RNase L Antiviral Activity Is Not a Critical Component of the Oas1b-Mediated Flavivirus Resistance Phenotype.

In mice, resistance to central nervous system (CNS) disease induced by members of the genus Flavivirus is conferred by an allele of the 2'-5' oligoadenylate synthetase 1b gene that encodes the inactive full-length protein (Oas1b-FL). The susceptibility allele encodes a C-terminally truncated protein (Oas1b-tr). We show that the efficiency of neuron infection in the brains of resistant and susceptible mice is similar after an intracranial inoculation of two flaviviruses, but amplification of viral proteins and double-stranded RNA (dsRNA) is inhibited in infected neurons in resistant mouse brains at later times. Active OAS proteins detect cytoplasmic dsRNA and synthesize short 2'-5'-linked oligoadenylates (2'-5'A) that interact with the latent endonuclease RNase L, causing it to dimerize and cleave single-stranded RNAs. To evaluate the contribution of RNase L to the resistance phenotype in vivo, we created a line of resistant RNase L-/- mice. Evidence of RNase L activation in infected RNase L+/+ mice was indicated by higher levels of viral RNA in the brains of infected RNase L-/- mice. Activation of type I interferon (IFN) signaling was detected in both resistant and susceptible brains, but Oas1a and Oas1b mRNA levels were lower in RNase L+/+ mice of both types, suggesting that activated RNase L also has a proflaviviral effect. Inhibition of virus replication was robust in resistant RNase L-/- mice, indicating that activated RNase L is not a critical factor in mediating this phenotype.IMPORTANCE The mouse genome encodes a family of Oas proteins that synthesize 2'-5'A in response to dsRNA. 2'-5'A activates the endonuclease RNase L to cleave single-stranded viral and cellular RNAs. The inactive, full-length Oas1b protein confers flavivirus-specific disease resistance. Although similar numbers of neurons were infected in resistant and susceptible brains after an intracranial virus infection, viral components amplified only in susceptible brains at later times. A line of resistant RNase L-/- mice was used to evaluate the contribution of RNase L to the resistance phenotype in vivo Activation of RNase L antiviral activity by flavivirus infection was indicated by increased viral RNA levels in the brains of RNase L-/- mice. Oas1a and Oas1b mRNA levels were higher in infected RNase L-/- mice, indicating that activated RNase L also have a proflaviviral affect. However, the resistance phenotype was equally robust in RNase L-/- and RNase L+/+ mice.

Increased early RNA replication by chimeric West Nile virus W956IC leads to IPS-1-mediated activation of NF-κB and insufficient virus-mediated counteraction of the resulting canonical type I interferon signaling.

Although infections with "natural" West Nile virus (WNV) and the chimeric W956IC WNV infectious clone virus produce comparable peak virus yields in type I interferon (IFN) response-deficient BHK cells, W956IC infection produces higher levels of "unprotected" viral RNA at early times after infection. Analysis of infections with these two viruses in IFN-competent cells showed that W956IC activated NF-κB, induced higher levels of IFN-ß, and produced lower virus yields than WNV strain Eg101. IPS-1 was required for both increased induction of IFN-ß and decreased yields of W956IC. In Eg101-infected cells, phospho-STAT1/STAT2 nuclear translocation was blocked at all times analyzed, while some phospho-STAT1/STAT2 nuclear translocation was still detected at 8 h after infection in W956IC-infected mouse embryonic fibroblasts (MEFs), and early viral protein levels were lower in these cells. A set of additional chimeras was made by replacing various W956IC gene regions with the Eg101 equivalents. As reported previously, for three of these chimeras, the low early RNA phenotype of Eg101 was restored in BHK cells. Analysis of infections with two of these chimeric viruses in MEFs detected lower early viral RNA levels, higher early viral protein levels, lower early IFN-ß levels, and higher virus yields similar to those seen after Eg101 infection. The data suggest that replicase protein interactions directly or indirectly regulate genome switching between replication and translation at early times in favor of translation to minimize NF-κB activation and IFN induction by decreasing the amount of unprotected viral RNA, to produce sufficient viral protein to block canonical type I IFN signaling, and to efficiently remodel cell membranes for exponential genome amplification.

