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Abstract: Genome cyclization is essential for flavivirus replication. We used RNases to probe the structures formed by the 5′-terminal 190 nucleotides and the 3′-terminal 111 nucleotides of the West Nile virus (WNV) genomic RNA. When analyzed individually, the two RNAs adopt stem-loop structures as predicted by the thermodynamic-folding program. However, when mixed together, the two RNAs form a duplex that is mediated through base-pairings of two sets of RNA elements (5′CS/3′CSI and 5′UAR/3′UAR). Formation of the RNA duplex facilitates a conformational change that leaves the 3′-terminal nucleotides of the genome (position −8 to −16) to be single-stranded. Viral NS5 binds specifically to the 5′-terminal stem-loop (SL1) of the genomic RNA. The 5′SL1 RNA structure is essential for WNV replication. The study has provided further evidence to suggest that flavivirus genome cyclization and NS5/5′SL1 RNA interaction facilitate NS5 binding to the 3′ end of the genome for the initiation of viral minus-strand RNA synthesis. [Copyright &y& Elsevier]

Abstract: A reporting replicon of West Nile virus (WN) was used to distinguish between the function of the 3′ untranslated region (UTR) in viral translation and RNA replication. Deletions of various regions of the 3′ UTR of the replicon did not significantly affect viral translation, but abolished RNA replication. A systematic mutagenesis showed that the flavivirus-conserved penta-nucleotide (5′-CACAG-3′ located at the top of the 3′ stem-loop of the genome) requires a specific sequence and structure for WN RNA synthesis, but not for viral translation. (i) Basepair structure and sequence at the 1st position of the penta-nucleotide are critical for RNA replication. (ii) The conserved nucleotides at the 2nd, 3rd, and 5th positions, but not at the 4th position of the penta-nucleotide, are essential for RNA synthesis. (iii) The nucleotide U (which is partially conserved in the genus Flavivirus) immediately downstream of the penta-nucleotide is not essential for viral replication. [Copyright &y& Elsevier]

Abstract: The role of the α-helical domain (MH) of dengue virus (DENV) precursor membrane protein in replication was investigated by site-directed mutagenesis. Proline substitutions of three residues (120, 123 and 127) at the C-terminus, but not those at the N-terminus of MH domain, reduced the virus-like particles of DENV1, DENV2 and DENV4 detected in supernatants. In a DENV2 replicon trans-packaging system, these three mutations suppressed particles detected; two of them (I123P and V127P) also affected viral entry. In the context of DENV2 genome-length RNA, all three mutations reduced virion assembly and virus spreading in cell culture. Analysis of revertants showed that mutation A120P could partially support viral infection cycle; in contrast, mutations I123P and V127P were lethal, and adaptations of I123P→I123L and V127P→V127L were required to restore the viral infection cycle. These findings demonstrate that the C-terminus of the MH domain is involved in both assembly and entry of DENV. [ABSTRACT FROM AUTHOR]

Abstract: We report a genetic interplay among three pairs of long-distance RNA interactions that are involved in West Nile virus (WNV) genome cyclization and replication: 5′CS/3′CSI (conserved sequence), 5′UAR/3′UAR (upstream AUG region), and 5′DAR/3′DAR (downstream AUG region). Deletion of the complete 3′CSI element is lethal for WNV replication, but the replication of the 3′CSI deletion virus could be rescued by second site mutations. Functional analysis, using a genome-length RNA and replicon, mapped the compensatory mutations to the 5′UAR/3′UAR and 5′DAR/3′DAR regions. Biochemical analysis showed that the 3′CSI deletion abolished the 5′ and 3′ RNA interaction of the genome; the compensatory mutations could partially restore the 5′ and 3′ genome cyclization. These results demonstrate, for the first time, that a flavivirus without 3′CSI could restore genome cyclization and viral replication through enhancement of the 5′UAR/3′UAR and 5′DAR/3′DAR interactions. [Copyright &y& Elsevier]

Abstract: We report that dengue virus (DENV) methyltransferase sequentially methylates the guanine N-7 and ribose 2′-O positions of viral RNA cap (GpppA→m7GpppA→m7GpppAm). The order of two methylations is determined by the preference of 2′-O methylation for substrate m7GpppA-RNA to GpppA-RNA, and the 2′-O methylation is not absolutely dependent on the prior N-7 methylation. A mutation that completely abolished the 2′-O methylation attenuated DENV replication in cell culture, whereas another mutation that abolished both methylations was lethal for viral replication, suggesting that N-7 methylation is more important than 2′-O methylation in viral replication. The latter mutant with lethal replication could be rescued by trans complementation using a wild-type DENV replicon. Furthermore, we found that chimeric DENVs containing the West Nile virus methyltransferase, polymerase, or full-length NS5 were nonreplicative, but the replication defect could also be rescued through trans complementation using the wild-type DENV replicon. [ABSTRACT FROM AUTHOR]

