Characterization of the Expression, Intracellular Localization, and Replication Complex Association of the Putative Mouse Hepatitis Virus RNA-Dependent RNA Polymerase

Abstract

Mouse hepatitis virus (MHV) RNA synthesis is mediated by a viral RNA-dependent RNA polymerase (RdRp) on membrane-bound replication complexes in the host cell cytoplasm. However, it is not known how the putative MHV RdRp (Pol) is targeted to and retained on cellular membranes. In this report, we show that a 100-kDa protein was stably detected by an anti-Pol antiserum as a mature product throughout the virus life cycle. Gradient fractionation and biochemical extraction experiments demonstrated that Pol was not an integral membrane protein but was tightly associated with membranes and coimmunoprecipitated with the replicase proteins 3CLpro, p22, and p12. By immunofluorescence confocal microscopy, Pol colocalized with viral proteins at replication complexes, distinct from sites of virion assembly, over the entire course of infection. To determine if Pol associated with cellular membranes in the absence of other viral factors, the pol domain of gene 1 was cloned and expressed in cells as a fusion with green fluorescent protein, termed Gpol. In Gpol-expressing cells that were infected with MHV, but not in mock-infected cells, Gpol relocalized from a diffuse distribution in the cytoplasm to punctate foci that colocalized with markers for replication complexes. Expression of Gpol deletion mutants established that the conserved enzymatic domains of Pol were dispensable for replication complex association, but a 38-amino-acid domain in the RdRp unique region of Pol was required. This study demonstrates that viral or virus-induced factors are necessary for Pol to associate with membranes of replication complexes, and it identifies a defined region of Pol that may mediate its interactions with those factors.For all known positive-strand RNA viruses, RNA synthetic activity occurs on viral replication complexes that are derived from cellular membranes and is mediated by viral RNA-dependent RNA polymerases (RdRps). Recent evidence suggests that viruses in the order Nidovirales, including coronaviruses and arteriviruses, replicate their RNA at double-membrane vesicles in the host cell cytoplasm (19,35). For the coronaviruses, a diverse family of positive-strand RNA viruses, it is not known how viral replication complexes are formed or how replicase proteins are recruited to and remain associated with cellular membranes. Although a core RdRp has been predicted based on sequence analysis, and membrane-associated RdRp activity has been detected in coronavirus-infected cells, the function of this putative enzyme has not been confirmed experimentally (1,6,16,18,27). As such, determination of the expression, intracellular localization, and membrane targeting of the coronavirus RdRp is central to understanding the functions of this protein during viral RNA synthesis.Mouse hepatitis virus (MHV) is a well-established model for studies of the replication and pathogenesis of coronaviruses. MHV contains a positive-strand RNA genome of approximately 32,000 nucleotides (nt) that serves as a template for the synthesis of multiple RNA species ...

Immunogenic characterization and epitope mapping of transmissible gastroenteritis virus RNA dependent RNA polymerase

“…Additionally, higher molecular mass polypeptides of around 150 kDa were also detected, which could represent RdRp homo-or hetero-oligomeric structures or pp1ab polyprotein processing precursors. Similar results have been reported in MHV, where RdRp protein precursors of about 150 and 300 kDa were detected (Brockway et al, 2003). RdRp was also recognized by immunofluorescence, showing a perinuclear vesicular pattern that is typical for CoV DMVs, where viral RNA synthesis takes place (Gosert et al, 2002;Knoops et al, 2008;Snijder et al, 2006).…”

Continuous and Discontinuous RNA Synthesis in Coronaviruses

“…In addition to the RdRp domain, nsp12 also contains an N-terminal domain that is essential for RdRp activity (122) and probably interacts with nsp5, nsp8, and nsp9 (123). In vitro, full-length nsp12 drives RNA synthesis in a primer-dependent manner on both homo- and heteropolymeric RNA templates (124, 125).…”

Characterization of viral proteins encoded by the SARS-coronavirus genome

“…In addition, the crystal structure of SARS-CoV nsp9, which has no designated function, has been solved and it has been shown to bind RNA as well as another non-structural protein, SARS-CoV nsp8 (Campanacci et al, 2003;Sutton et al, 2004). The SARS-CoV nsp9 may have a similar function as the nsp9 protein of mouse hepatitis virus (MHV), a Group 2 coronavirus, which colocalized and interacted with other proteins of the replication complex (Bost et al, 2000;Brockway et al, 2003). For the remaining non-structural proteins produced from pp1a or pp1ab, putative activities have been predicted based on the presence of functional domains in their sequences or by their structural similarities to other proteins (Gao et al, 2003a;Snijder et al, 2003;von Grotthuss et al, 2003;Fig.…”