Identification of novel host cell binding partners of Oas1b, the protein conferring resistance to flavivirus-induced disease in mice.

Oas1b was previously identified as the product of the Flv(r) allele that confers flavivirus-specific resistance to virus-induced disease in mice by an uncharacterized, RNase L-independent mechanism. To gain insights about the mechanism by which Oas1b specifically reduces the efficiency of flavivirus replication, cellular protein interaction partners were identified and their involvement in the Oas1b-mediated flavivirus resistance mechanism was analyzed. Initial difficulties in getting the two-hybrid assay to work with full-length Oas1b led to the discovery that this Oas protein uniquely has a C-terminal transmembrane domain that targets it to the endoplasmic reticulum (ER). Two peptides matching to oxysterol binding protein-related protein 1L (ORP1L) and ATP binding cassette protein 3, subfamily F (ABCF3), were identified as Oas1b interaction partners in yeast two-hybrid assays, and both in vitro-transcribed/translated peptides and full-length proteins in mammalian cell lysates coimmunoprecipitated with Oas1b. Knockdown of a partner involved in Oas1b-mediated antiflavivirus activity would be expected to increase flavivirus replication but not that of other types of viruses. However, RNA interference (RNAi) knockdown of ORP1L decreased the replication of the flavivirus West Nile virus (WNV) as well as that of other types of RNA viruses. This virus-nonspecific effect may be due to the recently reported dysregulation of late endosome movement by ORP1L knockdown. Knockdown of ABCF3 protein levels increased the replication of WNV but not that of other types of RNA viruses, and this effect on WNV replication was observed only in Oas1b-expressing cells. The results suggest that Oas1b is part of a complex located in the ER and that ABCF3 is a component of the Flv(r)-mediated resistance mechanism.

West nile virus infections suppress early viral RNA synthesis and avoid inducing the cell stress granule response.

West Nile virus (WNV) recently became endemic in the United States and is a significant cause of human morbidity and mortality. Natural WNV strain infections do not induce stress granules (SGs), while W956IC (a lineage 2/1 chimeric WNV infectious clone) virus infections produce high levels of early viral RNA and efficiently induce SGs through protein kinase R (PKR) activation. Additional WNV chimeric viruses made by replacing one or more W956IC genes with the lineage 1 Eg101 equivalent in the W956IC backbone were analyzed. The Eg-NS4b+5, Eg-NS1+3+4a, and Eg-NS1+4b+5 chimeras produced low levels of viral RNA at early times of infection and inefficiently induced SGs, suggesting the possibility that interactions between viral nonstructural proteins and/or between viral nonstructural proteins and cell proteins are involved in suppressing early viral RNA synthesis and membrane remodeling during natural WNV strain infections. Detection of exposed viral double-stranded RNA (dsRNA) in W956IC-infected cells suggested that the enhanced early viral RNA synthesis surpassed the available virus-induced membrane protection and allowed viral dsRNA to activate PKR.

West Nile virus infection does not induce PKR activation in rodent cells.

dsRNA-activated protein kinase (PKR) is activated by viral dsRNAs and phosphorylates eIF2a reducing translation of host and viral mRNA. Although infection with a chimeric West Nile virus (WNV) efficiently induced PKR and eIF2a phosphorylation, infections with natural lineage 1 or 2 strains did not. Investigation of the mechanism of suppression showed that among the cellular PKR inhibitor proteins tested, only Nck, known to interact with inactive PKR, colocalized and co-immunoprecipitated with PKR in WNV-infected cells and PKR phosphorylation did not increase in infected Nck1,2-/- cells. Several WNV stem-loop RNAs efficiently activated PKR in vitro but not in infected cells. WNV infection did not interfere with intracellular PKR activation by poly(I:C) and similar virus yields were produced by control and PKR-/- cells. The results indicate that PKR phosphorylation is not actively suppressed in WNV-infected cells but that PKR is not activated by the viral dsRNA in infected cells.

The Flvr-encoded murine oligoadenylate synthetase 1b (Oas1b) suppresses 2-5A synthesis in intact cells.

