The same results were obtained with an A30P expression cassette from MVEV (M

The same results were obtained with an A30P expression cassette from MVEV (M. of this genus include JEV, Murray Valley encephalitis virus 5-Bromo Brassinin (MVEV), WNV, and St. Louis encephalitis virus (SLEV). The characteristic feature of these viruses is that they may cause neuroinvasive disease. The flavivirus genome is 11 kb in length and contains a single long open reading frame that encodes three structural (C, prM, and E) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins (9,14,23). Flavivirus NS1 is a multifunctional protein shown to play a role in virus replication and assembly (13,15,18,20), as well as in the modulation of the 5-Bromo Brassinin innate immune response (6,7,24). However, the mechanism(s) of its contribution to viral pathogenesis is not fully understood. Interestingly, NS1 appears in Western blots with anti-NS1 antibodies as at least two heterogeneous clusters of proteins of different molecular masses (NS1 and NS1) (2,3,5,19). It was suggested that the slower migration of NS1 proteins may be due to either different glycosylation patterns or the generation of an alternative cleavage product with the cleavage site likely to reside within the downstream NS2A protein (3,10,15,19). Recently, it was proposedbased on computational analysis of RNA sequence and structure of the members of the Japanese encephalitis virus subgroupthat the NS1 protein is produced as the result of a 1 ribosomal frameshift (11). This analysis revealed the presence of conserved canonical frameshift-stimulating motifsnamely, a slippery heptanucleotide and a 3-adjacent potential pseudoknotnear the beginning of the NS2A gene (1,4,11,22) (Fig.1A). However, experimental evidence demonstrating 1 ribosomal frameshifting for NS1 production was lacking. == FIG. 1. == Disruption of the predicted pseudoknot structure by the alanine-to-proline mutation at position 30 of the NS2A gene abolishes NS1 production. (A) The frameshift motif and pseudoknot structure predicted for WT and A30P KUNV using pknotsRG software (21). The frameshift heptanucleotide and codon 30 of the NS2A gene are underlined, and the proposed interactions between bases in the predicted pseudoknot are shown as dashed lines. Altered nucleotides in codon 30 of the A30P mutant are highlighted. (B) Detection of NS1 and NS1 in lysates of BHK, Vero, A549, and C6/36 cells 18 h after infection with WT or A30P KUNV viruses at an MOI of 5. Infected cells were pulsed with 50 Ci of [35S]methionine in the methionine-free medium for 60 min, and then labeled medium was replaced with medium containing an excess of unlabeled methionine and cells were incubated for an additional 90 min. On completion of pulse-chase labeling, cells were lysed in 1% NP-40 lysis buffer and labeled NS1-containing proteins 5-Bromo Brassinin were immunoprecipitated with the anti-NS1 monoclonal antibody 4G4. Precipitated proteins were separated by SDS-PAGE and visualized on X-ray film. (C) Vero cells were transfected with DNA constructs coding for WT or A30P-mutated NS1-NS2A gene cassettes and incubated for 24 h prior to pulse-chase labeling and immunoprecipitation with 4G4 antibodies as in panel B. We previously described an Ala-to-Pro mutation at amino acid position 30 in the NS2A gene of the Kunjin strain (KUNV) of West Nile virus that allows persistent noncytopathic replication of replicon RNA in several mammalian cell lines (16,17). Introduction of this mutation into a KUNV infectious clone resulted in increased transcription of the beta interferon (IFN-) promoter in response to virus infection, and the mutant virus exhibited significantly reduced neuroinvasiveness in mice (16,17). However, the exact mechanism of how the A30P mutation in NS2A changed the properties of the mutant virus was not clear. Given the location of the mutation within the pseudoknot structure predicted to be involved in 1 ribosomal frameshifting (11), it was reasonable to assume that the mutation could abolish the formation of NS1, which may be at least partially responsible for the attenuated phenotype of the mutant virus. == The A30P IL1-BETA mutation in the NS2A gene abolishes production of NS1. == RNA structure and.