Post-entry Determinants of Mammalian Orthoreovirus Replication

Abstract

Coordination of viral replication requires successful and often complex interactions of viral proteins and cellular factors. Although much progress has been made in understanding the formation of specialized sites of replication where viral proteins and repurposed cellular factors direct genome multiplication and particle assembly, much remains unknown about the specific mechanisms of post-entry determinants of viral replication efficiency and cell tropism. Reoviruses are non-enveloped, double-stranded (ds) RNA viruses that display broad cell, tissue, and host tropism and serve as tractable models for the study of virus-cell interactions that dictate unique tropic properties. Reovirus replication protein μ2 has many functional and biochemical properties that are essential to post-entry steps in the viral life cycle. The goal of my dissertation research has been to determine how reovirus μ2, and its cooperation with other viral proteins, support progression of the viral replication cycle and contribute to reovirus cell tropism. Toward that goal, I characterized the genetic basis of strain-dependent reovirus replication in Madin Darby canine kidney (MDCK) cells. μ2 controls efficiency of reovirus replication in these cells, and amino acid residue 347 is the primary determinant of this phenotype. Furthermore, polymerase protein λ3 is a co-regulator of viral replication in a strain-dependent manner. I systematically tested post-entry steps in the reovirus life cycle to identify the critical step in reovirus tropism for MDCK cells. The replication block occurs following inclusion formation but prior to dsRNA synthesis, indicating that inclusions formed during non-permissive infection are morphologically normal but functionally abnormal. I also analyzed inclusion ultrastructure under permissive and non-permissive conditions of infection and found that particle assembly is inhibited in non-productively infected cells. These studies further an understanding of the role of μ2 in the viral life cycle to include initiation of particle assembly within viral inclusions and indicate that this step is subject to regulation by cellular factors.