Resistance to flavivirus-induced disease in mice is conferred by the autosomal gene Flv, identified as 2'-5' oligoadenylate synthetase 1b (Oas1b). Resistant mice express a full-length Oas1b protein while susceptible mice express the truncated Oas1btr. In this study, Oas1b was shown to be an inactive synthetase. Although the Oas/RNase L pathway was previously shown to have an antiviral role during flavivirus infections, Oas1b protein inhibited Oas1a in vitro synthetase activity in a dose-dependent manner and reduced 2-5A production in vivo in response to poly(I:C). These findings suggest that negative regulation of 2-5A by inactive Oas1 proteins may fine tune the RNase L response that if not tightly controlled could cause significant damage in cells. The results also indicate that flavivirus resistance conferred by Oas1b is not mediated by 2-5A. Instead, Oas1b inhibits flavivirus replication by an alternative mechanism that overrides the proviral effect of reducing 2-5A accumulation and RNase L activation.

Structure of equine 2'-5'oligoadenylate synthetase (OAS) gene family and FISH mapping of OAS genes to ECA8p15-->p14 and BTA17q24-->q25.

Mammalian 2'-5' oligoadenylate (2-5A) synthetases are important mediators of the antiviral activity of interferons. Both human and mouse 2-5A synthetase gene families encode four forms of enzymes: small, medium, large and ubiquitin-like. In this study, the structures of four equine OAS genes were determined using DNA sequences derived from fifteen cDNA and four BAC clones. Composition of the equine OAS gene family is more similar to that of the human OAS family than the mouse Oas family. Two OAS-containing bovine BAC clones were identified in GenBank. Both equine and bovine BAC clones were physically assigned by FISH to horse and cattle chromosomes, ECA8p15-->p14 and BTA17q24--> q25, respectively. The comparative mapping data confirm conservation of synteny between ungulates, humans and rodents.

Cell proteins TIA-1 and TIAR interact with the 3' stem-loop of the West Nile virus complementary minus-strand RNA and facilitate virus replication.

It was reported previously that four baby hamster kidney (BHK) proteins with molecular masses of 108, 60, 50, and 42 kDa bind specifically to the 3'-terminal stem-loop of the West Nile virus minus-stand RNA [WNV 3'(-) SL RNA] (P. Y. Shi, W. Li, and M. A. Brinton, J. Virol. 70:6278-6287, 1996). In this study, p42 was purified using an RNA affinity column and identified as TIAR by peptide sequencing. A 42-kDa UV-cross-linked viral RNA-cell protein complex formed in BHK cytoplasmic extracts incubated with the WNV 3'(-) SL RNA was immunoprecipitated by anti-TIAR antibody. Both TIAR and the closely related protein TIA-1 are members of the RNA recognition motif (RRM) family of RNA binding proteins. TIA-1 also binds to the WNV 3'(-) SL RNA. The specificity of these viral RNA-cell protein interactions was demonstrated using recombinant proteins in competition gel mobility shift assays. The binding site for the WNV 3'(-) SL RNA was mapped to RRM2 on both TIAR and TIA-1. However, the dissociation constant (K(d)) for the interaction between TIAR RRM2 and the WNV 3'(-) SL RNA was 1.5 x 10(-8), while that for TIA-1 RRM2 was 1.12 x 10(-7). WNV growth was less efficient in murine TIAR knockout cell lines than in control cells. This effect was not observed for two other types of RNA viruses or two types of DNA viruses. Reconstitution of the TIAR knockout cells with TIAR increased the efficiency of WNV growth, but neither the level of TIAR nor WNV replication was as high as in control cells. These data suggest a functional role for TIAR and possibly also for TIA-1 during WNV replication.

The 3' stem loop of the West Nile virus genomic RNA can suppress translation of chimeric mRNAs.

Cis-acting elements that regulate translation have been identified in the 3' noncoding regions (NCRs) of cellular and viral mRNAs. As one means of analyzing the effect on translation of the conserved 3' terminal RNA structure of the West Nile virus (WNV) genome, the translation efficiencies of chimeric mRNAs composed of a CAT reporter gene flanked by viral or nonviral 5' and 3' terminal sequences were compared. In vitro, the WNV 3'(+) stem loop (SL) RNA reduced the translation efficiencies of chimeric mRNAs with either viral or nonviral 5' NCRs, suggesting that a specific 3'-5' RNA-RNA interaction was not involved. In contrast, the 3' terminal sequence of a togavirus, rubella virus, enhanced translation efficiency. The WNV 3'(+)SL reduced translation efficiency both in cis and in trans and of both capped and uncapped chimeric mRNAs. We have previously reported that three cellular proteins bind specifically to the WNV 3'(+)SL RNA. Competition between the WNV 3'(+)SL and the 5' terminus of the chimeric mRNAs for proteins involved in translation initiation could explain the translation inhibition observed.

