HIV-1 passage through the nuclear pore complex

HIV-1穿过核孔复合体

• 批准号: 10014380
• 负责人: vineet n kewalramani
• 金额: $ 40.25万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10014380
• 关键词: Affect; Affinity; Antiviral Agents; Attenuated; Binding; Binding Proteins; C-terminal; Caliber; Cell Line; Cell Nucleus; Cell membrane; Cells; Complex; Cyclosporine; Cytoplasm; Dependence; Diffusion; Dipeptides; Docking; Exhibits; Filament; Genes; Genome; Glycine; HIV-1; Hela Cells; Host Factor 1 Protein; Human; Human Cell Line; Human immunodeficiency virus test; Impairment; Infection; Integration Host Factors; Interphase Cell; Jurkat Cells; Karyopherins; Mediating; Membrane Fusion; Modeling; Mutation; N-terminal; Night Monkey; Nuclear; Nuclear Envelope; Nuclear Import; Nuclear Pore; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Nucleoplasm; Pathway interactions; Phenotype; Play; Pore Proteins; Process; Proline; Property; Proteins; RNA; Reporter; Resistance; Retroviridae; Reverse Transcription; Role; Route; Series; Side; Site; Small Interfering RNA; Structure; TRIM5 gene; Testing; Variant; Vesicular stomatitis Indiana virus; Viral; Virus; Virus Replication; cell type; experimental study; genome wide screen; heterokaryon; inhibitor/antagonist; knock-down; macromolecule; molecular mass; mutant; nucleocytoplasmic transport; particle; prevent; receptor; trafficking

In the mature viral particle, two plus sense copies of the HIV-1 RNA genome and viral enzymatic proteins are enclosed in a conical core, which is composed of a lattice of CA hexamers and pentamers. After the HIV-1 Envelope fuses with the target-cell membrane, the viral core is released into the cytoplasm. Although previous models suggested that the core immediately disassembled after membrane fusion, the persisting integrity of the core in the cytoplasm and the presence of CA at the NPC indicate that CA is not an immediate castaway during early steps of replication and is instead functionally associated with the PIC facilitating critical steps in infection. HIV-1 infects dividing and nondividing cells equally well due to efficient utilization of NPCs . NPCs are transport channels that span the nuclear envelope and regulate bidirectional transport of macromolecules between the nucleus and cytoplasm. With an estimated molecular mass of more than 100 MDa, NPCs are composed of multiple copies of 30 different proteins called nucleoporins (Nups); including transmembrane Nups (Poms), structural Nups, and FG (phenyalanine-glycine) Nups. FG Nups are essential components of the nuclear diffusion barrier, and provide docking sites for transport receptors. NPCs allow the passive diffusion of molecules with a diameter of up to 9 nm, and active translocation of large cargoes with a diameter of up to 39 nm. Structural analysis suggests that the NPC can dilate up to 50 nm in diameter. How the PIC traverses the NPC has been a subject of great inquiry. Early experiments with HIV-1/MLV chimeric viruses revealed that replacement of HIV-1 CA with MLV CA impairs the ability of HIV-1 to infect nondividing cells. These findings were supported by studies showing specific mutations in CA also prevented HIV-1 infection of nondividing cells. Studies with host factors that regulate nuclear transport solidified a role for CA in this process. Genome-wide screens for HIV-1 host factors exhibited significant overlap in identifying karyopherins as well as nuclear pore protein components. In particular, transportin-3 (TNPO3 or TRN-SR2), Nup358 (also known as RNABP2), and Nup153 emerged as potent regulators of HIV-1 infection. Nup358, the largest FG Nup, is the main component of NPC filaments that extend towards the cytoplasmic side of the pore, and plays an essential role in regulating cargo trafficking by forming a physical meshwork of FG repeats. Another FG Nup, Nup153, is anchored in the nuclear side of the NPC and its FG enriched filaments extend into the nucleoplasm. Depletion of Nup358 or Nup153 impairs HIV-1 infection and reduces 2-LTR circle formation but does not affect reverse transcription. Parallel studies from our group on CPSF6 had revealed that mutation forms of the protein blocked HIV-1 nuclear entry. Notably, we selected for a virus resistant to this nuclear entry block and obtained N74D HIV-1, again implicating a role of CA in regulating HIV-1 nuclear entry. However, unlike previous HIV-1 CA mutants that were impaired for nuclear entry, the N74D