Localization of mouse hepatitis virus open reading frame 1A derived proteins

We have investigated the intracellular localization of proteolytic cleavage products encoded in the 5' portion of mouse hepatitis virus (MHV) gene 1, Immunofluorescent labeling of cells with an antiserum which recognizes p28, the ORF1a N-terminal cleavage product, resulted in widespread somewhat gra nular cytoplasmic staining, indicating that this protein is widely distribu ted in the cytoplasm of MIN-infected, but not control uninfected cells. Imm unofluorescent staining of infected cells with antisera which recognize the downstream polypeptides, p65, p240 and p290 labeled discrete vesicular per inuclear structures. Double immunofluorescent labeling of BHK cells express ing the MHV receptor (BHKMHVR1) and infected with MHV-A59 with a Golgi-spec ific anti-mannosidase II monoclonal antibody and with antiserum recognizing each of these anti-MIN ORF1a polypeptides, showed that the p240 and p290 p olypeptides were localized in discrete vesicular structures that overlapped the Golgi complex. Labeling with antibodies specific for p65 colocalized w ith the Golgi region, and showed staining of the perinuclear cytoplasm as w ell. Plasmids containing sequences contained in the first 6.75 kb of ORF1a have been expressed using the coupled vaccinia virus-T7 polymerase system. Immunofluorescent labeling of transfectants with the anti-ORF1a antisera sh owed patterns of antigen distribution similar to those observed in cells in fected with MHV-A59. A deletion analysis with constructs containing only po rtions of the ORF1a sequence indicated that 303 amino acids containing the first papain-like protease domain (PLP-1) was sufficient to associate this protein with the Golgi.