Host genetic variability and West Nile virus susceptibility

Efforts to understand the genetic basis of host susceptibility to viral infection and disease have led to the identification of genes whose products play key roles in determining the outcome of virus–host interactions. Examples of this include cellular genes that encode surface receptor components that function at the initiation stage of viral infection; genes that encode proteins that mediate adaptive cellular immune responses; and genes that affect the efficiency of virus multiplication by encoding proteins of the interferon (IFN) response pathway (1).

We now know considerable detail about the biology and biochemistry of the IFN system and its important role as an innate antiviral defense (2). Among the more than 100 cellular proteins whose expression is regulated by IFN are the 2′,5′-oligoadenylate synthetase (OAS) family of enzymes (2, 3). OAS was discovered as an IFN-inducible activity responsible in part for the inhibition of protein synthesis seen in response to double-stranded (ds) RNA in cell-free protein synthesizing systems. Transcription of Oas genes is up-regulated by IFN, and expressed OAS proteins are converted from an inactive to an enzymatically active form by a dsRNA-dependent process (Fig. 1). Activated OAS catalyzes the synthesis of ppp(A2′p)_nA oligoadenylates, which are abbreviated as “2–5A” because of their 2′,5′-phosphodiester bond linkage (4). The best understood actions of OAS occur through RNase L (5), a latent endoribonuclease that is activated after binding 2–5A and then degrades RNA by cleaving on the 3′-side of -UpXp- sequences. The OAS and RNase L proteins as well as other IFN-regulated proteins can display surprising virus-type selectivity in their antiviral actions (2). Combined biochemical and genetic analyses have firmly established important roles for OAS and RNase L in the antiviral actions of IFNs, particularly against picornaviruses (2, 3, 5). In recent issues of PNAS, Brinton (6), Desprès and Guénet (7), and their coworkers independently reported the discovery that a mutation in the Oas1b/L1 gene encoding a specific isoform of OAS is associated with West Nile (WN) virus susceptibility in the mouse model.

Figure 1.

Biochemical functions of IFN-inducible proteins, including the family of 2′-5′-oligoadenylate synthetases and RNase L nuclease that mediates RNA degradation and apoptosis, the family of Mx protein GTPases that seem to target viral nucleocapsids and inhibit RNA synthesis, the PKR kinase that inhibits translation initiation through phosphorylation of protein synthesis initiation factor eIF-2α, and the ADAR1 adenosine deaminase that edits RNA.

Variations in the host response to flavivirus infection has a long history (8–10). Selective breeding in mice led to the development of resistant and susceptible lines and the demonstration that a single, autosomal-dominant genetic locus, designated Flv, was responsible for differences in susceptibility to neurotrophic flaviviruses. The Flv gene confers resistance to flavivirus-induced disease in mice for several different mosquito-borne flaviviruses, including WN virus (9). In the studies reported by Mashimo et al. (7), WN virus caused encephalitis and 100% mortality in all classical laboratory inbred mouse strains that they tested, whereas no morbidity or mortality was seen in six unrelated inbred strains derived from wild ancestors of Mus m. domesticus, musculus, or spretus under similar conditions. Back-crosses led to the identification of a WN virus resistance/susceptibility locus within an interval of 0.4 cM on chromosome 5 that the authors designated Wnv (7) and that corresponds to the region where Flv previously had been mapped (9, 10). Perelygin et al. (6) used a positional cloning strategy to identify 22 genes from the Flv gene interval and then compared their sequences from congenic resistant C3H.PRI-Flvr and susceptible C3H/He mouse strains. Among the genes identified by both Perelygin et al. (6) and Mashimo et al. (7) within the interval defined by D5Mit408 and D5Mit242 that includes the Wnv/Flv locus are the gene cluster encoding the IFN-inducible OAS family of proteins. This region of mouse chromosome 5 is homologous to that in the region 12q24.1 of the human genome that includes the multiple Oas genes clustered over ≈130 kb (11).

