The putative helicase of the coronavirus mouse hepatitis virus is processed from the replicase gene polyprotein and localizes in complexes that are active in viral RNA synthesis

The coronavirus mouse hepatitis virus (MHV) translates its replicase gene ( gene 1) into two co-aminoterminal polyproteins, polyprotein la and polyprot ein lab. The gene I polyproteins are processed by viral proteinases to yiel d at least 15 mature products, including a putative RNA helicase from polyp rotein lab that is presumed to be involved in viral RNA synthesis. Antibodi es directed against polypeptides encoded by open reading frame Ib were used to characterize the expression and processing of the MHV helicase and to d efine the relationship of helicase to the viral nucleocapsid protein (N) an d to sites of viral RNA synthesis in MHV-infected cells, The antihelicase a ntibodies detected a 67-kDa protein in MHV-infected cells that was translat ed and processed throughout the virus life cycle. Processing of the 67-kDa helicase from polyprotein lab was abolished by E64d, a known inhibitor of t he MHV 3C-like proteinase, When infected cells were probed for helicase by immunofluorescence laser confocal microscopy, the protein was detected in p atterns that varied from punctate perinuclear complexes to large structures that occupied much of the cell cytoplasm. Dual-labeling studies of infecte d cells for helicase and bromo-UTP-labeled RNA demonstrated that the vast m ajority of helicase-containing complexes were active in viral RNA synthesis . Dual-labeling studies for helicase and the MHV N protein showed that the two proteins almost completely colocalized, indicating that N was associate d with the helicase-containing complexes. This study demonstrates that the putative RNA helicase is closely associated with MHV RNA synthesis and sugg ests that complexes containing helicase, N, and new viral RNA are the viral replication complexes.