An infectious clone of the West Nile flavivirus.

West Nile (WN) virus is the most widespread among flaviviruses, but until recently it was not known on the American continent. We describe here design of a subgenomic replicon, as well as a full-length infectious clone of the lineage II WN strain, which appeared surprisingly stable compared to other flavivirus infectious clones. This infectious clone was used to investigate effects of 5'- and 3'-nonrelated sequences on virus replication and infectivity of synthetic RNA. While a long nonrelated sequence at the 3'-end delayed but did not prevent establishment of the productive infectious cycle, a much shorter extra sequence at the 5'-end completely abrogated virus replication. Replacement of the conserved 5'-adenosine residue substantially delayed, but did not prevent, establishment of virus infection. In all cases, the recovered virus had restored its authentic 5'- and 3'-end genome sequences. However, the presence of extensive nonrelated sequences at both 5'- and 3'-ends could not be repaired.

Host factors involved in West Nile virus replication.

Viruses use cell proteins during many stages of their replication cycles, including attachment, entry, translation, transcription/replication, and assembly. Mutations in the cell proteins involved can cause disruptions of these critical host-virus interactions, which in turn can affect the efficiency of virus replication. These host-virus interactions also represent novel targets for the development of new antiviral agents. The different alleles of the murine Flv gene confer resistance or susceptibility to flavivirus-induced disease and provide a natural mutant system for the study of a host protein that can alter the outcome of a flavivirus infection. Since flaviviruses, such as West Nile virus, replicate in mosquitoes, mammals, and birds during their natural transmission cycles, it is expected that the critical cell proteins used by these viruses will be ones that are highly conserved between divergent host species. Our laboratory has focused on the identification and characterization of the flavivirus resistance gene product and of cell proteins that interact with the 3' terminal regions of the West Nile virus genomic and antigenomic RNAs. The 3' terminal regions of the viral RNAs function as promotors for viral RNA replication. Cell proteins that bind to the viral 3' RNAs were detected by gel shift and UV-induced cross-linking assays. Individual proteins were then purified and partially sequenced. Mutation of a mapped, protein-binding site within the 3' terminal region of the viral RNA in an infectious West Nile virus clone was used to demonstrate the functional importance of one of the cell proteins for efficient West Nile virus replication. Data from additional studies suggested possible roles for this viral RNA-cell protein interaction during the flavivirus replication cycle.

Cell proteins bind to a 67 nucleotide sequence within the 3' noncoding region (NCR) of simian hemorrhagic fever virus (SHFV) negative-strand RNA.

The 3'NCR of the SHFV negative-strand RNA [SHFV 3'(-)NCR RNA] is thought to be the initiation site of full-length and possibly also subgenomic positive-strand RNA and so is likely to contain cis-acting signals for viral RNA replication. Cellular and viral proteins may specifically interact with this region to form replication complexes. When in vitro transcribed SHFV 3'(-)NCR RNA was used as a probe in gel mobility shift assays, two RNA-protein complexes were detected with MA104 S100 cytoplasmic extracts. The specificity of thes RNA-protein interactions was demonstrated by competition gel mobility shift assays. Four MA104 protein (103, 86, 55, and 36 kDa) were detected by UV-induced cross-linking assays and three proteins (103, 55, and 36 kDa) were detected by northwestern blotting assays. The binding sites for these proteins were mapped to the region between nucleotides 117 to 184 on the SHFV 3'(-)NCR RNA. Four cellular proteins with identical molecular masses to those of the proteins that bind to the SHFV 3'(-)NCR RNA were detected by the 3'(-)NCR of another arterivirus, LDV-C, suggesting that divergent arteriviruses utilize the same set of conserved cell protein domains.

Immune mediated and inherited defences against flaviviruses.

