A Post-entry Step in the Mammalian Orthoreovirus Replication Cycle Is a Determinant of Cell Tropism

Abstract

Mammalian reoviruses replicate in a broad range of hosts, cells, and tissues. These viruses display strain-dependent variation in tropism for different types of cells in vivo and ex vivo. Early steps in the reovirus life cycle, attachment, entry, and disassembly, have been identified as pivotal points of virus-cell interaction that determine the fate of infection, either productive or abortive. However, in studies of the differential capacity of reovirus strains type 1 Lang and type 3 Dearing to replicate in Madin-Darby canine kidney (MDCK) cells, we found that replication efficiency is regulated at a late point in the viral life cycle following primary transcription and translation. Results of genetic studies using recombinant virus strains show that reovirus tropism for MDCK cells is primarily regulated by replication protein 2 and further influenced by the viral RNAdependent RNA polymerase protein, 3, depending on the viral genetic background. Furthermore, 2 residue 347 is a critical determinant of replication efficiency in MDCK cells. These findings indicate that components of the reovirus replication complex are mediators of cell-selective viral replication capacity at a post-entry step. Thus, reovirus cell tropism may be determined at early and late points in the viral replication program. Viral tropism, defined by the range of hosts and tissues productively infected, creates natural biologic groupings among viruses that correlate with infection pathology, clinical disease expression, and epidemiology. Delineating the molecular basis of viral cell tropism is fundamental to the elucidation of disease mechanisms and the identification of viral and cellular targets for treatment and prevention of infection. Growing threats posed by zoonotic viral diseases with global pandemic potential (1) underscore the necessity for an enhanced understanding of unifying principles that influence viral tropism and host range. We are conducting studies using mammalian reoviruses to better understand the nature of virus-cell interactions that dictate unique tropism properties.Mammalian orthoreoviruses (hereafter referred to as reoviruses) are an established model for studies of viral replication and pathogenesis (2-6). Reoviruses have contributed to the development of paradigms of viral disease based on discrete patterns of viral tropism for particular host cells and tissues. The reovirus virion is a nonenveloped, double layered, icosahedral particle consisting of an outer shell surrounding an inner core that contains 10 double-stranded (ds) RNA gene segments. The viral genome encodes eight structural and three nonstructural proteins. Viral RNA gene segments can be resolved into small, medium, and large size classes by SDS-PAGE, corresponding to , , and proteins, respectively. Four reovirus serotypes have been identified based on neutralization and hemagglutination studies (7-9). Strains type 1 Lang (T1) 3 and type 3 Dearing (T3) serve as prototypes for serotype 1 and 3 reoviruses, respectively. T1 and T3 display numerous phen...

A Single-Amino-Acid Polymorphism in Reovirus Protein μ2 Determines Repression of Interferon Signaling and Modulates Myocarditis

“…All viruses were generated from plasmids by reverse genetics as described previously (20,38). The recombinant viruses were plaque purified, amplified using L929 cells, purified using CsCl gradients (49), and stored as diluted aliquots at Ϫ80°C.…”

Reovirus Replication Protein μ2 Influences Cell Tropism by Promoting Particle Assembly within Viral Inclusions

“…Our previous studies of T3-infected MDCK cells revealed that inclusions produced by strain T3 display normal morphological and kinetic development despite an apparent functional abnormality leading to arrest of replication (33). To more precisely define viral inclusion composition and ultrastructure in T3-infected MDCK cells, we used both confocal immunofluorescence microscopy and thin-section TEM.…”

“…Strain T1 replicates efficiently, while T3 replicates poorly. Analysis of recombinant reovirus strains derived from reverse genetics revealed that the M1-encoded 2 protein of strain T1 in a T3 genetic background independently promotes reovirus replication in MDCK cells (33). The L1-encoded 3 protein is a modulator of 2 effects on replication efficiency, although 3 does not display an independent association with replication capacity in MDCK cells.…”

“…Reovirus inclusions are implicated functionally as determinants of viral cell tropism (33), where tropism is defined as the range of hosts, tissues, and cells productively infected. Reovirus strains type 1 Lang (T1) and type 3 Dearing (T3) display differen-tial capacities to replicate in Madin-Darby canine kidney (MDCK) cells (33,39).…”

“…Reovirus strains type 1 Lang (T1) and type 3 Dearing (T3) display differen-tial capacities to replicate in Madin-Darby canine kidney (MDCK) cells (33,39). Strain T1 replicates efficiently, while T3 replicates poorly.…”