mutant virus efficiently infected nondividing cells. We exploited this property to test if N74D HIV-1 had different nuclear pore requirements relative to WT HIV-1. Indeed we soon discovered that TNPO3, Nup358, and Nup153 depletion impaired WT HIV-1 but not N74D HIV-1 infection. WT HIV-1 and N74D HIV-1 do overlap in utilization of some Nups, and N74D HIV-1 appears to be more dependent on Nup85 and Nup155 relative to WT HIV-1. With this finding, we and others more carefully investigated the relationship between CA and NPC components. Nup358 and Nup153 are thought to have distinct interactions with CA. One group has shown that Nup358 interacts with CA through a CypA-homology domain in C-terminal domain of Nup358, while other studies have found that three FG repeats in the N-terminal domain are sufficient to support HIV-1 infection. In contrast, Nup153 has been shown to bind to a conserved pocket in CA, also targeted by CPSF6 and the antiviral compounds PF-74 and BI-2. Interestingly, both Nup153 and CPSF6 possess 'FG' dipeptides that are necessary for binding CA. Notably, HIV-1 with CA mutations that prevent CypA-interaction also appeared to exhibit reduced sensitivity to Nup153 depletion. CypA is a highly abundant cellular protein and binds to the HIV-1 CA residues glycine 89 and proline 90 on the proline-rich loop between helices 4 and 5. Disruption of this interaction by CA mutation (such as G89V or P90A), by cyclosporine A (CsA, a competitive inhibitor of CypA), by CypA knockdown, or by homozygous deletion of the CypA gene, impairs HIV-1 replication in most human cells. Although CypA is incorporated into HIV-1 particles, CypA-CA interaction in the target cell, rather than in the producer cell, is necessary to enhance viral replication. CypA can also have deleterious effects on HIV-1 replication. Passage of HIV-1 in a CD4+ HeLa cells in the presence of CsA selected two mutants (A92E and G94D) in the CypA-binding loop of CA, although neither mutation affected the affinity of CA for CypA. Viruses bearing either A92E or G94D, show attenuated HIV-1 infectivity in some human cell lines, such as HeLa and H9 cells; however, reducing the CypA-CA interaction by CsA treatment or by introducing an additional mutation at proline 90, rescued HIV-1 infectivity in these cells. In contrast, the A92E and G94D mutants are able to replicate in the presence or absence of CsA in other cell lines, such as Jurkat cells. How these CA mutants behave very differently following CsA treatment in different target cells is still a puzzle. One possible explanation for these phenotypes is that differential effects of CypA mutants on HIV-1 replication in different cell types might be due to variations in CypA expression levels. In support of this idea, some studies have shown that there is correlation between different CypA expression levels and CsA effects in different cell lines. Another possibility to explain the CsA-dependency of these mutants in some cell types is a CypA-dependent restriction factor activity. In owl monkey cells, TRIMCyp blocked HIV-1 infection, but restriction was released by CA mutants that disrupt the interaction with CypA, and by CsA treatment. Since A92E HIV-1 infection was also rescued by CA mutants or CsA, it was hypothesized that a CypA-dependent restriction factor was inhibiting A92E replication in these cells. Use of heterokaryons has shown that CsA-dependent infection by A92E and G94D mutants is due to a dominant cellular restriction factor, but the restriction is not by retrovirus restriction factor TRIM5alpha. Because A92E HIV-1 and G94D HIV-1 are impaired at the level of nuclear entry in nonpersmissive cell types, we sought to understand whether CypA interaction with the WT HIV-1 PIC also had the potential to restrict nuclear transport - but HIV-1 had adapted in use of the NPC to overcome the impediment. To investigate the role of nuclear pore subcomplexes in HIV-1 infection, we systematically depleted all thirty-two human nuclear pore proteins in HeLa cells with siRNA, and then infected with VSV-G pseudotyped reporter viruses. In this effort, we have identified HIV-1 dependency on nucleoporins that are regulated by CA interactions with CypA. Strikingly, CypA dictates the nuclear import route utilized by HIV-1, favoring an FG-receptor mediated pathway. We hypothesize that CypA regulates access the N74 pocket of HIV-1 CA and facilitates subsequent interaction with FG-nucleoporins.

