SUBCELLULAR-LOCALIZATION AND SOME BIOCHEMICAL-PROPERTIES OF THE FLAVIVIRUS KUNJIN NONSTRUCTURAL PROTEINS NS2A AND NS4A

In a previous study on the replication of Kunjin virus using immunoele ctron microscopy (E. G. Westaway, J. M. Mackenzie, M. T. Kenney, M. K. Jones, and A. A. Khromykh, 1997, J. Virol. 71, 6650-6661), NS1 and NS 3 were found associated with double-stranded RNA (dsRNA) within vesicl e packets (VP) in infected Vero cells, suggesting that these induced m embrane structures may be the cytoplasmic sites of RNA replication. NS 2B and NS3 (comprising the virus-encoded protease) were colocalized wi thin distinct paracrystalline (PC) or convoluted membranes (CM), also induced in the cytoplasm, suggesting that these membranes are the site s of proteolytic cleavage. In this study we found by immunofluorescenc e (IF) that the small hydrophobic nonstructural proteins NS2A and NS4A were located in discrete foci in the cytoplasm of infected cells at b oth 16 and 24 h postinfection, partially coincident with dsRNA fool. I n cryosections of infected cells at 24 h, NS2A was located by immunogo ld labeling primarily within VP, associated with labeled dsRNA. NS2A f used to glutathione S-transferase (GST) bound strongly to the 3' untra nslated region of Kunjin RNA and also to the proposed replicase compon ents NS3 and NS5 in cell lysates. NS4A was localized by immunogold lab eling within a majority of the virus-induced membranes, including VP, CM, and PC. GST-NS4A bound weakly to the 3' untranslated region of Kun jin RNA but was bound to NS4A strongly and to most of the other viral nonstructural proteins, including NS3 and NS5. Taken together the resu lts indicate that the flavivirus replication complex includes NS2A and NS4A in the VP in addition to the previously identified NS1 and NS3. (C) 1998 Academic Press.