Genetic Aspects Of Viral Oncogenesis In Wild Mouse Species

小鼠白血病病毒（mlv）是一种与肿瘤、神经系统和免疫缺陷疾病的诱导相关的γ -逆转录病毒。实验室小鼠和野生小鼠的近交系对小鼠γ -逆转录病毒感染和病毒引起的疾病的易感性不同，它们携带的mlv类型也不同。易感性差异是由于特定宿主基因的变化，我们一直在努力确定和表征几种宿主基因，这些基因要么参与病毒抗性，要么参与疾病过程。小鼠基因组包含小鼠γ -逆转录病毒基因组的拷贝，其中许多可以产生感染性和致病性病毒。还有一些宿主因素直接干扰病毒感染和复制，我们把精力集中在那些抑制病毒进入和病毒复制周期早期进入后阶段的因素上。在进入水平上，耐药可由细胞表面受体的多态性引起。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。我们目前的目标是表征这些耐药因子及其靶向的病毒，确定小鼠进化中抗病毒活性的起源和程度，并阐明相关机制。这项工作在很大程度上依赖于野生小鼠，因为实验室菌株只能提供小家鼠遗传多样性的有限样本。此外，野生小鼠物种使我们能够研究在自然种群中携带病毒的生存策略，并跟踪抗性基因的进化。这些小鼠还提供了新的抗性基因和病毒变体的来源。

Mouse leukemia viruses (MLVs) are gammaretroviruses linked to induction of neoplasms, and neurological and immunodeficiency diseases. Inbred strains of laboratory mice and wild mouse species differ in their susceptibility to mouse gammaretrovirus infection and to virus-induced diseases, and they also differ in the types of MLVs that they carry. Susceptibility differences are due to variations in specific host genes, and we have been engaged in an ongoing effort to identify and characterize several host genes that are either involved in virus resistance or that contribute to the disease process. The mouse genome contains copies of mouse gammaretrovirus genomes, many of which can produce infectious and pathogenic viruses. There are also host 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, resistance can be caused 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 aims to identify viral and cell receptor determinants responsible for virus binding and entry. We are currently working on the XPR1 receptor for the xenotropic/polytropic MLVs (XP-MLVs). We have determined that, in mouse populations exposed to infectious virus, virus resistance is mediated by polymorphisms of the cell surface receptor. We have identified a total of six XPR1 susceptibility variants in wild mice and described the geographic and species distribution of these Mus Xpr1 variants. Five of these receptors restrict entry by two or more of the virus host range variants that rely on XPR1, and all of these receptors evolved in populations exposed to X-MLVs. Virtually all mammalian species can be infected by X-MLVs, despite substantial sequence variation in the cell surface receptor they use for entry, XPR1. To determine why escape variants are rare in mammals, we examined the evolution of the entry determinants in XPR1, which lie in its third and fourth putative extracellular loops (ECLs). The critical ECL3 receptor determinant overlies a splice donor site and is evolutionarily conserved in vertegrate XPR1 genes. The 13 residue ECL4 is hypervariable, but this variability does not abolish receptor function, even when the entire ECL is replaced by the corresponding segment of the jellyfish gene. Deletions along the length of this ECL can influence but not abolish receptor function, and different deletions affect different XP-MLVs. Thus, receptor usage of a constrained splice site and a loop that tolerates mutations limits the likelihood of host escape mutations. We also examined susceptibility to X-MLVs in species outside the mammalian lineage because virus-infected mice are distributed globally and because XPR1 orthologs are found in all eukaryotes. We focused on birds because some birds and mice chare habitats, sometimes with a predator-prey relationship. We found that birds in contact with virus-infected mice have disabling mutations at the same two sites in XPR1 that are also disabled in virus-resistant laboratory mice. This indicates that birds and mice exposed to similar virus challenges evolve similar resistance strategies. In another series of experiments, we used phylogenetic and molecular biological methods to identify the wild mouse origins of the endogenous XP-MLVs found in the sequenced C57BL mouse genome. We traced 12 X-MLVs to Asian mouse species, but failed to identify any of the P-MLV endogenous retroviruses (ERVs) in any wild mice, including species that carry multiple copies of P-MLV ERVs suggesting a high level of insertional polymorphism. This level of insertional polymorphism is surprising for a set of ERVs not known to be expressed as infectious virus, and is under further investigation. We are now looking at the ability of these ERVs to generate infectious virus. We are in the process of sequencing 25 well characterized virus isolates and find that all are products of recombination of ERV-derived MLVs of different host range subgroups. The isolates that do not cause leukemogenesis in inoculated neonates result from recombination of P-MLVs and ecotropic (mouse-tropic) MLVs (E-MLVs). The leukemogenic isolates, however, are recombinants of E-MLVs, P-MLVs and X-MLVs. We are also characterizing viruses from wild mouse species to document the evolutionary history of these viruses in natural populations and to determine their origins and how viral genes evolve in the presence of specific host restriction factors. Finally, we examined the role of MLV ERVs in the use of xenograft experiments designed to provide a mouse model for primary myelofibrosis. Immunodeficient mice are crucial tools for identifying cancer stem cells and dissecting the molecular mechanisms that drive the evolution of malignancies. In studies done to define neoplastic stem cells of primary muelofibrosis (PMF), we observed a high frequency of acute myeloid leukemia (AML) in immunodeficient mice homozygous for the PRKDCscid and IL2RGnull alleles. We found that an endogenous ecotropic ERV is induced and is pathogenic in the PMF-microenvironment, likely due to induced proliferation of mouse cells by human PMF-derived cells. These proliferating cells are targets of retroviral transformation. This study corroborates a role of paracrine stimulation in PMF disease progression, underlines the imporatnce of target cell type and numbers in MLV-induced disease, and mandates awareness of replicating MLV in NOD immunodeficient mice,which can significantly influence experimental results and their interpretation.