Abstract: West Nile virus (WNV) is a mosquito-borne pathogen. During replication, WNV acquires different carbohydrates and lipid membranes, depending on its mosquito or vertebrate hosts. Consequently, WNV derived from mosquito and vertebrate cell lines differ in their infectivity for dendritic cells (DCs) and induction of type I interferon (IFN-α/β) in vitro. We evaluated the pathogenesis of WNV derived from mosquito (WNVC6/36) and vertebrate (WNVBHK) cell lines in mice. The tissue tropism, infectivity, clinical disease, and mortality did not differ for mice inoculated with WNVC6/36 or WNVBHK, and there were only minor differences in viral load and serum levels of IFN-α/β. The replication kinetics of WNVC6/36 and WNVBHK were equivalent in primary DCs and skin cells although primary DCs were more susceptible to WNVC6/36 infection than to WNVBHK infection, suggesting that less virus is produced per infected cell for WNVC6/36. In conclusion, viral source has minimal effect on WNV pathogenesis in vivo. [Copyright &y& Elsevier]

Abstract: Lycorine potently inhibits flaviviruses in cell culture. At 1.2-μM concentration, lycorine reduced viral titers of West Nile (WNV), dengue, and yellow fever viruses by 102- to 104-fold. However, the compound did not inhibit an alphavirus (Western equine encephalitis virus) or a rhabdovirus (vesicular stomatitis virus), indicating a selective antiviral spectrum. The compound exerts its antiviral activity mainly through suppression of viral RNA replication. A Val→Met substitution at the 9th amino acid position of the viral 2K peptide (spanning the endoplasmic reticulum membrane between NS4A and NS4B proteins) confers WNV resistance to lycorine, through enhancement of viral RNA replication. Initial chemistry synthesis demonstrated that modifications of the two hydroxyl groups of lycorine can increase the compound''s potency, while reducing its cytotoxicity. Taken together, the results have established lycorine as a flavivirus inhibitor for antiviral development. The lycorine-resistance results demonstrate a direct role of the 2K peptide in flavivirus RNA synthesis. [Copyright &y& Elsevier]

Abstract: West Nile virus methyltransferase catalyzes N7 and 2′-O methylations of the viral RNA cap (GpppA-RNA→m7GpppAm-RNA). The two methylation events are independent, as evidenced by efficient N7 methylation of GpppA-RNA→m7GpppA-RNA and GpppAm-RNA→m7GpppAm-RNA, and by the 2′-O methylation of GpppA-RNA→GpppAm-RNA and m7GpppA-RNA→m7GpppAm-RNA. However, the 2′-O methylation activity prefers substrate m7GpppA-RNA to GpppA-RNA, thereby determining the dominant methylation pathway as GpppA-RNA→m7GpppA-RNA→m7GpppAm-RNA. Mutant enzymes with different methylation defects can trans complement one another in vitro. Furthermore, sequential treatment of GpppA-RNA with distinct methyltransferase mutants generates fully methylated m7GpppAm-RNA, demonstrating that separate molecules of the enzyme can independently catalyze the two cap methylations in vitro. [Copyright &y& Elsevier]

Abstract: An adaptive mutation (E249G) within West Nile virus (WNV) NS4B gene was consistently recovered from replicon RNAs in C3H/He mouse cells. The E249G is located at the C-terminal tail of NS4B predicted to be on the cytoplasmic side of the endoplasmic reticulum membrane. The E249G substitution reduced replicon RNA synthesis. Compared with the wild-type NS4B, the E249G mutant protein exhibited a similar efficiency in evasion of interferon-β response. Recombinant E249G virus exhibited smaller plaques, slower growth kinetics, and lower RNA synthesis than the wild-type virus in a host-dependent manner, with the greatest difference in rodent cells (C3H/He and BHK-21) and the least difference in mosquito cells (C3/36). Selection of revertants of E249G virus identified a second site mutation at residue 246, which could compensate for the low replication phenotype in cell culture. These results demonstrate that distinct residues within the C-terminal tail of flavivirus NS4B are critical for viral replication. [Copyright &y& Elsevier]