title: Mouse hepatitis virus replicase proteins associate with two distinct populations of intracellular membranes.
abstract: The coronavirus replicase gene (gene 1) is translated into two co-amino-terminal polyproteins that are proteolytically processed to yield more than 15 mature proteins. Several gene 1 proteins have been shown to localize at sites of viral RNA synthesis in the infected cell cytoplasm, notably on late endosomes at early times of infection. However, both immunofluorescence and electron microscopic studies have also detected gene 1 proteins at sites distinct from the putative sites of viral RNA synthesis or virus assembly. In this study, mouse hepatitis virus (MHV)-infected cells were fractionated and analyzed to determine if gene 1 proteins segregated to more than one membrane population. Following differential centrifugation of lysates of MHV-infected DBT cells, gene 1 proteins as well as the structural N and M proteins were detected almost exclusively in a high-speed small membrane pellet. Following fractionation of the small membrane pellet on an iodixanol density gradient, the gene 1 proteins p28 and helicase cofractionated with dense membranes (1.12 to 1.13 g/ml) that also contained peak concentrations of N. In contrast, p65 and p1a-22 were detected in a distinct population of less dense membranes (1.05 to 1.09 g/ml). Viral RNA was detected in membrane fractions containing helicase, p28, and N but not in the fractions containing p65 and p1a-22. LAMP-1, a marker for late endosomes and lysosomes, was detected in both membrane populations. These results demonstrate that multiple gene 1 proteins segregate into two biochemically distinct but tightly associated membrane populations and that only one of these populations appears to be a site for viral RNA synthesis. The results further suggest that p28 is a component of the viral replication complex whereas the gene 1 proteins p1a-22 and p65 may serve roles during infection that are distinct from viral RNA transcription or replication.
authors.affiliation: Department of Microbiology and Immunology, Vanderbilt University, Nashville, Tennessee 37232, USA.
authors.forename: A C
authors.initials: AC
authors.lastname: Sims
authors.forename: J
authors.initials: J
authors.lastname: Ostermann
authors.forename: M R
authors.initials: MR
authors.lastname: Denison
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D002458:Cell Fractionation; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D000086862:Coronavirus M Proteins; D004265:DNA Helicases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000821:virology; D011992:Endosomes / Q000378:metabolism / Q000821:virology; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000378:metabolism / Q000821:virology; D007425:Intracellular Membranes / Q000378:metabolism* / Q000821:virology; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000378:metabolism / Q000821:virology; D008562:Membrane Glycoproteins / Q000032:analysis; D051379:Mice; D018613:Microscopy, Confocal; D008970:Molecular Weight; D006517:Murine hepatitis virus / Q000201:enzymology*; D019251:Nucleocapsid / Q000378:metabolism; D019590:Nucleocapsid Proteins; D012367:RNA, Viral / Q000096:biosynthesis / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D014283:Triiodobenzoic Acids / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D015703:Antigens, CD; D000086862:Coronavirus M Proteins; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D019590:Nucleocapsid Proteins; D012367:RNA, Viral; D014283:Triiodobenzoic Acids; D014763:Viral Matrix Proteins; D014764:Viral Proteins; C087637:gene 1 protein, Coronavirus; C099600:nucleocapsid protein, Hepatitis virus; D012324:RNA-Dependent RNA Polymerase; D004265:DNA Helicases; C044834:iodixanol