Colocalization and membrane association of murine hepatitis virus gene 1 products and de novo-synthesized viral RNA in infected cells

Murine hepatitis virus (MHV) gene 1, the 22-kb polymerase (pol) gene, is fi rst translated into a polyprotein and subsequently processed into multiple proteins by viral autoproteases, Genetic complementation analyses suggest t hat the majority of the gene 1 products are required for viral RNA synthesi s. However, there is no physical evidence supporting the association of any of these products with viral RNA synthesis. We have now performed immunofl uorescent-staining studies with four polyclonal antisera to localize variou s MHV-A59 gene 1 products in virus-infected cells. Immunoprecipitation expe riments showed that these antisera detected proteins representing the two p apain-like proteases and the JC-like protease encoded by open reading frame (ORF) la, the putative polymerase (p100) and a p35 encoded by ORF Ib, and their precursors, De novo-synthesized viral RNA was labeled with bromouridi ne triphosphate in lysolecithin-permeabilized MHV-infected cells. Confocal microscopy revealed that all of the viral proteins detected by these antise ra colocalized with newly synthesized viral RNA in the cytoplasm, particula rly in the perinuclear region of infected cells. Several cysteine and serin e protease inhibitors, i.e,, E64d, leupeptin, and zinc chloride, inhibited viral RNA synthesis without affecting the localization of viral proteins, s uggesting that the processing of the MHV gene 1 polyprotein is tightly asso ciated with viral RNA synthesis. Dual labeling with antibodies specific for cytoplasmic membrane structures showed that MHV gene 1 products and RNA co localized 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 reticulu m, Our results provide clear physical evidence that several MHV gene 1 prod ucts, including the proteases and the polymerase, are associated with the v iral RNA replication-transcription machinery, which may localize to differe nt membrane structures in different cell lines.