Distribution and Redistribution of HIV-1 Nucleocapsid Protein in Immature, Mature, and Integrase-Inhibited Virions: a Role for Integrase in Maturation
Distribution and Redistribution of HIV-1 Nucleocapsid Protein in Immature, Mature, and Integrase-Inhibited Virions: a Role for Integrase in Maturation
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DOI：
10.1128/jvi.01522-15
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发表时间：
2015-10-01
影响因子：
5.4
通讯作者：
Steven, Alasdair C.
中科院分区：
文献类型：
--
作者：
Fontana, Juan;Jurado, Kellie A.;Steven, Alasdair C.
在病毒粒子成熟过程中，HIV-1衣壳蛋白组装成一个锥形核心，内含病毒核糖核蛋白(VRNP)复合体，被认为主要由病毒RNA和核衣壳蛋白(NC)组成。病毒感染后，病毒RNA被逆转录成双链DNA，然后在整合酶(IN)催化下被整合到宿主染色体中。某些IN突变(II类)和抗病毒药物(变构IN抑制剂[ALLINI])会对成熟产生不利影响，导致含有“偏心凝聚体”的病毒粒子，即位于看似空壳外的电子致密聚集体。在这里，我们证明了除了电子密度的这种错位化之外，II类IN突变和ALLINI都增加了衣壳畸形的病毒粒子的比例(从类似的12%增加到类似的53%)。偏心凝析油具有很高的NC含量，这是一种利用NC对辐射损伤的敏感性的新型标记技术--断层气泡图成像。断层气泡图还定位了野生型核心内的NC，并将未成熟病毒粒子中的球状搞笑外壳衬里。我们的结论是，偏心冷凝物代表未包装的vRNP，IN的遗传或药物抑制都会损害vRNP与成熟核心的整合。提供反式IN作为VPR-IN融合蛋白的一部分，部分恢复了具有内部电子密度的锥形核心的形成和II类IN缺失突变病毒的感染性。此外，ALLINI诱导偏心冷凝物形成的能力既需要IN，也需要病毒RNA。基于这些观察，我们提出了IN在HIV-1成熟过程中启动核心形态形成和vRNP掺入成熟核心的作用。重要的是，成熟是HIV-1感染性所必需的过程，涉及核心组装，病毒核糖核蛋白(vRNP，由vRNA和核衣壳蛋白[NC]组成)被包装成锥形衣壳。变构整合酶抑制剂(ALLINI)影响多种病毒过程。我们已经鉴定了具有相同表型的ALLINI和整合酶突变体。首先，通过比较ALLINI对病毒周期几个步骤的影响，我们表明抑制成熟是复合效力的原因。其次，通过低温电子断层扫描，我们发现ALLINI破坏了锥形衣壳的组装。第三，通过发展断层气泡图成像，它专门标记NC蛋白，我们发现ALLINI阻止vRNP包装；相反，vRNP在核心外形成“偏心凝聚体”。第四，当整合酶以反式形式提供时，典型的整合酶缺失病毒的畸形核心被锥形核心部分取代。第五，vRNA是Allini诱导偏心凝析形成所必需的。这些观察结果表明整合酶参与了衣壳的形态发生和vRNP包装。
During virion maturation, HIV-1 capsid protein assembles into a conical core containing the viral ribonucleoprotein (vRNP) complex, thought to be composed mainly of the viral RNA and nucleocapsid protein (NC). After infection, the viral RNA is reverse transcribed into double-stranded DNA, which is then incorporated into host chromosomes by integrase (IN) catalysis. Certain IN mutations (class II) and antiviral drugs (allosteric IN inhibitors [ALLINIs]) adversely affect maturation, resulting in virions that contain "eccentric condensates," electron-dense aggregates located outside seemingly empty capsids. Here we demonstrate that in addition to this mislocalization of electron density, a class II IN mutation and ALLINIs each increase the fraction of virions with malformed capsids (from similar to 12% to similar to 53%). Eccentric condensates have a high NC content, as demonstrated by "tomo-bubblegram" imaging, a novel labeling technique that exploits the susceptibility of NC to radiation damage. Tomo-bubblegrams also localized NC inside wild-type cores and lining the spherical Gag shell in immature virions. We conclude that eccentric condensates represent nonpackaged vRNPs and that either genetic or pharmacological inhibition of IN can impair vRNP incorporation into mature cores. Supplying IN in trans as part of a Vpr-IN fusion protein partially restored the formation of conical cores with internal electron density and the infectivity of a class II IN deletion mutant virus. Moreover, the ability of ALLINIs to induce eccentric condensate formation required both IN and viral RNA. Based on these observations, we propose a role for IN in initiating core morphogenesis and vRNP incorporation into the mature core during HIV-1 maturation.IMPORTANCEMaturation, a process essential for HIV-1 infectivity, involves core assembly, whereby the viral ribonucleoprotein (vRNP, composed of vRNA and nucleocapsid protein [NC]) is packaged into a conical capsid. Allosteric integrase inhibitors (ALLINIs) affect multiple viral processes. We have characterized ALLINIs and integrase mutants that have the same phenotype. First, by comparing the effects of ALLINIs on several steps of the viral cycle, we show that inhibition of maturation accounts for compound potency. Second, by using cryoelectron tomography, we find that ALLINIs impair conical capsid assembly. Third, by developing tomo-bubblegram imaging, which specifically labels NC protein, we find that ALLINIs block vRNP packaging; instead, vRNPs form "eccentric condensates" outside the core. Fourth, malformed cores, typical of integrase-deleted virus, are partially replaced by conical cores when integrase is supplied in trans. Fifth, vRNA is necessary for ALLINI-induced eccentric condensate formation. These observations suggest that integrase is involved in capsid morphogenesis and vRNP packaging.