Interactions of APOBEC3 Proteins with Murine Leukemia Virus

在过去的几年里，人们已经清楚地认识到，人类细胞含有一种酶，APOBEC3G (hA3G)，它对某些逆转录病毒的感染产生了深刻的抵抗力。hA3G蛋白具有胞嘧啶脱氨酶活性，其抗病毒作用的机制之一是对负链DNA中的胞嘧啶残基进行脱氨，在原病毒的编码链中产生G-to-A突变。HIV-1编码一种名为Vif的蛋白质，该蛋白质通过与hA3G结合并促进其在蛋白酶体中的降解来阻断其作用。尽管经过多年的深入研究，hA3G与病毒粒子结合的机制以及除脱氨外是否存在抗病毒作用仍是未解决的问题。虽然人类A3G与HIV-1的相互作用一直是许多实验室研究的中心对象，但很明显，APOBEC3家族的其他成员也具有抗病毒作用，APOBEC3家族成员存在于许多哺乳动物物种中，不同的病毒对不同的APOBEC3亚型具有不同的敏感性模式。小鼠只含有一个家族成员，APOBEC3 （mA3）。据报道，MLV对mA3具有抗性，因为它们不将mA3整合到组装病毒粒子中，而其他研究表明，mA3被整合到MLV颗粒中，但没有明显的抗病毒作用。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，与已发表的报告相反，MLV和相关载体对mA3敏感，尽管它们对hA3G敏感得多。其他实验表明mA3对delta-vif HIV-1的效力与hA3G相当。我们无法检测到MLV感染后mA3诱导的G:A高突变，尽管在被hA3G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超突变并不是APOBEC蛋白干扰逆转录病毒感染的唯一机制。相反，mA3已被报道在delta-vif HIV-1中诱导G:A高突变。我们惊讶地发现，我们的结果与其他实验室发表的数据相冲突。差异必须是由于我们所使用的试剂或实验设计的一个或多个差异。我们将尝试找出相关的差异；这项研究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，这些数据表明MLV对mA3的抗病毒活性有部分抗性。MLV是一种“简单的”逆转录病毒，只编码三种多蛋白，这些多蛋白被组装成具有传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。我们打算尝试选择具有更高抗性水平的MLV变体，以阐明没有vif样蛋白的病毒可以阻断APOBEC蛋白的抗病毒作用的可能途径。我们将通过在表达亲生态MLV受体的293T细胞中传代具有复制能力的MLV来进行选择。mA3将在诱导启动子的控制下表达，因此其水平可以随着实验的进行而调节。如果证明mA3对细胞中的慢性表达毒性太大，则实验将以一系列瞬时步骤进行，而不是连续传代。如果获得MLV的ma3抗性变体，则将确定导致抗性的突变。[与2007年4月HIV耐药性项目的实地考察报告中的Rein项目3相对应]在过去几年中，人们已经清楚地认识到，人类细胞含有一种酶，APOBEC3G (hA3G)，它能诱导对某些逆转录病毒的感染产生深刻的抵抗力。hA3G蛋白具有胞嘧啶脱氨酶活性，其抗病毒作用的机制之一是对负链DNA中的胞嘧啶残基进行脱氨，在原病毒的编码链中产生G-to-A突变。HIV-1编码一种名为Vif的蛋白质，该蛋白质通过与hA3G结合并促进其在蛋白酶体中的降解来阻断其作用。尽管经过多年的深入研究，hA3G与病毒粒子结合的机制以及除脱氨外是否存在抗病毒作用仍是未解决的问题。虽然人类A3G与HIV-1的相互作用一直是许多实验室研究的中心对象，但很明显，APOBEC3家族的其他成员也具有抗病毒作用，APOBEC3家族成员存在于许多哺乳动物物种中，不同的病毒对不同的APOBEC3亚型具有不同的敏感性模式。小鼠只含有一个家族成员，APOBEC3 （mA3）。据报道，MLV对mA3具有抗性，因为它们不将mA3整合到组装病毒粒子中，而其他研究表明，mA3被整合到MLV颗粒中，但没有明显的抗病毒作用。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，与已发表的报告相反，MLV和相关载体对mA3敏感，尽管它们对hA3G敏感得多。其他实验表明mA3对delta-vif HIV-1的效力与hA3G相当。我们无法检测到MLV感染后mA3诱导的G:A高突变，尽管在被hA3G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超突变并不是APOBEC蛋白干扰逆转录病毒感染的唯一机制。相反，mA3已被报道在delta-vif HIV-1中诱导G:A高突变。我们惊讶地发现，我们的结果与其他实验室发表的数据相冲突。差异必须是由于我们所使用的试剂或实验设计的一个或多个差异。我们将尝试找出相关的差异；这项研究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，这些数据表明MLV对mA3的抗病毒活性有部分抗性。MLV是一种“简单的”逆转录病毒，只编码三种多蛋白，这些多蛋白被组装成具有传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。我们打算尝试选择具有更高抗性水平的MLV变体，以阐明没有vif样蛋白的病毒可以阻断APOBEC蛋白的抗病毒作用的可能途径。我们将通过在表达亲生态MLV受体的293T细胞中传代具有复制能力的MLV来进行选择。mA3将在诱导启动子的控制下表达，因此其水平可以随着实验的进行而调节。如果证明mA3对细胞中的慢性表达毒性太大，则实验将以一系列瞬时步骤进行，而不是连续传代。如果获得抗ma3的MLV变体，则mut[摘要截短为7800个字符]

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 intend to try to select MLV variants with higher levels of resistance, in order to elucidate possible ways that viruses without a Vif-like protein can block the antiviral effects of APOBEC proteins. We will perform this selection by passaging replication-competent MLV in 293T cells expressing the ecotropic MLV receptor. mA3 will be expressed under the control of an inducible promoter so that its levels can be regulated as the experiment proceeds. If mA3 proves too toxic for chronic expression in the cells, the experiment will be performed in a series of transient steps rather than continuous passage. If an mA3-resistant variant of MLV is obtained, the mutation(s) responsible for the resistance will be identified. [Corresponds to Rein Project 3 in the April 2007 site visit report of the HIV Drug Resistance Program]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 intend to try to select MLV variants with higher levels of resistance, in order to elucidate possible ways that viruses without a Vif-like protein can block the antiviral effects of APOBEC proteins. We will perform this selection by passaging replication-competent MLV in 293T cells expressing the ecotropic MLV receptor. mA3 will be expressed under the control of an inducible promoter so that its levels can be regulated as the experiment proceeds. If mA3 proves too toxic for chronic expression in the cells, the experiment will be performed in a series of transient steps rather than continuous passage. If an mA3-resistant variant of MLV is obtained, the mut [summary truncated at 7800 characters]