ORF1a-Encoded Replicase Subunits Are Involved in the Membrane Association of the Arterivirus Replication Complex
| Autor: | Eric J. Snijder, Yvonne van der Meer, Hans van Tol, Jacomine Krijnse Locker |
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| Rok vydání: | 1998 |
| Předmět: |
Proteases
Polyproteins Octoxynol Immunology RNA-dependent RNA polymerase Biology Endoplasmic Reticulum Virus Replication Microbiology Cell Line Polyethylene Glycols Arterivirus Open Reading Frames chemistry.chemical_compound Equartevirus Cricetinae Virology RNA polymerase Chlorocebus aethiops Animals Vero Cells Integral membrane protein Cell Nucleus Endoplasmic reticulum Cell Membrane RNA-Dependent RNA Polymerase biology.organism_classification Molecular biology RNA Helicase A Virus-Cell Interactions Cell biology chemistry Insect Science RNA Viral Rabbits |
| Zdroj: | Journal of Virology. 72:6689-6698 |
| ISSN: | 1098-5514 0022-538X |
| DOI: | 10.1128/jvi.72.8.6689-6698.1998 |
| Popis: | Among the functions of the replicase of equine arteritis virus (EAV; family Arteriviridae , order Nidovirales ) are important viral enzyme activities such as proteases and the putative RNA polymerase and RNA helicase functions. The replicase is expressed in the form of two polyproteins (open reading frame 1a [ORF1a] and ORF1ab), which are processed into 12 nonstructural proteins by three viral proteases. In immunofluorescence assays, the majority of these cleavage products localized to the perinuclear region of the cell. A dense granular and vesicular staining was observed, which strongly suggested membrane association. By using confocal microscopy and double-label immunofluorescence, the distribution of the EAV replicase was shown to overlap with that of PDI, a resident protein of the endoplasmic reticulum and intermediate compartment. An in situ labeling of nascent viral RNA with bromo-UTP demonstrated that the membrane-bound complex in which the replicase subunits accumulate is indeed the site of viral RNA synthesis. A number of ORF1a-encoded hydrophobic domains were postulated to be involved in the membrane association of the arterivirus replication complex. By using various biochemical methods (Triton X-114 extraction, membrane purification, and sodium carbonate treatment), replicase subunits containing these domains were shown to behave as integral membrane proteins and to be membrane associated in infected cells. Thus, contribution to the formation of a membrane-bound scaffold for the viral replication-transcription complex appears to be an important novel function for the arterivirus ORF1a replicase polyprotein. |
| Databáze: | OpenAIRE |
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