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Title page for ETD etd-08132012-112658

Type of Document Dissertation
Author Bokiej, Magdalena
URN etd-08132012-112658
Title The role of σ1 length and flexibility in reovirus replication
Degree PhD
Department Microbiology and Immunology
Advisory Committee
Advisor Name Title
Earl Ruley Committee Chair
Benjamin Spiller Committee Member
Jens Meiler Committee Member
John V. Williams Committee Member
Terence S. Dermody Committee Member
  • reovirus
  • attachment
  • sigma1
  • internalization
  • disassembly
  • escape
  • assembly
Date of Defense 2012-08-03
Availability unrestricted
This dissertation research was focused on elucidating mechanisms at the reovirus-host interface that allow successful completion of the initial replicative steps and lead to viral protein synthesis. During the course of the study, I discovered new functional domains in the reovirus attachment protein σ1. Sigma 1 is an elongated trimer with head-and-tail morphology that engages cell-surface carbohydrate and junctional adhesion molecule-A (JAM-A). This protein is comprised of three domains partitioned by two flexible linkers termed inter-domain regions (IDRs). To determine the importance of σ1 length and flexibility at different stages of reovirus infection, I 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. I 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 ∆IDR1 and ∆IDR2, respectively. Decreasing length or flexibility of σ1 resulted in delayed reovirus infection and reduced viral titers. L1, L2, and ∆IDR1 but not ∆IDR2 displayed reduced cell attachment, but altering σ1 length or flexibility did not diminish the efficiency of virion internalization. Replication of ∆IDR2 was hindered at a post-disassembly 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 L and IDR viruses. However, ∆IDR1 harbored substantially less σ1 on the outer capsid. Taken together, these findings 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 post-uncoating replication step. Thus, I discovered that σ1 functions are not restricted to mediating reovirus attachment.
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