Genetic Aspects Of Viral Oncogenesis In Wild Mouse Species

野生小鼠种类和各种近亲繁殖的实验室小鼠品系在对小鼠γ -逆转录病毒和逆转录病毒诱导的癌症的易感性方面彼此不同。这些差异是由于特定宿主基因的变化，我们一直在努力识别和表征几种与病毒抗性有关的小鼠基因。我们的主要兴趣一直是直接干扰病毒感染和复制的因素，我们把精力集中在那些抑制病毒进入和病毒复制周期进入后早期阶段的因素上。在进入水平上，有两种类型的抗性基因以受体-病毒相互作用为目标。受体可被内源性逆转录病毒产生的病毒包膜糖蛋白阻断，或由细胞表面受体的多态性引起耐药性。逆转录病毒进入受体细胞后，病毒抗性因子Fv1、mApobec/Rfv3和TRIM5alpha可抑制或改变逆转录和向细胞核的易位。

Wild mouse species and the various inbred laboratory mouse strains differ from one another in their susceptibility to the mouse gammaretroviruses and retrovirus-induced cancers. These differences are due to variations in specific host genes, and we have been engaged in an ongoing effort to identify and characterize several mouse genes involved in virus resistance. Our major interest has been on factors that interfere directly with virus infection and replication, and we focus our efforts on those factors that inhibit virus entry and the early post-entry stages of the virus replicative cycle. At the level of entry, there are two types of resistance genes that target the receptor-virus interaction. Receptors can be blocked by virus envelope glycoprotein produced by endogenous retroviruses, or resistance can be cause by polymorphisms in the cell surface receptor. After the gammaretrovirus enters the receptive cell, reverse transcription and translocation to the nucleus can be inhibited or altered by virus resistance factors Fv1, mApobec/Rfv3, and TRIM5alpha. Our current aim is to characterize these resistance factors and the viruses they target, define the origin and extent of antiviral activity in Mus evolution, and elucidate the responsible mechanisms. This work relies heavily on wild mice because laboratory strains provide only a limited sampling of the genetic diversity in Mus. Also, wild mouse species allow us to examine survival strategies in natural populations that harbor virus and to follow the evolution of the resistance genes. These mice additionally provide a source of novel resistance genes and virus variants. One set of projects is concerned with cell surface virus receptors and these studies focus on identifying viral and cell receptor determinants responsible for virus binding, entry, and receptor mediated cytopathicity. One series of experiments focuses on two unusual variants of the ecotropic gammaretroviruses that are cytopathic in M. dunni cells and also have altered host range. These phenotypes are due to different amino acid substitutions at the same site in viral envelope gene of the two viruses. This substitution alters one of the 3 amino acids that form the cell surface receptor binding site. The fact that these 2 viruses cause cytopathic effects in a cell line with a variant receptor gene suggests that the virus-receptor interaction mediates cytopathicity. This conclusion was confirmed by the observation that cytopathicity due to virus infection is seen in stable transfectants expressing this variant receptor but not in transfectants expressing the prototypical receptor. We have now inoculated mice with this cytopathic virus to determine if the lymphoma-type cancer induced by the progenitor virus is altered by the mutations that cause cytopathic cell fusion. In another series of experiments, we have been using phylogenetic methods to identify and characterize host genes that have had an anti-viral role in the genus Mus. Among the mouse genes responsible for resistance to mouse leukemia viruses is Rfv3 (recovery from Friend virus). This gene was recently shown to be encoded by the mouse APOBEC (mA3) gene, a cytidine deaminase gene known to restrict other retroviruses in mice and in humans. We sequenced mA3 from multiple laboratory and wild mice to examine its evolution. We discovered that the mA3 allele in virus resistant mice is disrupted by insertion of the regulatory signals of a mouse leukmia virus that may be responsible for enhanced mA3 expression and altered splicing. We also subjected the mA3 protein coding sequences to phylogenetic analysis. We identified 10 sites under positive selection, 6 of which are in two clusters that distinguish the virus restrictive and nonrestrictive mouse variants, and that are known to be important for human APOBEC3G function. We also showed that these two clusters are positioned opposite each other along the groove that forms the mA3 active site. We thus show that mA3 has had an antiviral role throughout mouse evolution, and we identify an inserted regulatory sequence and two clusters in the protein coding sequence that may contribute to this antiviral function. We are also interested in determining the extent to which virus resistance is mediated by polymorphisms of the cell surface receptor. We seek to analyze the XPR1 receptor for the xenotropic/polytropic gammaretroviruses and for XMRV, a xenotropic virus-like virus isolated from humans with prostate cancer or chronic fatigue syndrome. We have now identified a total of five XPR1 susceptibility variants in wild mice. We have characterized the latest of these to be identified, Xpr1m. We described the geographic and species distribution of the Mus Xpr1 variants, but failed to find the laboratory mouse allele of this gene in any wild mouse; this allele encodes a receptor that is uniquely resistant to xenotropic gammaretroviruses. We used mutagenesis and phylogenetic analysis to evaluate the functional contributions made by constrained, variable and deleted residues in this receptor. Rodent Xpr1 is under positive selection indicating a history of host-pathogen conflicts; several codons under selection have known roles in virus entry. All non-Mus mammals are susceptible to mouse X-MLV, but some restrict other members of this family of viruses and the resistance of hamster and gerbil cells to the human-derived XMRV indicates that XMRV has unique receptor requirements. We also showed that the hypervariable fourth extracellular XPR1 loop (ECL4) contains 3 evolutionarily constrained residues that do not contribute to receptor function, we identified two novel residues important for virus entry, and we described a unique pattern of ECL4 variation in the 3 virus-restrictive Xpr1 variants found in MLV-infected house mice; these mice carry different deletions in ECL4 suggesting either that these sites or loop size affects receptor function.