using reassortant viruses to investigate mechanisms of reovirus pathogenesis indicate that the S1 gene segregates with strain-specific differences in reovirus growth in the intestine (4,24), pathway of spread in the host (24,25,45), tropism for neural tissues (25,50,51), inhibition of DNA synthesis, (39,46), and induction of apoptosis (35,46,47). In addition, mutations in the S1 gene are selected during persistent reovirus infections of cultured cells (23,52,55). For most properties linked to the S1 gene, a direct association with the 1 protein has been deduced by the demonstration that a particular phenotype is determined by viral attachment (9,31,42,51) or by the identification of mutations in the deduced amino acid sequence of 1 without an attendant change in 1s (2).…”

“…Additional work suggests that 1s also is capable of translocation into the nucleus (3). The 1s ORF is maintained in every S1 gene sequence determined to date and varies from 119 to 125 amino acids in length (1,2,7,9,12,14,29,30,32,55). Alignments of 1sdeduced amino acid sequences of prototype reovirus strains type 1 Lang (T1L), type 2 Jones, and type 3 Dearing (T3D) demonstrate that 1s is highly divergent among strains of the three reovirus serotypes, sharing only 18 identical amino acid positions (14).…”