Optimum Length and Flexibility of Reovirus Attachment Protein σ1 Are Required for Efficient Viral Infection

Abstract

Reovirus attachment protein σ1 is an elongated trimer with head-and-tail morphology that engages cell-surface carbohydrate and junctional adhesion molecule A (JAM-A). The σ1 protein is comprised of three domains partitioned by two flexible linkers termed interdomain regions (IDRs). To determine the importance of σ1 length and flexibility at different stages of reovirus infection, we generated viruses with mutant σ1 molecules of altered length and flexibility and tested these viruses for the capacity to bind the cell surface, internalize, uncoat, induce protein synthesis, assemble, and replicate. We reduced the length of the α-helical σ1 tail to engineer mutants L1 and L2 and deleted midpoint and head-proximal σ1 IDRs to generate ΔIDR1 and ΔIDR2 mutant viruses, respectively. Decreasing length or flexibility of σ1 resulted in delayed reovirus infection and reduced viral titers. L1, L2, and ΔIDR1 viruses but not ΔIDR2 virus displayed reduced cell attachment, but altering σ1 length or flexibility did not diminish the efficiency of virion internalization. Replication of ΔIDR2 virus was hindered at a postdisassembly step. Differences between wild-type and σ1 mutant viruses were not attributable to alterations in σ1 folding, as determined by experiments assessing engagement of cell-surface carbohydrate and JAM-A by the length and IDR mutant viruses. However, ΔIDR1 virus harbored substantially less σ1 on the outer capsid. Taken together, these data suggest that σ1 length is required for reovirus binding to cells. In contrast, IDR1 is required for stable σ1 encapsidation, and IDR2 is required for a postuncoating replication step. Thus, the structural architecture of σ1 is required for efficient reovirus infection of host cells.

Amino acids substitutions in σ1 and μ1 outer capsid proteins of a Vero cell-adapted mammalian orthoreovirus are required for optimal virus binding and disassembly

“…Although somewhat variable from one preparation to another, the ratio of the number of virus particles to viral infectivity titer was approximately 100 particles/PFU for both viruses. This is, in fact, quite similar to previous reports by other groups (Bokiej and Dermody, 2012;Doyle et al, 2012;Frierson et al, 2012;Hand and Tamm, 1973;Mendez et al, 2000).…”

“…This is not surprising considering that binding assay are routinely performed at 50,000 particles per cell by other investigators (for example Bokiej and Dermody, 2012). An intermediate MOI of 80 was thus used thereafter to avoid saturation of the cellular receptors.…”

Characterizing Functional Domains for TIM-Mediated Enveloped Virus Entry

“…5C). Consistent with this possibility, both adenoviruses and reoviruses have been shown to require flexibility and length in the stalk of their attachment proteins for receptor binding (49,50). Taken together, these results suggest that adequate distance and flexibility on either the host or virus side are necessary for effective receptor-virus interactions and infection.…”

The Nogo Receptor NgR1 Mediates Infection by Mammalian Reovirus

“…While there are no reports of cellular receptors for BRV, the NgR1-encoding gene is conserved in primates. Alternatively, conformational changes in σ1 associated with the transition of virions to ISVPs have been hypothesized to alter the capacity of σ1 to interact with cell-surface receptors (Bokiej et al, 2012; Dryden et al, 1993). …”

“…Reovirus T3SA+ virions or ISVPs serially diluted in TBS containing 0.1% BSA and 0.05% Tween were added to the wells and incubated at 4°C overnight. Wells were washed prior to adding reovirus-specific monoclonal antibody 9BG5 (Burstin et al, 1982) at room temperature for 2 h. Wells were washed and incubated with fluorescent secondary antibodies (LI-COR) at room temperature for 2 h (Bokiej et al, 2012). Following washing, signal intensity was quantified using the Odyssey Imaging System.…”