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首页> 外文期刊>MBio >Membrane Binding and Subcellular Localization of Retroviral Gag Proteins Are Differentially Regulated by MA Interactions with Phosphatidylinositol-(4,5)-Bisphosphate and RNA
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Membrane Binding and Subcellular Localization of Retroviral Gag Proteins Are Differentially Regulated by MA Interactions with Phosphatidylinositol-(4,5)-Bisphosphate and RNA

机译：膜结合和逆转录病毒gag蛋白的亚细胞定位受与磷脂酰肌醇-（4,5）-双磷酸和RNA的MA相互作用的差异调节。

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ABSTRACT The matrix (MA) domain of HIV-1 mediates proper Gag localization and membrane binding via interaction with a plasma-membrane (PM)-specific acidic phospholipid, phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P_(2)]. HIV-1 MA also interacts with RNA, which prevents Gag from binding to membranes containing phosphatidylserine, a prevalent cellular acidic phospholipid. These results suggest that the MA-bound RNA promotes PM-specific localization of HIV-1 Gag by blocking nonspecific interactions with cellular membranes that do not contain PI(4,5)P_(2). To examine whether PI(4,5)P_(2)dependence and RNA-mediated inhibition collectively determine MA phenotypes across a broad range of retroviruses and elucidate the significance of their interrelationships, we compared a panel of Gag-leucine zipper constructs (GagLZ) containing MA of different retroviruses. We found that in vitro membrane binding of GagLZ via HIV-1 MA and Rous sarcoma virus (RSV) MA is both PI(4,5)P_(2)dependent and susceptible to RNA-mediated inhibition. The PM-specific localization and virus-like particle (VLP) release of these GagLZ proteins are severely impaired by overexpression of a PI(4,5)P_(2)-depleting enzyme, polyphosphoinositide 5-phosphatase IV (5ptaseIV). In contrast, membrane binding of GagLZ constructs that contain human T-lymphotropic virus type 1 (HTLV-1) MA, murine leukemia virus (MLV) MA, and human endogenous retrovirus K (HERV-K) MA is PI(4,5)P_(2)independent and not blocked by RNA. The PM localization and VLP release of these GagLZ chimeras were much less sensitive to 5ptaseIV expression. Notably, single amino acid substitutions that confer a large basic patch rendered HTLV-1 MA susceptible to the RNA-mediated block, suggesting that RNA readily blocks MA containing a large basic patch, such as HIV-1 and RSV MA. Further analyses of these MA mutants suggest a possibility that HIV-1 and RSV MA acquired PI(4,5)P_(2)dependence to alleviate the membrane binding block imposed by RNA. IMPORTANCE MA basic residues in the HIV-1 structural protein Gag interact with phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P_(2)] and RNA. RNA inhibits HIV-1 MA binding to non-PI(4,5)P_(2)acidic lipids. This inhibition may promote PM specificity of Gag membrane binding, an early essential step in virus assembly. However, whether and how relationships between these interactions have developed among retroviruses are poorly understood. In this study, by comparing diverse retroviral MA domains, we elucidated a strong correlation among PI(4,5)P_(2)dependence, susceptibility to RNA-mediated inhibition, and cellular behaviors of Gag. Mutagenesis analyses suggest that a large basic patch on MA is sufficient to confer susceptibility to RNA-mediated inhibition but not for PI(4,5)P_(2)-dependent membrane binding. Our findings highlight RNA’s role as a general blocker of large basic patches and suggest a possibility that some retroviruses, including HIV-1, have evolved to bind PI(4,5)P_(2), while others have adopted smaller basic patches on their MA domains, to overcome the RNA-mediated restriction of membrane binding.
机译：摘要HIV-1的基质（MA）结构域通过与血浆膜（PM）特异性酸性磷脂，磷脂酰肌醇-（4,5）-双磷酸[PI（4,5）P_ （2）]。 HIV-1 MA还与RNA相互作用，从而阻止Gag结合到含有磷脂酰丝氨酸（一种普遍的细胞酸性磷脂）的膜上。这些结果表明，与MA结合的RNA通过阻止与不含PI（4,5）P_（2）的细胞膜的非特异性相互作用来促进HIV-1 Gag的PM特异性定位。为了检查PI（4,5）P_（2）的依赖性和RNA介导的抑制作用是否共同决定了广泛的逆转录病毒的MA表型，并阐明了它们之间的相互关系，我们比较了一组Gag-亮氨酸拉链构建体（GagLZ）包含不同逆转录病毒的MA。我们发现通过HIV-1 MA和劳斯肉瘤病毒（RSV）MA的GagLZ的体外膜结合既是PI（4,5）P_（2）依赖性的，也容易受到RNA介导的抑制作用。这些GagLZ蛋白的PM特异性定位和病毒样颗粒（VLP）释放由于PI（4,5）P_（2）耗尽酶多磷酸肌醇5磷酸酶IV（5ptaseIV）的过度表达而受到严重损害。相反，包含人类T型淋巴病毒1型（HTLV-1）MA，鼠白血病病毒（MLV）MA和人类内源性逆转录病毒K（HERV-K）MA的GagLZ构建体的膜结合是PI（4,5） P_（2）独立且不受RNA阻断。这些GagLZ嵌合体的PM定位和VLP释放对5ptaseIV表达的敏感性低得多。值得注意的是，赋予大的基本补丁的单个氨基酸取代使HTLV-1 MA容易受RNA介导的阻断作用，表明RNA容易阻断含有大的基本补丁的MA，例如HIV-1和RSV MA。这些MA突变体的进一步分析表明HIV-1和RSV MA获得PI（4,5）P_（2）依赖性以减轻RNA施加的膜结合阻滞的可能性。重要说明HIV-1结构蛋白Gag中的MA基本残基与磷脂酰肌醇-（4,5）-双磷酸[PI（4,5）P_（2）]和RNA相互作用。 RNA抑制HIV-1 MA与非PI（4,5）P_（2）酸性脂质的结合。这种抑制作用可能会促进Gag膜结合的PM特异性，这是病毒装配过程中的早期重要步骤。但是，人们对逆转录病毒之间这些相互作用之间是否建立关系以及如何建立关系的了解很少。在这项研究中，通过比较不同的逆转录病毒MA域，我们阐明了PI（4,5）P_（2）依赖性，对RNA介导的抑制的敏感性以及Gag的细胞行为之间有很强的相关性。诱变分析表明MA上的一个大的基本补丁足以赋予对RNA介导的抑制作用的敏感性，但不足以依赖PI（4,5）P_（2）的膜结合。我们的研究结果突显了RNA作为大的基本补丁的一般阻断剂的作用，并暗示了某些逆转录病毒（包括HIV-1）已经进化为结合PI（4,5）P_（2）的可能性，而另一些则在其基础上采用了较小的基本补丁MA域，以克服RNA介导的膜结合限制。

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