Interactions of APOBEC3 Proteins with Murine Leukemia Viruses

在过去的几年里，人们已经清楚地发现，人类细胞含有一种酶，APOBEC 3G（hA 3G），它可以诱导对某些逆转录病毒感染的深刻抵抗力。hA 3G蛋白具有胞苷脱氨酶活性，并且负责其抗病毒作用的一个机制是负链DNA中的胞嘧啶残基的脱氨基作用，在原病毒的编码链中产生G至A的突变。 HIV-1编码一种名为Vif的蛋白质，通过与hA 3G结合并促进其在蛋白酶体中的降解来阻断hA 3G的作用。尽管经过多年的深入研究，hA 3G掺入病毒体的机制以及除脱氨基作用以外的抗病毒作用的存在仍然是未解决的问题。 虽然人A3 G与HIV-1的相互作用一直是许多实验室研究的中心目标，但很明显APOBEC 3家族的其他成员也可以具有抗病毒作用，APOBEC 3家族成员存在于许多哺乳动物物种中，并且不同的病毒对不同的APOBEC 3亚型具有不同的敏感性模式。小鼠仅含有一个家族成员APOBEC 3（mA 3）。据报道，MLV对mA 3具有抗性，因为它们不会将其掺入组装的病毒体中，而其他研究表明mA 3掺入MLV颗粒中而没有显著的抗病毒作用。 我们与大卫·德尔塞博士合作，重新检查了MLV和MLV衍生载体对mA 3的反应，沿着它们对hA 3G的敏感性。我们发现，与已发表的报告相反，MLV和相关载体对mA 3敏感，尽管它们对hA 3G更敏感。其他实验表明，mA 3对delta-vif HIV-1的效力与hA 3G的效力相等。我们无法检测到MLV感染后mA 3诱导的G：A超突变，尽管在hA 3G灭活的MLV中观察到高水平的突变。这一观察结果支持了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，已报道mA 3在delta-vif HIV-1中诱导G：A超突变。 我们惊讶地发现，我们的结果与其他实验室发表的数据相矛盾。这些差异必须是由于我们使用的试剂或实验设计中的一个或多个差异造成的。我们将尝试确定相关的差异，这一搜索的结果可能会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mA 3的抗病毒活性具有部分抗性。MLV是一种简单的逆转录病毒，仅编码组装形成感染性子代病毒体的三种多蛋白。因此，确定其对mA 3的抗性机制将具有相当大的意义。我们正在通过构建嵌合MLV并测试其对mA 3的敏感性来实现这一目标，以努力定位抗性的决定因素。我们还正在生产在昆虫细胞中生产重组mA 3蛋白的试剂;该试剂在分析mA 2限制性机制和MLV对该限制性的抗性方面将是非常宝贵的。[对应于2007年4月艾滋病毒耐药性项目现场访问报告中的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 have re-examined the response of MLV and MLV-derived vectors to mA3, along with their sensitivity to hA3G. We find, 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 are 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. [Corresponds to Rein Project 3 in the April 2007 site visit report of the HIV Drug Resistance Program]