Svetlana V. Scherbik

Positional cloning of the murine flavivirus resistance gene

Inbred mouse strains exhibit significant differences in their susceptibility to viruses in the genus Flavivirus, which includes human pathogens such as yellow fever, Dengue, and West Nile virus. A single gene, designated Flv, confers this differential susceptibility and was mapped previously to a region of mouse chromosome 5. A positional cloning strategy was used to identify 22 genes from the Flv gene interval including 10 members of the 2′-5′-oligoadenylate synthetase gene family. One 2′-5′-oligoadenylate synthetase gene, Oas1b, was identified as Flv by correlation between genotype and phenotype in nine mouse strains. Susceptible mouse strains produce a protein lacking 30% of the C-terminal sequence as compared with the resistant counterpart because of the presence of a premature stop codon. The Oas1b gene differs from all the other murine Oas genes by a unique four-amino acid deletion in the P-loop located within the conserved RNA binding domain. Expression of the resistant allele of Oas1b in susceptible embryo fibroblasts resulted in partial inhibition of the replication of a flavivirus but not of an alpha togavirus.

RNase L Plays a Role in the Antiviral Response to West Nile Virus

ABSTRACT Alleles at the Flv locus determine disease outcome after a flavivirus infection in mice. Although comparable numbers of congenic resistant and susceptible mouse embryo fibroblasts (MEFs) are infected by the flavivirus West Nile virus (WNV), resistant MEFs produce ∼100- to 150-fold lower titers than susceptible ones and flavivirus titers in the brains of resistant and susceptible animals can differ by >10,000-fold. The Flv locus was previously identified as the 2′-5′ oligoadenylate synthetase 1b (Oas1b) gene. Oas gene expression is up-regulated by interferon (IFN), and after activation by double-stranded RNA, some mouse synthetases produce 2-5A, which activates latent RNase L to degrade viral and cellular RNAs. To determine whether the lower levels of intracellular flavivirus genomic RNA from resistant mice detected in cells at all times after infection were mediated by RNase L, RNase L activity levels in congenic resistant and susceptible cells were compared. Similar moderate levels of RNase L activation by transfected 2-5A were observed in both types of uninfected cells. After WNV infection, the mRNAs of IFN-β and three Oas genes were up-regulated to similar levels in both types of cells. However, significant levels of RNase L activity were not detected until 72 h after WNV infection and the patterns of viral RNA cleavage products generated were similar in both types of cells. When RNase L activity was down-regulated in resistant cells via stable expression of a dominant negative RNase L mutant, ∼5- to 10-times-higher yields of WNV were produced. Similarly, about ∼5- to 10-times-higher virus yields were produced by susceptible C57BL/6 RNase L−/− cells compared to RNase L+/+ cells that were either left untreated or pretreated with IFN and/or poly(I) · poly(C). The data indicate that WNV genomic RNA is susceptible to RNase L cleavage and that RNase L plays a role in the cellular antiviral response to flaviviruses. The results suggest that RNase L activation is not a major component of the Oas1b-mediated flavivirus resistance phenotype.

Differential Expression of Interferon (IFN) Regulatory Factors and IFN-Stimulated Genes at Early Times after West Nile Virus Infection of Mouse Embryo Fibroblasts

ABSTRACT Although lineage I West Nile virus (WNV) strain Eg101 induced beta interferon (IFN-β) production as early as 12 h after infection in primary mouse embryo fibroblasts and did not inhibit the JAK-STAT signaling pathway, it was still able to replicate efficiently. To gain insights about possible viral countermeasures used by this virus to suppress the host response, the cell transcriptional profile and the kinetics of IFN regulatory factor (IRF) expression and activation were examined at early times after infection. By 12 h after WNV infection, the majority of the up-regulated genes were ones involved in IFN pathways. However, comparison of IFN-stimulated gene (ISG) expression levels in mock-infected, IFN-treated, and virus-infected cells indicated that WNV infection suppressed the up-regulation of a subset of ISGs, including genes involved in transcriptional regulation, apoptosis, and stress responses, prior to 24 h after infection. Analysis of mRNA and protein levels for representative genes indicated that suppression was at the transcriptional and posttranscriptional levels. Translocation of IRF-3 to the nucleus was observed beginning at 8 h, IRF-7 expression was detected by 12 h, but IRF-1 expression was not detected until 24 h after infection. Virus-induced gene suppression was sufficient to overcome the effect of exogenous IFN pretreatment for 1 h but not for 4 h prior to infection. These data indicate that WNV can selectively counteract the host response at early times after infection by previously unreported mechanisms.

The mammalian 2'-5' oligoadenylate synthetase gene family: evidence for concerted evolution of paralogous Oas1 genes in Rodentia and Artiodactyla.