BACKGROUND: Flavivirus infection elicits an abundant immune response in the host which is directed against a number of the viral proteins. Resistance to flavivirus-induced disease can also be controlled via a non-immune mechanism involving the product of a naturally occurring murine gene, Flv. OBJECTIVES: To review studies that have reported the mapping of epitopes on flavivirus proteins that elicit T- or B-cell immune responses in mice or humans and to discuss a possible mechanism for flavivirus-specific genetic resistance. STUDY DESIGN: Purified viral proteins and synthetic peptides were used to map B-cell epitopes. Purified proteins, vaccinia-expressed viral protein fragments and synthetic peptides were used to map T-cell epitopes. Congenic-resistant, C3H/RV and congenic susceptible, C3H/He mice and cell cultures were used to study the mechanism of genetic resistance to flavivirus infection. RESULTS: T- and B-cell epitopes have been mapped to the E, NS1 and NS3 proteins of several flaviviruses. Immune responses to the C, PreM, NS2a, NS4a, and NS5 proteins have also been documented. Data suggest that the Flv gene product acts intracellularly to suppress the synthesis of viral genomic RNA. CONCLUSIONS: Although flavivirus infection elicits an abundant immune response, this response is not always rapid enough to protect the host from developing encephalitis. During secondary infections both the humoral and cellular flavivirus-specific responses can confer protection. Dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) appear to be caused by an overly vigorous immune response. In genetically resistant animals reduced production of virus results in a slower spread of the infection, which in turn allows time for the immune response to develop and to clear the infection before disease symptoms appear.

A 68-nucleotide sequence within the 3' noncoding region of simian hemorrhagic fever virus negative-strand RNA binds to four MA104 cell proteins.

The 3' noncoding region (NCR) of the negative-strand RNA [3'(-)NCR RNA] of the arterivirus simian hemorrhagic fever virus (SHFV) is 209 nucleotides (nt) in length. Since this 3' region, designated 3'(-)209, is the site of initiation of full-length positive-strand RNA and is the template for the synthesis of the 5' leader sequence, which is found on both full-length and subgenomic mRNAs, it is likely to contain cis-acting signals for RNA synthesis and to interact with cellular and viral proteins to form replication complexes. Gel mobility shift assays showed that cellular proteins in MA104 S100 cytoplasmic extracts formed two complexes with the SHFV 3'(-)209 RNA, and results from competition gel mobility shift assays demonstrated that these interactions were specific. Four proteins with molecular masses of 103, 86, 55, and 36 kDa were detected in UV-induced cross-linking assays, and three of these proteins (103, 55, and 36 kDa) were also detected by Northwestern blotting assays. Identical gel mobility shift and UV-induced cross-linking patterns were obtained with uninfected and SHFV-infected extracts, indicating that the four proteins detected are cellular, not viral, proteins. The binding sites for the four cellular proteins were mapped to the region between nt 117 and 184 (68-nt sequence) from the 3' end of the SHFV negative-strand RNA. This 68-nt sequence was predicted to form two stem-loops, SL4 and SL5. The 3'(-)NCR RNA of another arterivirus, lactate dehydrogenase-elevating virus C (LDV-C), competed with the SHFV 3'(-)209 RNA in competition gel mobility shift assays. UV-induced cross-linking assays showed that four MA104 cellular proteins with the same molecular masses as those that bind to the SHFV 3'(-)209 RNA also bind to the LDV-C 3'(-)NCR RNA and equine arteritis virus 3'(-)NCR RNA. However, each of these viral RNAs also bound to an additional MA104 protein. The binding sites for the MA104 cellular proteins were shown to be located in similar positions in the LDV-C 3'(-)NCR and SHFV 3'(-)209 RNAs. These data suggest that the binding sites for a set of the cellular proteins are conserved in all arterivirus RNAs and that these cell proteins may be utilized as components of viral replication complexes.

Definition of an epitope on NS3 recognized by human CD4+ cytotoxic T lymphocyte clones cross-reactive for dengue virus types 2, 3, and 4.

