APOBEC3G inhibits elongation of HIV-1 reverse transcripts.
APOBEC3G inhibits elongation of HIV-1 reverse transcripts.
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DOI：
10.1371/journal.ppat.1000231
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发表时间：
2008-12
期刊：
影响因子：
6.7
通讯作者：
Malim, Michael H.
中科院分区：
文献类型：
--
作者：
Bishop, Kate N.;Verma, Mohit;Kim, Eun-Young;Wolinsky, Steven M.;Malim, Michael H.
APOBEC 3G（A3 G）是一种宿主胞苷脱氨酶，在不存在Vif的情况下，可以限制HIV-1复制并减少细胞中积累的病毒DNA的量。初步研究表明，A3 G在逆转录过程中诱导新生HIV-1 cDNA的广泛突变。有人提出，这引发了病毒DNA的降解，但现在有越来越多的证据表明，这种机制可能是不正确的。在这里，我们使用一种天然的内源性逆转录酶试验表明，在无细胞病毒颗粒，A3 G能够抑制HIV-1的cDNA积累，不仅在超突变的情况下，但也没有明显需要任何靶细胞因子。我们发现，虽然逆转录启动A3 G的存在下，延伸的cDNA产物受阻。这些数据支持A3 G通过抑制合成而不是诱导降解来降低HIV-1 cDNA水平的模型。APOBEC蛋白是细胞编码的因子，其抑制许多逆转录病毒如HIV-1和逆转录转座子的复制。在许多情况下，抑制明显与病毒或转座子DNA的胞苷至尿苷编辑相关。另一方面，使用特定APOBEC蛋白/底物组合或编辑缺陷的工程化蛋白的许多研究已经表明，与编辑不同的抑制机制也是有效的。在这里，我们分析了APOBEC 3G，一种有效的HIV-1抑制剂，对病毒逆转录使用无细胞病毒（天然内源性逆转录酶测定）的影响。我们报告说，APOBEC 3G以剂量依赖性方式抑制病毒DNA合成，并且不需要编辑能力。由于添加的第一个核苷酸的tRNA引物是不受A3 G和抑制的幅度增加，后来的逆转录中间体的测量，我们认为，APOBEC 3 G的作用是通过阻碍逆转录酶的转运酶沿着其RNA模板，也许是通过直接结合到RNA。这些结果为这类宿主抗病毒蛋白的生物活性提供了新的见解。
APOBEC3G (A3G) is a host cytidine deaminase that, in the absence of Vif, restricts HIV-1 replication and reduces the amount of viral DNA that accumulates in cells. Initial studies determined that A3G induces extensive mutation of nascent HIV-1 cDNA during reverse transcription. It has been proposed that this triggers the degradation of the viral DNA, but there is now mounting evidence that this mechanism may not be correct. Here, we use a natural endogenous reverse transcriptase assay to show that, in cell-free virus particles, A3G is able to inhibit HIV-1 cDNA accumulation not only in the absence of hypermutation but also without the apparent need for any target cell factors. We find that although reverse transcription initiates in the presence of A3G, elongation of the cDNA product is impeded. These data support the model that A3G reduces HIV-1 cDNA levels by inhibiting synthesis rather than by inducing degradation. APOBEC proteins are cell-encoded factors that inhibit the replication of numerous retroviruses, such as HIV-1, and retrotransposons. In many cases, inhibition is clearly associated with cytidine-to-uridine editing of viral or transposon DNA. On the other hand, a number of studies with particular APOBEC protein/substrate combinations, or engineered proteins that are editing-deficient, have indicated that inhibitory mechanism(s) distinct from editing are also operative. Here, we have analyzed the effects of APOBEC3G, a potent HIV-1 inhibitor, on viral reverse transcription using cell-free viruses (natural endogenous reverse transcriptase assays). We report that APOBEC3G inhibits viral DNA synthesis in a dose-dependent fashion, and does not require editing capabilities to do so. Because the addition of the first nucleotide to the tRNA primer is unaffected by A3G and the magnitude of inhibition increases as later reverse transcription intermediates are measured, we suggest that APOBEC3G acts by impeding the translocation of the reverse transcriptase enzyme along its RNA template, perhaps by binding directly to the RNA. These results provide novel insight into the biological activities of this class of host anti-viral proteins.
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