Three size forms of OAS have been characterized in mouse and human cells through study of cDNA and genomic clones and their encoded RNAs and proteins (2, 3, 11). Multiple Oas1 genes in mice but one in humans, together with alternative splicing, give rise to several isoforms of the small-sized “p40” form of OAS, designated OAS1. Middle-sized “p70” forms of OAS, designated OAS2, are generated by differential splicing of transcripts from a single Oas2 gene. The large-sized “p100” form is encoded by a single Oas3 gene that has three adjacent, repeated OAS1-like domains. Evidence suggests that mammalian Oas genes underwent successive gene duplication events, resulting in the three sizes of OAS proteins containing one (Oas1), two (Oas2), or three (Oas3) homologous domains (3, 11). The different-sized OAS1, OAS2, and OAS3 proteins found in mouse and human cells are associated with different subcellular fractions, including membranes, cytoplasm, and nucleus; they differ in the concentration of dsRNA required for their activation, the reaction conditions required for their optimal enzymatic activity, and the size pattern of the 2–5A products produced (2, 3). The full physiological significance of these differences is not yet established, nor is it understood why rodent genomes contain multiple copies of Oas1 genes. However, the results of Perelygin et al. (6) and Mashimo et al. (7) suggest that a specific isoform (OAS1b, L1) is especially important in the host response to WN virus and, furthermore, that this OAS isoform plays a uniquely essential role in determining WN virus pathogenesis in mice. Mouse strains that are susceptible to WN virus were found by both groups (6, 7) to have a C-to-T transition in the fourth exon sequence of the Oas1b/L1 gene. This substitution results in a codon change, from CGA (arg) in resistant strains to UGA (stop) in susceptible strains. The stop codon is predicated to cause a truncated OAS1b/L1 protein to be produced in susceptible mice. All resistant mice analyzed by the authors (6, 7) had a normal Oas1b/L1 gene with extended ORF. This absolute correlation between Oas1b/L1 genotype and resistance/susceptibility phenotype was found in all mouse strains analyzed (6, 7).

WN virus replicates in mammals, birds, and mosquitoes. Comparison of congenic susceptible BALB/c and resistant BALB/c-MBT mice indicates that survival of WN-infected resistant animals correlates with restriction of virus replication within the central nervous system (7). The principal features of the flavivirus multiplication cycle essential to understanding the possible role of OAS in affecting the WN virus–host interaction are summarized in Fig. 2. WN virions are enveloped, possess a positive-strand ssRNA genome, and multiply in the cytoplasm of infected cells (1). The spherical enveloped virion includes the E protein, which is presumed to interact with the host cell receptor during virion attachment. The nucleocapsid includes an ≈11-kb RNA genome complexed with the capsid C protein. Virion penetration is believed to occur by means of receptor-mediated endocytosis and is followed by uncoating and release of the genome RNA that functions as messenger RNA. The single long ORF of the uncoated viral genome first is translated by the host cell protein synthesizing machinery into a large viral polyprotein. Proteolytic processing of the viral polyprotein precursor occurs by a pathway involving viral and cellular protease activities to generate mature viral structural (E, C, and M) and nonstructural (NS) proteins, including the NS3 serine protease and NS5 RNA polymerase. In addition to its initial role as mRNA, the positive-stranded genome RNA also serves as the template for RNA replication catalyzed by viral enzymes to generate the complementary minus-strand RNA product. Minus-stranded RNA then serves as the template for synthesis of additional positive-stranded RNA molecules. Positive-stranded RNA is estimated to account for >90% of the viral RNA produced in infected cells, with the minus-stranded RNA found predominantly in double-stranded replicative intermediate structures. Progeny virions mature in cytoplasmic vesicles, and release is believed to occur by vesicle fusion at the plasma membrane (1).

Figure 2.

Schematic diagram of flavivirus multiplication cycle.