The role of dengue virus-specific serotype-cross-reactive T lymphocytes in recovery from and pathogenesis of dengue virus infections is not known. In the present paper, we have defined a dengue serotype-cross-reactive epitope recognized by two CD4+ CD8- cytotoxic T lymphocyte (CTL) clones, JK36 and JK46. These T cell clones were established from the peripheral blood T lymphocytes of a dengue-3-immune donor, using a limiting dilution method. JK36 and JK46 were cross-reactive for dengue virus types 2, 3, and 4, but not for type 1, and recognized the NS3 protein. The smallest synthetic peptide recognized by JK36 was an 8-amino acid peptide that contains amino acids (aa) 226 to 233 (VVAAEMEE) of NS3. The smallest peptide recognized by JK46 was an 11-amino acid peptide that contains aa 224 to 234 (TRVVAAEMEEA). HLA-DR15 was the restriction allele for recognition of these peptides by both JK36 and JK46. This is the first epitope to be defined that is recognized by human CD4+ CTL cross-reactive for dengue virus types 2, 3, and 4.

Translation elongation factor-1 alpha interacts with the 3' stem-loop region of West Nile virus genomic RNA.

The conserved 3'-terminal stem-loop (3' SL) of the West Nile virus (WNV) genomic RNA was previously used to probe for cellular proteins that may be involved in flavivirus replication and three cellular proteins were detected that specifically interact with the WNV 3' SL RNA (J. L. Blackwell and M. A. Brinton, J. Virol. 69:5650-5658, 1995). In this study, one of these cellular proteins was purified to apparent homogeneity by ammonium sulfate precipitation and liquid chromatography. Amino acid sequence Western blotting, and supershift analyses identified the cellular protein as translation elongation factor-1 alpha (EF-1 alpha). Competition gel mobility shift assays demonstrated that the interaction between EF-1 alpha and WNV 3' SL RNA was specific. Dephosphorylation of EF-1 alpha by calf intestinal alkaline phosphatase inhibited its binding to WNV 3' SL RNA. The apparent equilibrium dissociation constant for the interaction between EF-1 alpha and WNV 3' SL RNA was calculated to be 1.1 x 10(-9) M. Calculation of the stoichiometry of the interaction indicated that one molecule of EF-1 alpha binds to each molecule of WNV 3' SL RNA. Using RNase footprinting and nitrocellulose filter binding assays, we detected a high-activity binding site on the main stem of the WNV 3' SL RNA. Interaction with EF-1 alpha at the high-activity binding site was sequence specific, since nucleotide substitution in this region reduced the binding activity of the WNV 3' SL RNA for EF-1 alpha by approximately 60%. Two low-activity binding sites were also detected, and each accounted for approximately 15 to 20% of the binding activity. Intracellular association between the host protein and the viral RNA was suggested by coimmunoprecipitation of WNV genomic RNA and EF-1 alpha, using an anti-EF-1 alpha antibody.

Dominant recognition by human CD8+ cytotoxic T lymphocytes of dengue virus nonstructural proteins NS3 and NS1.2a.

A severe complication of dengue virus infection, dengue hemorrhagic fever (DHF), is hypothesized to be immunologically mediated and virus-specific cytotoxic T lymphocytes (CTLs) may trigger DHF. It is also likely that dengue virus-specific CTLs are important for recovery from dengue virus infections. There is little available information on the human CD8+ T cell responses to dengue viruses. Memory CD8+CTL responses were analyzed to determine the diversity of the T cell response to dengue virus and to identify immunodominant proteins using PBMC from eight healthy adult volunteers who had received monovalent, live-attenuated candidate vaccines of the four dengue serotypes. All the donors had specific T cell proliferation to dengue and to other flaviviruses that we tested. CTLs were generated from the stimulated PBMC of all donors, and in the seven donors tested, dengue virus-specific CD8+CTL activity was demonstrated. The nonstructural (NS3 and NS1.2a) and envelope (E) proteins were recognized by CD8+CTLs from six, five, and three donors, respectively. All donors recognized either NS3 or NS1.2a. In one donor who received a dengue 4 vaccine, CTL killing was seen in bulk culture against the premembrane protein (prM). This is the first demonstration of a CTL response against the prM protein. The CTL responses using the PBMC of two donors were serotype specific, whereas all other donors had serotype-cross-reactive responses. For one donor, CTLs specific for E, NS1.2a, and NS3 proteins were all HLA-B44 restricted. For three other donors tested, the potential restricting alleles for recognition of NS3 were B38, A24, and/or B62 and B35. These results indicate that the CD8+CTL responses of humans after immunization with one serotype of dengue virus are diverse and directed against a variety of proteins. The NS3 and NS1.2a proteins should be considered when designing subunit vaccines for dengue.

