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.I nnate resistance to flavivirus-induced morbidity and mortality was demonstrated in mice in the 1920s (1) and showed monogenic autosomal dominant inheritance (2). The alleles that determined resistance and susceptibility were designated Flv r and Flv s , respectively (3). Resistant mice are susceptible to infections with other types of viruses but are resistant to all flaviviruses (4). Resistant mice can be infected by flaviviruses, but the virus titers in their tissues are lower by 1,000-10,000 times than those in the tissues of susceptible animals, and the spread of the infection in resistant mice is slower (5, 6). Cell cultures derived from many different tissues of resistant mice also produce lower yields of virus; peak titers from resistant cultures are 100-1,000 times lower than those from susceptible cultures (7-9). Previous studies indicate that the Flv gene product acts intracellularly on flavivirus replication.The flavivirus-resistant allele was demonstrated in wild Mus musculus domesticus populations in both the U.S. and Australia, and flavivirus genetic resistance was reported in other Mus species (10-12). Most commonly used inbred laboratory mouse strains were derived from a small number of progenitors, and the majority of them have a homozygous flavivirus-susceptible genotype. Only the Det, BSVR, BRVR, CASA͞Rk, CAST͞Ei, MOLD͞Rk, and PRI inbred strains have the resistant allele (13). The characteristics of a resistant-like allele (designated Flv r -like) in CASA͞Rk and CAST͞Ei strains were similar to those of the PRI Flv r allele. The MOLD͞Rk animals carry an allele designated minor resistance, Flv mr , that can protect carriers from disease after infection with the attenuated 17D strain of yellow fever virus but not from the virulent Murray Valley encephalitis virus (10).The resistant allele from donor PRI mice was introduced onto the susceptible C3H͞He background to produce the co...

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 ϳ100-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 upregulated 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-␤ 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, ϳ5-to 10-times-higher yields of WNV were produced. Similarly, about ϳ5-to 10-times-higher virus yields were produced by susceptible C57BL/6 RNase L Variation in susceptibility to flavivirus-induced disease among mice was first observed in the 1920s and subsequently shown to be controlled by a single locus (56,72). This virus-specific resistance is dominant. Studies with the C3H.PRI-Flv r and C3H/HeJ congenic pair of mouse strains (26) showed that although resistant mice support the replication of flaviviruses, virus titers in their tissues are significantly lower and the spread of infection is slower compared to that in susceptible mice (4,24,32,65). Cell cultures prepared from various tissues obtained from resistant mice also produce lower yields of flaviviruses than do comparable cell cultures from susceptible mice (13; reviewed in reference 8). Coinheritance of the Flv alleles with those of the Ric locus on chromosome 5 identified the chromosomal location of the Flv locus (36). The Flv locus was then mapped on mouse chromosome 5 by linkage analysis first with known flanking genes (61) and then with microsatellite markers (67). The Oas1b gene was identified as the Flv locus by a positional cloning strategy (51). Mashimo et al. (45) confirmed the identification of this gene. The transcript of the Oas1b allele in susceptible mice contains a premature stop codon and encodes a truncated protein. The mechanism(s) through which the products of the different Oas1b alleles confer differential susceptibility to flavivirus-induced ...

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.

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 T he genus Flavivirus, in the family Flaviviridae, consists of ϳ70 viruses and includes human pathogens such as dengue virus, yellow fever virus, tick-borne encephalitis virus, Japanese encephalitis virus, and West Nile virus (WNV) (20). WNV was first isolated in Uganda in 1937 and was previously reported to be endemic in Africa, Australia, and southern Asia (3); it has recently emerged in the Americas, with over 23,000 human infections reported in the United States as of late 2006 (2, 39). WNV is arthropod borne, with a natural transmission cycle typically between Culex mosquito species and birds, with occasional virus transmission by mosquitoes to horses and humans (3). Usually, WNV infections in humans are asymptomatic or cause mild flu-like symptoms. However, some infections cause more severe disease with symptoms such as meningitis, encephalitis, or paralysis, which can be fatal (3).The 2=-5= oligoadenylate synthetase (OAS) pathway functions as an innate host defense response against viral infections. OAS gene expression is upregulated by the signaling of interferons produced by cells in response to a viral infection (34). Viral doublestranded RNA (dsRNA) binds to and activates OAS, causing it to polymerize ATP into short 2=-5=-linked oligomers (2-5A) (14). These 2-5A oligomers bind to and activate latent endoribonuclease L (RNase L,) which is constitutively expressed in cells. Activated RNase L cleaves viral and cellular single-stranded RNAs.Data from numerous studies indicate that both host factors and virus virulence factors determine the outcome of a virus infection. Genetically controlled resistance to flavivirus-induced central nervous system (CNS) disease in mice was first discovered in the 1920s and rediscovered several times in the 1930s because it was not appreciated that all of the viruses being tested belonged to the same virus genus and family (4). Breeding studies with mice displaying differential susceptibility to flavivirus-induced disease showed that the alleles of a single gene, Flv, control...