The subcellular location of the nonstructural proteins NS1, NS2B, and NS3 in Vero cells infected with the flavivirus Kunjin was investigated using indirect immunofluorescence and cryoimmunoelectron microscopy with monospecific antibodies. Comparisons were also made by dual immunolabelling using antibodies to double-stranded RNA (dsRNA), the putative template in the flavivirus replication complex. At 8 h postinfection, the immunofluorescent patterns showed NS1, NS2B, NS3, and dsRNA located in a perinuclear rim with extensions into the peripheral cytoplasm. By 16 h, at the end of the latent period, all patterns had changed to some discrete perinuclear foci associated with a thick cytoplasmic reticulum. By 24 h, this localization in perinuclear foci was more apparent and some foci were dual labelled with antibodies to dsRNA. In immunogold-labelled cryosections of infected cells at 24 h, all antibodies were associated with clusters of induced membrane structures in the perinuclear region. Two important and novel observations were made. First, one set of induced membranes comprised vesicle packets of smooth membranes dual labelled with anti-dsRNA and anti-NS1 or anti-NS3 antibodies. Second, adjacent masses of paracrystalline arrays or of convoluted smooth membranes, which appeared to be structurally related, were strongly labelled only with anti-NS2B and anti-NS3 antibodies. Paired membranes similar in appearance to the rough endoplasmic reticulum were also labelled, but less strongly, with antibodies to the three nonstructural proteins. Other paired membranes adjacent to the structures discussed above enclosed accumulated virus particles but were not labelled with any of the four antibodies. The collection of induced membranes may represent virus factories in which translation, RNA synthesis, and virus assembly occur.

The subcellular locations in infected Vero cells of Kunjin (KUN) virus core protein C and NS4B were analyzed by immunofluorescence (IF) and by immunoelectron microscopy using monospecific antibodies. Selection of appropriate fixation methods for IF showed that both proteins were associated at all times with perinuclear membranes spreading outward in a reticular pattern and they entered the nucleus late during the latent period. Subsequently NS4B was also dispersed through the nucleoplasm, while C appeared in the nucleolus and the nucleoplasm. These nuclear locations were confirmed by immunogold labeling of cryosections of infected cells at 24 hr postinfection. Labeling of NS4B in cryosections was especially enriched in the perinuclear membranes of the endoplasmic reticulum. When C and NS4B were each expressed separately in stably transformed cell lines, both cytoplasmic and nuclear localization was observed by IF and confirmed by immunoelectron microscopy. Thus the two proteins translocated to the nucleus independently of each other and of other viral proteins. Dual IF with antibodies to double-stranded RNA showed that cytoplasmic locations of C and NS4B were apparently associated in part with the sites of viral RNA synthesis which were resistant to solubilization by Triton X-100.

A BHK cell line persistently expressing a Kunjin (KUN) virus replicon RNA (repBHK, similar to our recently described ME/76Neo BHK cell line [A. A. Khromykh and E. G. Westaway, J. Virol. 71:1497–1505, 1997]) was used for rescue and propagation of KUN viruses defective in the RNA polymerase gene (NS5). A new infectious full-length KUN virus cDNA clone, FLSDX, prepared from our previously described cDNA clone pAKUN (A. A. Khromykh and E. G. Westaway, J. Virol. 68:4580–4588, 1994) and possessing ∼105-fold higher specific infectivity than that of pAKUN, was used for preparation of defective mutants. Deletions of the predicted RNA polymerase motif GDD (producing FLdGDD) and of one of the predicted methyltransferase motifs (S-adenosylmethionine [SAM] binding site, producing FLdSAM) were introduced separately into FLSDX. Transcription and transfection of FLdGDD and FLdSAM RNAs into repBHK cells but not into normal BHK cells resulted in their replication and the recovery of defective viruses able to replicate only in repBHK cells. Reverse transcription-PCR and sequencing analyses showed retention of the introduced deletions in the genomes of the recovered viruses. Retention of these deletions, as well as our inability to recover viruses able to replicate in normal BHK cells after prolonged incubation (for 7 days) of FLdGDD- or FLdSAM-transfected repBHK cells, excluded the possibility that recombination had occurred between the deleted defective NS5 genes present in transfected RNAs and the functional NS5 gene present in the repBHK cells. An RNA with a point mutation in the GDD motif (FLGVD) was also complemented in transfected repBHK cells, and defective virus was recovered by day 3 after transfection. However, in contrast to the results with FLdGDD and FLdSAM RNAs, prolonged (4 days or more) incubation of FLGVD RNA in normal BHK cells allowed recovery of a virus in which theGVD mutation had reverted via a single base change to the wild-type GDD sequence. Overall, these results represent the first demonstration of trans-complementation of defective flavivirus RNAs with deleterious deletions in the flavivirus RNA polymerase gene NS5. The complementation system described here may prove to be useful for the in vivo complementation of deletions and mutations affecting functional domains or the essential secondary structure in any of the other flavivirus nonstructural proteins.