Cell proteins bind specifically to West Nile virus minus-strand 3' stem-loop RNA.

The first 96 nucleotides of the 5'noncoding region (NCR) of West Nile virus (WNV) genomic RNA were previously reported to form thermodynamically predicted stem-loop (SL) structures that are conserved among flaviviruses. The complementary minus-strand 3' NCR RNA, which is thought to function as a promoter for the synthesis of plus-strand RNA, forms a corresponding predicted SL structure. RNase probing of the WNV 3' minus-strand stem-loop RNA [WNV (-)3' SL RNA] confirmed the existence of a terminal secondary structure. RNA-protein binding studies were performed with BHK S100 cytoplasmic extracts and in vitro-synthesized WNV (-)3' SL RNA as the probe. Three RNA-protein complexes (complexes 1,2, and 3) were detected by a gel mobility shift assay, and the specificity of the RNA-protein interactions was confirmed by gel mobility shift and UV-induced cross-linking competition assays. Four BHK cell proteins with molecular masses of 108, 60, 50, and 42 kDa were detected by UV-induced cross-linking to the WNV (-)3' SL RNA. A preliminary mapping study indicated that all four proteins bound to the first 75 nucleotides of the WNV 3' minus-strand RNA, the region that contains the terminal SL. A flavivirus resistance phenotype was previously shown to be inherited in mice as a single, autosomal dominant allele. The efficiencies of infection of resistant cells and susceptible cells are similar, but resistant cells (C3H/RV) produce less genomic RNA than congenic, susceptible cells (C3H/He). Three RNA-protein complexes and four UV-induced cross-linked cell proteins with mobilities identical to those detected in BHK cell extracts with the WNV (-)3' SL RNA were found in both C3H/RV and C3H/He cell extracts. However, the half-life of the C3H/RV complex 1 was three times longer than that of the C3H/He complex 1. It is possible that the increased binding activity of one of the resistant cell proteins for the flavivirus minus-strand RNA could result in a reduced synthesis of plus-strand RNA as observed with the flavivirus resistance phenotype.

Identification of amino acids involved in recognition by dengue virus NS3-specific, HLA-DR15-restricted cytotoxic CD4+ T-cell clones.

The majority of T-cell clones derived from a donor who experienced dengue illness following receipt of a live experimental dengue virus type 3 (DEN3) vaccine cross-reacted with all four serotypes of dengue virus, but some were serotype specific or only partially cross-reactive. The nonstructural protein, NS3, was immuno-dominant in the CD4+ T-cell response of this donor. The epitopes of four NS3-specific T-cell clones were analyzed. JK15 and JK13 recognized only DEN3 NS3, while JK44 recognized DEN1, DEN2, and DEN3 NS3 and JK5 recognized DEN1, DEN3, and West Nile virus NS3. The epitopes recognized by these clones on the DEN3 NS3 protein were localized with recombinant vaccinia viruses expressing truncated regions of the NS3 gene, and then the minimal recognition sequence was mapped with synthetic peptides. Amino acids critical for T-cell recognition were assessed by using peptides with amino acid substitutions. One of the serotype-specific clones (JK13) and the subcomplex- and flavivirus-cross-reactive clone (JK5) recognized the same core epitope, WITDFVGKTVW. The amino acid at the sixth position of this epitope is critical for recognition by both clones. Sequence analysis of the T-cell receptors of these two clones showed that they utilize different VP chains. The core epitopes for the four HLA-DR15-restricted CD4+ CTL clones studied do not contain motifs similar to those proposed by previous studies on endogenous peptides eluted from HLA-DR15 molecules. However, the majority of these dengue virus NS3 core epitopes have a positive amino acid (K or R) at position 8 or 9. Our results indicate that a single epitope can induce T cells with different virus specificities despite the restriction of these T cells by the same HLA-DR15 allele. This finding suggests a previously unappreciated level of complexity for interactions between human T-cell receptors and viral epitopes with very similar sequences on infected cells.