Processing of the coronavirus MHV-JHM polymerase polyprotein: identification of precursors and proteolytic products spanning 400 kilodaltons of ORF1a.
The replicase of mouse hepatitis virus strain JHM (MHV-JHM) is encoded by two overlapping open reading frames, ORF1a and ORF1b, which are translated to produce a 750-kDa precursor polyprotein. The polyprotein is proposed to be processed by viral proteinases to generate the functional replicase complex. To date, only the MHV-JHM amino-terminal proteins p28 and p72, which is processed to p65, have been identified. To further elucidate the biogenesis of the MHV-JHM replicase, we cloned and expressed five regions of ORF1a in bacteria and prepared rabbit antisera to each region. Using the immune sera to immunoprecipitate radiolabeled proteins from MHV-JHM infected cells, we determined that the MHV-JHM ORF1a is initially processed to generate p28, p72, p250, and p150. Pulse-chase analysis revealed that these intermediates are further processed to generate p65, p210, p40, p27, the MHV 3C-like proteinase, and p15. A putative replicase complex consisting of p250, p210, p40, p150, and a large protein (> 300 kDa) coprecipitate from infected cells disrupted with NP-40, indicating that these proteins are closely associated even after initial proteolytic processing. Immunofluorescence studies revealed punctate labeling of ORF1a proteins in the perinuclear region of infected cells, consistent with a membrane-association of the replicase complex. Furthermore, in vitro transcription/translation studies of the MHV-JHM 3Cpro and flanking hydrophobic domains confirm that 3C protease activity is significantly enhanced in the presence of canine microsomal membranes. Overall, our results demonstrate that the MHV-JHM ORF1a polyprotein: (1) is processed into more than 10 protein intermediates and products, (2) requires membranes for efficient biogenesis, and (3) is detected in discrete membranous regions in the cytoplasm of infected cells.
Schiller
J J
JJ
Department of Microbiology and Immunology, Loyola University of Chicago, Stritch School of Medicine, Maywood, Illinois 60153, USA.
Kanjanahaluethai
A
A
Baker
S C
SC
D000818:Animals; D000906:Antibodies; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D004285:Dogs; D019084:Fluorescent Antibody Technique, Indirect; D007425:Intracellular Membranes / Q000378:metabolism; D051379:Mice; D008861:Microsomes / Q000378:metabolism; D006517:Murine hepatitis virus / Q000737:chemistry / Q000378:metabolism*; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests / Q000379:methods; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*
D000906:Antibodies; D014764:Viral Proteins; C087637:gene 1 protein, Coronavirus