Multiple 2′-5′ oligoadenylate (2-5A) synthetases are important components of innate immunity in mammals. Gene families encoding these proteins have previously been studied mainly in humans and mice. To reconstruct the evolution of this gene family in mammals, a search for additional 2-5A synthetase genes was performed in rat, cattle, pig, and dog. Twelve 2′-5′ oligoadenylate synthetase (Oas) genes were identified in the rat genome, including eight Oas1 genes, two Oas1 pseudogenes, single copies of Oas2 and Oas3, and two Oas-like genes, Oasl1 and Oasl2. Four OAS genes were detected in the pig genome and five OAS genes were found in both the cattle and dog genomes. An OAS3 gene was not found in either the cattle or the pig genome. While two tandemly duplicated OAS-like (OASL) genes were identified in the dog genome, only a single OASL orthologue was found in both the cattle and the pig genomes. The bovine and porcine OASL genes contain premature stop codons and encode truncated proteins, which lack the typical C-terminal double ubiquitin domains. The cDNA sequences of the rat, cattle, pig, and dog OAS genes were amplified, sequenced and compared with each other and with those in the human, mouse, horse, and chicken genomes. Evidence of concerted evolution of paralogous 2′-5′ oligoadenylate synthetase 1 genes was obtained in rodents (Rodentia) and even-toed ungulates (Artiodactyla). Calculations using the nonparametric Kolmogorov-Smirnov test suggested that the homogenization of paralogous OAS1 sequences was due to gene conversion rather than stabilizing selection.

Virus-Induced Ca2+ Influx Extends Survival of West Nile Virus-Infected Cells

ABSTRACT West Nile virus (WNV) infection leads to rapid and sustained Ca2+ influx. This influx was observed with different strains of WNV and in different types of cells. Entry during virion endocytosis as well as through calcium channels contributed to the Ca2+ influx observed in WNV-infected cells. Ca2+ influx was not detected after infection with vesicular stomatitis virus (VSV) and occurred only through endocytosis in Sindbis virus-infected cells. Caspase 3 cleavage and activation of several kinases, including focal adhesion kinase (FAK), mitogen-activated extracellular signal-regulated protein kinase (ERK1/2), and protein-serine kinase B alpha (Akt), at early times after WNV infection were shown to be dependent on Ca2+ influx. Although the activation of these kinases was sustained in virus-infected cells throughout infection, UV-inactivated WNV induced only a transient activation of FAK and ERK1/2 at early times after infection. The Ca2+-dependent FAK activation observed in WNV-infected cells was not mediated by αvβ3 integrins. Reduction of Ca2+ influx at early times of infection by various treatments decreased the viral yield and delayed both the early transient caspase 3 cleavage and the activation of FAK, Akt, and ERK signaling. The results indicate that Ca2+ influx is required for early infection events needed for efficient viral replication, possibly for virus-induced rearrangement of the endoplasmic reticulum (ER) membrane. Increased caspase 3 cleavage at both early (transient) and late times of infection correlated with decreased activation of the FAK and ERK1/2 pathways, indicating a role for these kinases in extending the survival of flavivirus-infected cells.

West Nile Virus Infections Suppress Early Viral RNA Synthesis and Avoid Inducing the Cell Stress Granule Response

ABSTRACT 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.

Identification of Novel Host Cell Binding Partners of Oas1b, the Protein Conferring Resistance to Flavivirus-Induced Disease in Mice

ABSTRACT 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.

Type 1 IFN-independent activation of a subset of interferon stimulated genes in West Nile virus Eg101-infected mouse cells.

Although infection of mouse embryofibroblasts (MEFs) with WNV Eg101 induced interferon (IFN) beta production and STAT1 and STAT2 phosphorylation, these transcription factors (TFs) were not detected in the nucleus or on the promoters of four IRF-3-independent interferon stimulated genes (ISGs): Oas1a and Irf7 (previously characterized as IFN/ISGF3-dependent), Oas1b and Irf1. These ISGs were upregulated in WNV Eg101-infected STAT1-/-, STAT2-/-, and IFN alpha/beta receptor-/- MEFs. Although either IRF-3 or IRF-7 could amplify/sustain Oas1a and Oas1b upregulation at later times after infection, these factors were not required for the initial gene activation. The lack of upregulation of these ISGs in WNV Eg101-infected IRF-3/9-/- MEFs suggested the involvement of IRF-9. Activation of Irf1 in infected MEFs did not depend on any of these IRFs. The data indicate that additional alternative activation mechanisms exist for subsets of ISGs when a virus infection has blocked ISG activation by the canonical IFN-mediated pathway.

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.

Phylogenetic Relationships of the Siberian Iris Species Inferred from Noncoding Chloroplast DNA Sequences

A study of phylogenetic relationships of Iris species has been complicated because of extreme morpho‐ecological diversity, wide distribution of the genus, multiple hybridizations, and convergent evolution processes in the genus. In order to get an insight into the evolutionary history of Iris and to clarify some contradictions of contemporary classifications, we performed a molecular analysis of the phylogenetic relationships of a heterogeneous group of Iris species occurring in Siberia and covering major taxonomic groups of the genus Iris. According to contemporary classifications, these species belong to the subgenera Limniris (Tausch) Spach, Xyridion (Tausch) Spach, Iris, and Pardanthopsis (Hance) Baker. However, the position of Pardanthopsis within the genus Iris and the position of Xyridion as a distinct subgenus or a part of Limniris are disputable. Based on an analysis of 56 RAPD markers in 12 Siberian Iris species and comparative analysis of trnL intron and trnL‐trnF intergenic spacer noncoding chloroplast DNA sequences in all 22 Siberian Iris species, we reconstructed the phylogenetic relationships of Siberian Iris species. In general, the species grouping coincides with contemporary classifications. According to our results, Pardanthopsis dichotoma forms a separate branch on the phylogenetic trees based on sequences of chloroplast DNA and RAPD analysis, which supports its position as a distinct genus. All of the Siberian Iris species are clustered into four phylogenetic groups. Our results indicate that the phylogeny and taxonomic structure of the genus Iris may need reconsideration.