Four proteins processed from the replicase gene polyprotein of mouse hepatitis virus colocalize in the cell periphery and adjacent to sites of virionassembly

The replicase gene (gene 1) of the coronavirus mouse hepatitis virus (MHV) encodes two co-amino-terminal polyproteins presumed to incorporate all the virus-encoded proteins necessary for viral RNA synthesis. The polyproteins are cotranslationally processed by viral proteinases into at least 15 matur e proteins, including four predicted cleavage products of less than 25 kDa that together would comprise the final 59 kDa of protein translated from op en reading frame la. Monospecific antibodies directed against the four dist inct domains detected proteins of 10, 12, and 15 kDa (p1a-10, p1a-12, and p 1a-15) in MHV-A59-infected DBT cells, in addition to a previously identifie d 22-kDa protein (p1a-22). When infected cells were probed by immunofluores cence laser confocal microscopy, p1a-10, -22, -12, and -15 were detected in discrete foci that were prominent in the perinuclear region but were widel y distributed throughout the cytoplasm as well. Dual-labeling experiments d emonstrated colocalization of the majority of p1a-22 in replication complex es with the helicase, nucleocapsid, and 3C-like proteinase, as well as with p1a-10, -12, and -15. p1a-22 was also detected in separate foci adjacent t o the replication complexes. The majority of complexes containing the gene 1 proteins were distinct from sites of accumulation of the M assembly prote in. However, in perinuclear regions the gene 1 proteins and nucleocapsid we re intercalated with sites of M protein localization. These results demonst rate that the complexes known to be involved in RNA synthesis contain multi ple gene 1 proteins and are closely associated with structural proteins at presumed sites of virion assembly.