Interactions of APOBEC3 Proteins with Murine Leukemia Viruses

在过去的几年里，人类细胞含有一种名为APOBEC3G(HA3G)的酶，这种酶对某些逆转录病毒的感染具有深刻的抵抗力，这一点在过去几年中已经变得明显。HA3G蛋白具有胞苷脱氨酶活性，其抗病毒作用的机制之一是使负链DNA中的胞嘧啶残基脱氨基，导致前病毒编码链发生G-to-A突变。HIV-1编码一种名为Vif的蛋白质，通过与hA3G结合并促进其在蛋白酶体中的降解来阻断hA3G的影响。尽管经过了几年的深入研究，但hA3G在病毒粒子中的掺入机制以及除脱氨基作用外是否存在抗病毒作用仍是悬而未决的问题。虽然人类A3G与HIV-1的相互作用一直是许多实验室的中心研究对象，但很明显，APOBEC3家族的其他成员也可以具有抗病毒作用，APOBEC3家族成员存在于许多哺乳动物中，并且不同的病毒对不同的APOBEC3亚型有不同的敏感性模式。小鼠只有一个家族成员，APOBEC3(MA3)。据报道，MLV对MA3具有抵抗力，因为它们不会将MA3掺入到组装病毒粒子中，而其他研究表明，MA3被掺入MLV颗粒中，而没有显著的抗病毒作用。与David Derse博士合作，我们重新检查了MLV和MLV衍生载体对MA3的反应，以及它们对hA3G的敏感性。我们发现，与已发表的报道相反，MLV和相关载体对MA3敏感，尽管它们对hA3G更敏感。其他实验表明，MA3对Delta-vif HIV-1的效力与hA3G相当。我们一直无法检测到MLV感染后由MA3诱导的G：超突变，尽管在MLV被hA3G灭活的情况下观察到了高水平的突变。这一观察结果支持G：A超突变不是APOBEC蛋白干扰逆转录病毒感染的唯一机制的概念。相比之下，MA3已被报道可诱导G：一种Delta-vif HIV-1的高度突变。我们惊讶地发现，我们的结果与其他实验室公布的数据相矛盾。这些差异肯定是由于我们所使用的试剂或实验设计中的一个或多个不同所致。我们将试图确定相关的差异；这一搜索的结果可能会很好地阐明该领域的重要问题，包括APOBEC蛋白抑制逆转录病毒感染的机制。综上所述，数据表明MLV对MA3的抗病毒活性具有部分抗性。MLV是一种简单的逆转录病毒，只编码三种多蛋白，这些多蛋白组装成具有感染性的子代病毒粒子。因此，确定其对MA3的抗性机制将是相当有意义的。我们正在通过构建嵌合MLV并测试它们对MA3的敏感性来实现这一目标，以努力定位抗性的决定因素。我们还在生产在昆虫细胞中产生重组MA3蛋白的试剂；这种试剂将在分析MA2限制的机制和MLV对这种限制的抗性方面发挥非常重要的作用。最近的成就和研究现状：a.MA3 Moloney MLV的灭活和抗性机制分析MLV在很大程度上对MA3产生抗性，并完全抵抗MA3诱导的超突变。我们用内源多嗜性MLV的Gag基因替换了Moloney MLV的Gag基因。产生的病毒对MA3的反应与Moloney MLV对MA3的反应相似，但仍然对MA3的超突变具有抗性。我们正在继续尝试确定MLV中负责耐药性的部位，部分是通过在敏感和耐药病毒之间建立其他嵌合体来实现的。我们的数据还表明，MA3在逆转录之前或开始时阻止MLV感染。重组MA3的生化研究我们正在提纯具有酶活性的重组MA3蛋白。我们将对其进行生化表征，并寻找与Moloney MLV蛋白的相互作用，这些相互作用可能参与Moloney MLV对MA3的抗性。[对应于2011年11月艾滋病毒耐药计划现场访问报告中的Rein项目3]

It has become clear in the last few years that human cells contain an enzyme, APOBEC3G (hA3G), that induces profound resistance to infection by certain retroviruses. hA3G protein possesses cytidine deaminase activity, and one mechanism responsible for its antiviral effects is deamination of cytosine residues in minus-strand DNA, producing G-to-A mutation in the coding strand of the provirus. HIV-1 encodes a protein, Vif, that blocks the effects of hA3G by binding to it and promoting its degradation in the proteasome. Despite several years of intensive study, the mechanism of hA3G incorporation into virions and the existence of antiviral effects other than deamination are still unresolved questions. While the interaction of human A3G with HIV-1 has been a central object of investigation in many laboratories, it is clear that other members of the APOBEC3 family can also have antiviral effects, that APOBEC3 family members are present in many mammalian species, and that different viruses have distinct patterns of sensitivity to the different APOBEC3 isoforms. Mice contain only one family member, APOBEC3 (mA3). It has been reported that MLVs are resistant to mA3 because they do not incorporate it into assembling virions, whereas other studies indicate that mA3 is incorporated into MLV particles without a significant antiviral effect. In collaboration with Dr. David Derse, we re-examined the response of MLV and MLV-derived vectors to mA3, along with their sensitivity to hA3G. We found, contrary to the published reports, that MLV and related vectors are sensitive to mA3, although they are considerably more sensitive to hA3G. Other experiments showed that the potency of mA3 against delta-vif HIV-1 is equal to that of hA3G. We have been unable to detect G:A hypermutation induced by mA3 following MLV infections, although high levels of the mutations are observed with MLV inactivated by hA3G. This observation supports the concept that G:A hypermutation is not the only mechanism by which APOBEC proteins interfere with retroviral infections. In contrast, mA3 has been reported to induce G:A hypermutation in delta-vif HIV-1. We were surprised to find that our results are in conflict with published data from other laboratories. The discrepancies must be due to one or more differences in the reagents or experimental designs we have used. We will attempt to identify the relevant differences; the results of this search might well shed light on important questions in the field, including the mechanism by which APOBEC proteins inhibit retroviral infections. Taken together, the data show that MLV is partially resistant to the antiviral activity of mA3. MLV is a simple retrovirus, encoding only the three polyproteins that are assembled to form infectious progeny virions. Thus, it would be of considerable interest to determine the mechanism of its resistance to mA3. We are pursuing this goal by constructing chimeric MLVs and testing their sensitivity to mA3, in an effort to locate the determinants of resistance. We are also generating the reagents to produce recombinant mA3 protein in insect cells; this reagent will be invaluable in analysis of the mechanism of mA2 restriction and of MLV resistance to this restriction. Recent Accomplishments and Current Research: a. Analysis of mechanisms of MLV inactivation by, and resistance to, mA3 Moloney MLV is largely resistant to mA3 and completely resistant to mA3-induced hypermutation. We replaced the gag gene of Moloney MLV with that of an endogenous polytropic MLV. The response of the resulting virus to mA3 is similar to that of Moloney MLV to mA3, but is still resistant to hypermutation by mA3. We are continuing to try to identify sites in MLV responsible for resistance, in part by making other chimeras between sensitive and resistant viruses. Our data also show that mA3 blocks MLV infection before or at the beginning of reverse transcription. b. Biochemical studies on recombinant mA3 We are purifying recombinant, enzymatically active mA3 protein. We will characterize it biochemically and look for interactions with Moloney MLV proteins that might be involved in the mA3 resistance of Moloney MLV. [Corresponds to Rein Project 3 in the November 2011 site visit report of the HIV Drug Resistance Program]