Positional cloning of the murine flavivirus resistance gene

Andrey Perelygin

Svetlana Scherbik

Igor Zhulin

Bronislava Stockman

Yan Li

Margo Brinton

^{Department of Biology, Georgia State University, Atlanta, GA 30303; and}^{School of Biology, Georgia Institute of Technology, Atlanta, GA 30332}

^{To whom reprint requests should be addressed at: Georgia State University, Department of Biology, P.O. Box 4010, Atlanta, GA 30302-4010. E-mail:}ude.usg.rehtnap@bamoib.

Communicated by Hilary Koprowski, Thomas Jefferson University, Philadelphia, PA

Received 2002 Mar 8; Accepted 2002 May 14.

Abstract

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.

Abstract

Innate 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, CASA/Rk, CAST/Ei, MOLD/Rk, and PRI inbred strains have the resistant allele (13). The characteristics of a resistant-like allele (designated Flv^r-like) in CASA/Rk and CAST/Ei strains were similar to those of the PRI Flv^r allele. The MOLD/Rk 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 C3H/He background to produce the congenic inbred C3H.PRI-Flv^r (formerly C3H.RV) strain by a standard backcross protocol followed by brother-sister matings with selection at each generation for the resistance phenotype (12). These congenic strains also carry different alleles of the Ric gene, which controls susceptibility to Rickettsia tsutsugamushi and is located on mouse chromosome 5 (14). These data suggested linkage between the Flv and the Ricloci, and the congenic strains were subsequently used to map the Flv locus on mouse chromosome 5 by linkage with the Ric and rd loci (15). Subsequently, 12 microsatellite markers from mouse chromosome 5 were genotyped relative to the Flv gene in 1,325 backcross animals. Two of the microsatellite markers, D5Mit408 and D5Mit242, exhibited map distances with the Flv locus of 0.30 and 0.15 centimorgans, respectively, whereas one additional marker, D5Mit159, showed no recombination with Flv, indicating linkage of <0.15 centimorgans (16).

To isolate the Flv gene we used a positional cloning strategy. The loci located near the D5Mit159 marker were identified first, and then their sequences were compared in cells from congenic resistant and susceptible mice. We report identification of the Flv gene as mouse 2′-5′-oligoadenylate synthetase 1B (Oas1b).

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Acknowledgments

This work was supported by Public Health Service grants: National Institute of General Medical Sciences Grant GM54896 and National Institute of Allergy and Infectious Diseases, National Institutes of Health Grant AI45135.

Acknowledgments

Abbreviations

Flv	flavivirus resistance gene
Oas	2′-5′-oligoadenylate synthetase
BHK	baby hamster kidney
WNV	West Nile virus
BAC	bacterial artificial chromosome
NCR	noncoding region
CPE	cytopathic effect

Abbreviations

Footnotes

Footnotes

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