Using West Nile virus strain Kunjin virus (WNV KUN ) as a model system for flavivirus replication, we showed that the virus replication complex (RC) is associated with the dsRNA template located in induced membranes only in the cytoplasm. In this report we established for the first time that the RNA-dependent RNA polymerase NS5 is located in flavivirus-induced membranes, including the site of viral RNA replication. We found no evidence for nuclear localization of the essential RC components NS5 and its dsRNA template for WNV KUN or the closely related WNV strain Sarafend, by immuno-electron microscopy or by immunofluorescence. Metabolic radiolabelling with [32 P]orthophosphate revealed that WNV KUN NS5 was phosphorylated and this was confirmed by Western blotting with antibodies specific for phosphorylated serine and threonine only. These observations of a cytoplasmic location for the WNV polymerase and its phosphorylation state correspond to the characteristics of the hepatitis C virus RNA polymerase NS5B.Using West Nile virus strain Kunjin virus (WNV KUN ) as a model system for flavivirus replication, we observed nuclear localization of core protein (C) and of the nonstructural protein NS4B (Westaway et al., 1997a) but found no evidence of the replication complex (RC) in the nuclei (Khromykh et al., 2001a, b;Westaway et al., 2002). In early studies, the replication site of flavivirus species Japanese encephalitis virus (JEV) and dengue-2 virus (DEN2V) appeared perinuclear by immunofluorescence (IF) (Cardiff et al., 1973;Edward & Takegami, 1993;Ng & Corner, 1989), and the flavivirus double-stranded RNA (dsRNA) template for WNV KUN was shown to be located in the cytoplasm but absent from the nucleus (Ng et al., 1983). Nuclear localization of the RNAdependent RNA polymerase (RdRp) NS5 was shown by IF in yellow fever virus (YFV)-infected Vero cells (Buckley et al., 1992), DEN2V-infected CV-1 cells (Kapoor et al., 1995) and DEN2V-infected Huh-7 cells (Miller et al., 2006). In JEV-infected PS cells, NS3 and NS5 appeared to co-localize with sites of viral RNA synthesis along the inner periphery of the nucleus by IF and immuno-electron microscopy (IEM) (Uchil et al., 2006).NS5 is included in the consensus composition of the RC (NS1, NS3, NS5, NS2A and NS4A) defined for WNV KUN (Mackenzie et al., 1998;Westaway et al., 1997aWestaway et al., , 2002 and is highly conserved (Coia et al., 1988). Recombinant dengue-1 virus (DEN1V) and WNV NS5 species have displayed RdRp activity in vitro (Guyatt et al., 2001;Steffens et al., 1999;Tan et al., 1996). Location of replication sites only in the cytoplasm of WNV KUN -infected cells was established by RdRp assays of heavy membrane fractions (Chu & Westaway, 1992), by IF using antibodies that co-localized dsRNA with specific non-structural proteins (Mackenzie et al., 1998;Westaway et al., 1997a), by showing that bromo-substituted uridine was incorporated in nascent viral RNA during pulse labelling and by cryo-IEM of thin sections of cells (Mackenzie et al., 1998Westaway et al.,...