在成熟的病毒颗粒中，HIV-1 RNA基因组和病毒酶蛋白的两个加上感官拷贝被封闭在锥形核心中，该核心由Ca Hexamers和Pentamers的晶格组成。 HIV-1包膜与靶细胞膜融合后，病毒核被释放到细胞质中。尽管以前的模型表明，核心在膜融合后立即分解，但核心在细胞质中的持续完整性和NPC处的CA存在表明CA在复制的早期步骤中并不是立即抛弃，而是与PIC促进感染的关键步骤相关的功能。由于有效利用NPC，HIV-1感染了分裂和非分裂细胞的非分裂细胞。 NPC是跨越核包膜并调节核和细胞质之间大分子的双向转运的传输通道。 NPC的估计分子质量超过100 MDa，由30种不同蛋白质的副本（NUP）组成。包括跨膜NUP（POM），结构NUP和FG（苯甲氨酸 - 甘氨酸）NUP。 FG NUP是核扩散屏障的重要组成部分，并为传输受体提供对接位点。 NPC允许直径高达9 nm的分子的被动扩散，并且直径高达39 nm的大型货物的主动易位。结构分析表明，NPC的直径可扩张高达50 nm。图片如何穿越NPC一直是一个很好的询问的主题。 HIV-1/MLV嵌合病毒的早期实验表明，用MLV CA替换HIV-1 Ca会损害HIV-1感染非分散细胞的能力。这些发现得到的研究表明，在CA中特异性突变也阻止了非各个细胞的HIV-1感染。调节核转运的宿主因素的研究巩固了CA在此过程中的作用。 HIV-1宿主因子的全基因组筛选在鉴定核蛋白和核孔蛋白成分方面表现出显着的重叠。特别是，Transperin-3（TNPO3或TRN-SR2），NUP358（也称为RNABP2）和NUP153成为HIV-1感染的有效调节剂。 NUP358是最大的FG NUP，是NPC丝的主要组成部分，延伸到孔的细胞质侧，并通过形成FG重复的物理网络来调节货物贩运方面起着至关重要的作用。另一个FG NUP NUP153锚定在NPC的核侧，其FG富集的丝延伸到核质中。 NUP358或NUP153的耗竭会损害HIV-1的感染并减少2-LTR圆的形成，但不会影响逆转录。我们小组对CPSF6的平行研究表明，蛋白质的突变阻断了HIV-1核进入。值得注意的是，我们选择了对这种核进入阻滞有抗药性的病毒，并获得了N74D HIV-1，这再次暗示了CA在调节HIV-1核进入中的作用。但是，与以前的HIV-1 Ca突变体受到核进入的损害不同，N74D突变体病毒有效地感染了非分散细胞。我们利用该特性来测试N74D HIV-1相对于WT HIV-1是否具有不同的核孔要求。的确，我们很快发现TNPO3，NUP358和NUP153耗竭受损WT HIV-1，但没有N74D HIV-1感染。 WT HIV-1和N74D HIV-1在某些NUP的利用中确实重叠，而N74D HIV-1似乎更依赖于NUP85和NUP155相对于WT HIV-1。有了这一发现，我们和其他人更仔细地研究了CA和NPC组件之间的关系。 NUP358和NUP153被认为与CA有不同的相互作用。一组表明NUP358通过NUP358的C末端结构域中的CYPA - 同源结构域与CA相互作用，而其他研究发现N末端结构域中的三个FG重复序列足以支持HIV-1感染。