HIV Drug Resistance Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, USA.
J Virol. 2014 Apr;88(7):3850-60. doi: 10.1128/JVI.03456-13. Epub 2014 Jan 22.
Many murine leukemia viruses (MLVs) are partially resistant to restriction by mouse APOBEC3 (mA3) and essentially fully resistant to induction of G-to-A mutations by mA3. In contrast, Vif-deficient HIV-1 (ΔVif HIV-1) is profoundly restricted by mA3, and the restriction includes high levels of G-to-A mutation. Human APOBEC3G (hA3G), unlike mA3, is fully active against MLVs. We produced a glutathione S-transferase-mA3 fusion protein in insect cells and demonstrated that it possesses cytidine deaminase activity, as expected. This activity is localized within the N-terminal domain of this 2-domain protein; the C-terminal domain is enzymatically inactive but required for mA3 encapsidation into retrovirus particles. We found that a specific arginine residue and several aromatic residues, as well as the zinc-coordinating cysteines in the C-terminal domain, are necessary for mA3 packaging; a structural model of this domain suggests that these residues line a potential nucleic acid-binding interface. Mutation of a few potential phosphorylation sites in mA3 drastically reduces its antiviral activity by impairing either deaminase activity or its encapsidation. mA3 deaminates short single-stranded DNA oligonucleotides preferentially toward their 3' ends, whereas hA3G exhibits the opposite polarity. However, when packaged into infectious ΔVif HIV-1 virions, both mA3 and hA3G preferentially induce deaminations toward the 5' end of minus-strand viral DNA, presumably because of the sequence of events during reverse transcription in vivo. Despite the fact that mA3 in MLV particles does not induce detectable deaminations upon infection, its deaminase activity is easily detected in virus lysates. We still do not understand how MLV resists mA3-induced G-to-A mutation.
One way that mammalian cells defend themselves against infection by retroviruses is with APOBEC3 proteins. These proteins convert cytidine bases to uridine bases in retroviral DNA. However, mouse APOBEC3 protein blocks infection by murine leukemia viruses without catalyzing this base change, and the mechanism of inhibition is not understood in this case. We have produced recombinant mouse APOBEC3 protein for the first time and characterized it here in a number of ways. Our mutational studies shed light on the mechanism by which mouse APOBEC3 protein is incorporated into retrovirus particles. While mouse APOBEC3 does not catalyze base changes in murine leukemia virus DNA, it can be recovered from these virus particles in enzymatically active form; it is still not clear why it fails to induce base changes when these viruses infect new cells.
许多鼠白血病病毒（MLV）对小鼠 APOBEC3（mA3）的限制具有部分抗性，并且基本上对 mA3 诱导的 G 到 A 突变具有完全抗性。相比之下，缺乏 Vif 的 HIV-1（ΔVif HIV-1）受到 mA3 的强烈限制，并且这种限制包括高水平的 G 到 A 突变。人 APOBEC3G（hA3G）与 mA3 不同，对 MLV 完全有效。我们在昆虫细胞中产生了谷胱甘肽 S-转移酶-mA3 融合蛋白，并证明它具有胞嘧啶脱氨酶活性，这是预期的。这种活性位于该 2 结构域蛋白的 N 端结构域内；C 端结构域无酶活性，但需要将 mA3 包装到逆转录病毒颗粒中。我们发现，特定的精氨酸残基和几个芳香族残基以及 C 端结构域中的锌配位半胱氨酸对于 mA3 包装是必需的；该结构域的结构模型表明，这些残基排列在潜在的核酸结合界面上。mA3 中少数潜在磷酸化位点的突变通过损害脱氨酶活性或其包装而严重降低其抗病毒活性。mA3 优先在其 3'末端使短单链 DNA 寡核苷酸脱氨，而 hA3G 则表现出相反的极性。然而，当包装到感染性 ΔVif HIV-1 病毒粒子中时，mA3 和 hA3G 都优先在负链病毒 DNA 的 5'末端诱导脱氨，这可能是由于体内逆转录过程中的一系列事件。尽管 MLV 颗粒中的 mA3 在感染时不会诱导可检测到的脱氨，但在病毒裂解物中很容易检测到其脱氨酶活性。我们仍然不理解 MLV 如何抵抗 mA3 诱导的 G 到 A 突变。
哺乳动物细胞抵御逆转录病毒感染的一种方法是使用 APOBEC3 蛋白。这些蛋白质将胞嘧啶碱基转化为逆转录病毒 DNA 中的尿嘧啶碱基。然而，小鼠 APOBEC3 蛋白阻止了鼠白血病病毒的感染，而没有催化这种碱基变化，并且在这种情况下，抑制机制尚不清楚。我们首次生产了重组小鼠 APOBEC3 蛋白，并在此基础上对其进行了多种特性的描述。我们的突变研究阐明了小鼠 APOBEC3 蛋白被掺入逆转录病毒颗粒的机制。虽然小鼠 APOBEC3 不会在鼠白血病病毒 DNA 中催化碱基变化，但它可以从这些病毒颗粒中以酶活性形式回收；当这些病毒感染新细胞时，它为什么不能诱导碱基变化仍然不清楚。
