Colocalization and Membrane Association of Murine Hepatitis Virus Gene 1 Products and De Novo-Synthesized Viral RNA in Infected Cells

Abstract

Murine hepatitis virus (MHV) gene 1, the 22-kb polymerase (pol) gene, is first translated into a polyprotein and subsequently processed into multiple proteins by viral autoproteases. Genetic complementation analyses suggest that the majority of the gene 1 products are required for viral RNA synthesis. However, there is no physical evidence supporting the association of any of these products with viral RNA synthesis. We have now performed immunofluorescent-staining studies with four polyclonal antisera to localize various MHV-A59 gene 1 products in virus-infected cells. Immunoprecipitation experiments showed that these antisera detected proteins representing the two papain-like proteases and the 3C-like protease encoded by open reading frame (ORF) 1a, the putative polymerase (p100) and a p35 encoded by ORF 1b, and their precursors. De novo-synthesized viral RNA was labeled with bromouridine triphosphate in lysolecithin-permeabilized MHV-infected cells. Confocal microscopy revealed that all of the viral proteins detected by these antisera colocalized with newly synthesized viral RNA in the cytoplasm, particularly in the perinuclear region of infected cells. Several cysteine and serine protease inhibitors, i.e., E64d, leupeptin, and zinc chloride, inhibited viral RNA synthesis without affecting the localization of viral proteins, suggesting that the processing of the MHV gene 1 polyprotein is tightly associated with viral RNA synthesis. Dual labeling with antibodies specific for cytoplasmic membrane structures showed that MHV gene 1 products and RNA colocalized with the Golgi apparatus in HeLa cells. However, in murine 17CL-1 cells, the viral proteins and viral RNA did not colocalize with the Golgi apparatus but, instead, partially colocalized with the endoplasmic reticulum. Our results provide clear physical evidence that several MHV gene 1 products, including the proteases and the polymerase, are associated with the viral RNA replication-transcription machinery, which may localize to different membrane structures in different cell lines.

Comparative in vivo analysis of the nsp15 endoribonuclease of murine, porcine and severe acute respiratory syndrome coronaviruses

“…1). This observation is consistent with its putative role in viral replication and suggests that nsp15 is likely associated with other viral nsps and viral RNA in the replication/transcription complexes found in the perinuclear region during viral infection (Bost et al, 2001;Shi et al, 1999). However, we also found a substantial amount of nsp15 present outside the putative viral replication/transcription complexes and in some cases throughout the cytoplasm (Fig.…”

Biogenesis and architecture of arterivirus replication organelles

“…Interestingly, this in vitro RTC activity was lost after detergent treatment, strongly suggesting that the RTC's functionality depends on the integrity of the membranes (van Hemert et al, 2008a). Similar data has also been published for coronaviruses (Bost et al, 2000;Brockway et al, 2003;Denison et al, 1999;Harcourt et al, 2004;Prentice et al, 2004b;Shi et al, 1999;Snijder et al, 2006;van der Meer et al, 1999;van Hemert et al, 2008b).…”

Bioinformatics and functional analyses of coronavirus nonstructural proteins involved in the formation of replicative organelles

“…Membrane-bound replicative organelles are a widespread but not universal intracellular feature associated with positive-strand RNA virus replication (Neuman et al, 2014a). Coronaviruses form a double-membrane organelle (DMO) derived from the endoplasmic reticulum (ER) that contains viral RNA and replicase proteins (Deming et al, 2007;Hagemeijer et al, 2010;Oostra et al, 2007;Shi et al, 1999;van der Meer et al, 1999). In the DMO, two lipid bilayers are held at a constant distance of about 20 nm (Angelini et al, 2013).…”