相比之下，NUP153已显示与CA中的保守口袋结合，也是CPSF6和抗病毒化合物PF-74和BI-2的靶向。有趣的是，NUP153和CPSF6都具有结合Ca所必需的“ FG”二肽。值得注意的是，具有预防CYPA交流的Ca突变的HIV-1似乎也表现出对NUP153耗竭的敏感性降低。 CYPA是一种高度丰富的细胞蛋白，与HIV-1 Ca残基89和脯氨酸90在螺旋线4和5之间的脯氨酸环上结合。通过Ca突变（例如G89V或P90A）（例如G89V或P90A）（例如CSA A（CSA）（CSA）（CSA），CYPA，CIPA型疾病型疾病，通过CA突变（例如G89V或P90A）破坏这种相互作用。 CYPA基因，会损害大多数人类细胞中的HIV-1复制。尽管CYPA被掺入HIV-1颗粒中，但对于增强病毒复制是必要的，而靶细胞中的CYPA-CA相互作用，而不是生产者细胞中的相互作用。 CYPA也可能对HIV-1复制产生有害影响。在CSA存在的情况下，HIV-1在CSA存在的情况下通过CA的CAPA结合环中选择了两个突变体（A92E和G94D），尽管这两个突变都不影响CA对CYPA的亲和力。携带A92E或G94D的病毒在某些人细胞系（例如HELA和H9细胞）中显示出HIV-1感染性的减弱；但是，通过CSA处理降低CYPA-CA相互作用，或通过在Proline 90上引入额外的突变，从而挽救了这些细胞中的HIV-1感染性。相反，在其他细胞系（例如Jurkat细胞）中，A92E和G94D突变体能够在存在或不存在CSA的情况下复制。在不同靶细胞中CSA处理后，这些CA突变体的行为如何仍然是一个难题。这些表型的一种可能解释是，CYPA突变体对不同细胞类型中HIV-1复制的差异影响可能是由于CYPA表达水平的变化所致。为了支持这一想法，一些研究表明，不同细胞系中不同的CYPA表达水平与CSA效应之间存在相关性。解释这些突变体在某些细胞类型中的CSA依赖性的另一种可能性是依赖CYPA的限制因子活性。在猫头鹰猴细胞中，三膜细胞阻断了HIV-1的感染，但CA突变体释放了限制，从而破坏了与CYPA的相互作用以及通过CSA处理。由于A92E HIV-1感染也被CA突变体或CSA挽救，因此假设CYPA依赖性限制因子抑制了这些细胞中的A92E复制。异源性的使用表明，A92E和G94D突变体的CSA依赖性感染是由于主要的细胞限制因子引起的，但限制不是逆转录病毒限制因子Trim5alpha。由于A92E HIV-1和G94D HIV-1在非授予细胞类型的核进入水平上受到损害，因此我们试图了解CYPA与WT HIV-1 PIC的相互作用是否也有可能限制核运输 - 但是HIV-1适用于NPC的使用来胜过障碍。为了研究核孔亚复合物在HIV-1感染中的作用，我们系统地耗尽了所有三十二个人类核孔蛋白在HeLa细胞中使用siRNA，然后被VSV-G-pseudotypecty型报告的病毒感染。在这项工作中，我们已经确定了HIV-1对与CA相互作用的核孔蛋白的依赖性。令人惊讶的是，CYPA决定了HIV-1使用的核进口途径，有利于FG受体介导的途径。我们假设CYPA调节访问HIV-1 CA的N74口袋，并促进随后与FG-核孔蛋白的相互作用。