In WN virus-infected cells, viral dsRNA structures might arise through single-stranded transcripts that possess significant double-stranded character, in addition to the potential source of dsRNA structures such as the RNA duplexes formed during replication (Fig. 2). Interestingly, more viral dsRNA is found in brains from flavivirus-infected resistant than susceptible mice (12). Among cellular proteins (in addition to OAS) for which dsRNA plays an important role are the RNA-dependent protein kinase PKR and ADAR1 adenosine deaminase (Fig. 1). PKR inhibits translation through phosphorylation of protein synthesis initiation factor eIF-2 (13), and ADAR1 edits RNA through C6 deamination of adenosine to yield inosine (14). Double-stranded RNA is an effector molecule for both OAS and PKR and can either activate or antagonize enzymatic activity depending upon the specific RNA structure and RNA concentration. For ADAR1, by contrast, the dsRNA structure is the substrate (2). In most cases in virally infected cells, the activator RNA for either OAS or PKR still has not been precisely defined. However, the results of Mashimo et al. (7) and Perelygin et al. (6) imply a level of differential RNA selectivity among the dsRNA-dependent enzymes. Several isoforms of OAS other than 1b/L1, and also PKR, are presumably expressed in susceptible mouse strains that possess the mutant 1b/L1 isoform. These enzymes apparently are not sufficient to mediate resistance to WN virus. Thus, the interesting possibility arises as to whether the WN virus susceptibility seen in laboratory mice possessing the mutant OAS1b/L1 is caused by the impairment of a function of OAS other than the dsRNA-activated synthesis of 2,5A oligomers. That is, does the wild-type 1b/L1 OAS protein in resistant mice have a dsRNA-independent function? Pertinent to this possibility, the 9–2 isoform of mouse OAS1 recently has been shown to be a dual-function protein that independently can synthesize 2–5A oligomers and promote cellular apoptosis (15). The proapoptotic activity, which is OAS-isoform specific, depends upon a functional Bcl-2 homology 3 domain present in the C-terminal region that interacts with the Bcl-2 and BclxL anti-apoptotic proteins (15). Curiously, it is the C-terminal region of the OAS1 and OAS2 size class isoforms that are unique, in part because of alternative splicing (3, 11). Targeted disruption of the mouse RNase L gene provides one form of a functional knock-out of the 2–5A pathway. Both apoptosis and the antiviral effects of IFN against encephalomyocarditis virus are defective in RNase L^−/− MEF cells and mutant mice devoid of RNase L (5). However, the 9–2 isoform of OAS1 shows comparable apoptotic activity in RNase L^−/− cells as wild-type fibroblasts (15). It will be interesting to learn whether the presence or absence of RNase L affects the flavivirus resistance/susceptibility phenotype.

Earlier studies using antibody to IFN suggested that flavivirus resistance was independent of IFN-α/β (16). However, basal and inducible levels of OAS differ significantly, depending upon the type of cell, type of tissue, and type of IFN (2, 3). It is conceivable that the OAS1b/L1 isoform in resistant mice is IFN-inducible and was still expressed even in the presence of antibody against IFN (16) to a level sufficient to confer resistance, or alternatively, that basal Oas1b/L1 gene expression indeed is IFN-independent and occurs at a level sufficient to establish the resistance phenotype. Possibly studies with mice deficient in components of the IFN signal-transduction pathway through targeted gene disruption (2) might help to resolve this issue.

Constitutive expression of transcripts for Oas1b/L1 was detected in all 14 BALB/c mouse tissues examined by Northern blotting (6). When OAS1b/L1 isoform-specific antibody becomes available, it will be important to establish whether the accumulation of truncated OAS1b/L1 protein is detectable in tissues including brain of susceptible mice, compared with the full-length form of the protein expected in resistant mice. As a beginning to assess function of the OAS1b/L1 protein, low-level expression of the cDNA for full-length OAS1b in cell lines from susceptible C3H/He mice partially inhibited WN virus replication (6). But, increased levels of OAS1b expression curiously did not inhibit WN multiplication (6). Clearly, further examination of the resistance/susceptibility phenotype by quantitative overexpression analyses of mutant and wild-type forms of OAS1b/L1 in cultured cells, and more importantly, in transgenic mice derived from susceptible strains, may help to confirm and extend the very interesting observations of Perelygin et al. (6) and Mashimo et al. (7). Other examples of genetic variants of IFN-responsive genes are known in mice that confer altered susceptibility to viral infection, as exquisitely illustrated by myxoviruses and the Mx genes. Resistance to myxoviruses is conferred by the Mx1 gene in inbred A2G and SL/NiA mouse strains and some wild mice (17). Additionally, in transgenic mice deficient in endogenous Mx1 protein and also lacking functional IFN-α/β receptors, the human MxA protein is sufficient to establish an antiviral state (18).

WN virus is one of several members of the Flaviviridae family that are associated with human disease. WN virus is widely distributed throughout Africa, the Middle East, and parts of Asia. In 1999, WN virus emerged in the eastern seaboard of the United States. An initial WN outbreak in the New York City area resulted in several cases of encephalitis and a small number of deaths. WN virus subsequently has reappeared in the USA each summer since 1999, with increasing geographic distribution (19). Migrating birds are presumed to play a significant role in facilitating dispersal of the virus to mosquito populations over distant geographic locations. Given the low background of immunity, WN virus spread and amplified transmission has the potential to result in future summertime epidemics. Hence, understanding the genetic determinants that affect WN virus susceptibility and resistance is of utmost importance for defining the molecular mechanisms responsible for WN virus pathogenesis.