Assembly and Complementation of Viral Proteins during Retroviral Replication

使用简单的逆转录病毒作为模型系统，我们研究了病毒组装的三个方面。我们已经解剖了一个对病毒组装很重要的基序，它位于小鼠白血病病毒(MLV)衣壳(CA)的C-末端区域。从我们的突变分析中产生的数据，我们假设这个基序中的序列形成了一个-螺旋；螺旋结构和螺旋的相位的保持对其功能至关重要。我们正在进行实验，以进一步研究这个基序，以深入了解CA在病毒组装中的功能域。我们已经进行了实验，以确定在MLV和脾坏死病毒(SNV)Gag共组装中重要的结构域(S)。MLV和SNV是远亲病毒。利用杂交病毒，我们确定了MLV和SNV Gag的功能互补需要同源的CA。我们还研究了MLV Gag组件，这些组件对早期病毒复制事件很重要。我们发现，有效的病毒感染需要同源的CA和p12结构域。这项研究表明，成熟的CA和p12蛋白在早期MLV复制过程中协同工作，很可能是为了逆转录/前整合复合体的运输。我们还检测了Gag突变对病毒粒子RNA二聚体成熟的影响。我们检测了两个Gag突变体：一个MLV CA缺失突变和一个HIV-1 PTAP替代突变。我们发现，尽管这些病毒粒子中的大部分Gag蛋白被切割，但病毒粒子RNA二聚体并不成熟。这些数据表明，病毒粒子RNA二聚体的成熟不仅需要GAG的蛋白水解性切割，而且可能与病毒粒子的形态变化有关。我们现在正在研究基于慢病毒的系统，即HIV-1和HIV-2的功能互补和共组装。使用遗传互补分析和成像方法，我们已经证明了HIV-1和HIV-2 Gag可以共组装；而且，这些共组装的病毒可以具有传染性。据我们所知，这是第一个证明两个慢病毒可以有这样的相互作用的同类研究。在这项研究中，我们目前正在跟踪几条线索。我们打算定义异源Gag相互作用的程度，并确定这种混合颗粒在病毒复制中的优势。例如，我们打算确定混合颗粒是否可以更好地对抗药物治疗，并通过绕过细胞防御蛋白来适应不同宿主细胞的复制。此外，我们正在研究HIV-1和其他慢病毒的RNA包装机制。逆转录病毒可以通过互补相互作用。来自两种不同病毒的蛋白质和RNA可以在双重感染的细胞中相互作用。据估计，有100万人双重感染艾滋病毒-1和艾滋病毒-2，这为这两种病毒在人类群体中可能的相互作用提供了基础。使用两种不同的系统，我们检测了异源Gag蛋白是否可以共组装并相互补充功能。我们观察到MLV和SNV Gag可以相互作用，但它们的基因组必须在Gag中编码一个同源衣壳(CA)结构域来挽救病毒复制。相比之下，HIV-1和HIV-2的Gag蛋白可以通过功能互补共组装并挽救病毒复制。我们将研究同时含有HIV-1和HIV-2 Gag的颗粒的性质，并进一步确定不同逆转录病毒Gag蛋白的相互作用和互补程度。我们将量化病毒复制所需的功能核衣壳(NC)和晚期结构域基序，并将确定病毒复制不同步骤对各种病毒蛋白功能的需求。利用我们最新开发的成像系统，我们将研究活细胞中Gag-Gag和Gag-RNA的相互作用。我们还将探索凝聚核心的形成和稳定的RNA二聚体之间的联系。这些研究的结果将提供对逆转录病毒通过互补相互作用的见解，并将进一步了解病毒组装过程和Gag蛋白在病毒复制的不同阶段的功能。[对应于2007年4月HIV耐药计划实地考察报告中的HU项目3]

Using simple retroviruses as a model system, we have examined three aspects of virus assembly. We have dissected a motif that is important for virus assembly and located at the C-terminal region of murine leukemia virus (MLV) capsid (CA). From data generated in our mutational analyses, we hypothesize that the sequences in this motif form an α-helix; maintenance of the helical structure and the phase of the helix are critical to its function. We are performing experiments to further investigate this motif to gain insight into the functional domains of CA in virus assembly. We have performed experiments to determine the domain(s) in Gag that is important for the coassembly of MLV and spleen necrosis virus (SNV) Gag. MLV and SNV are distantly related viruses. Using hybrid viruses, we determined that homologous CA is needed for functional complementation of MLV and SNV Gag. We have also studied the MLV Gag components that are important for early virus replication events. We found that homologous CA and p12 domains are required for efficient virus infection. This study indicated that the mature CA and p12 proteins cooperate during early MLV replication, most likely for the transport of the reverse transcription/preintegration complex. We have also examined the effects of Gag mutations on virion RNA dimer maturation. We examined two Gag mutants: an MLV CA deletion mutation and an HIV-1 PTAP substitution mutant. We found that although most of Gag proteins from these virions were cleaved, virion RNA dimers did not mature. These data suggest that virion RNA dimer maturation requires more than proteolytic cleavage of Gag and is likely to be associated with virion morphological changes. We are now studying functional complementation and coassembly in lentivirus-based systems namely, HIV-1 and HIV-2. Using a genetic complementation assay and an imaging approach, we have demonstrated that HIV-1 and HIV-2 Gag can coassemble; furthermore, these coassembled viruses can be infectious. To our knowledge, this is the first study of its kind to demonstrate that two lentiviruses can have such interactions. We are currently following several leads in this study. We intend to define the extent of heterologous Gag interaction, and determine the advantage of such mixed particles in virus replication. For example, we intend to determine whether mixed particles can better counter drug treatment and adapt to replication of a different host cell via bypassing of the cellular defense proteins. Additionally, we are studying the RNA packaging mechanisms of HIV-1 and other lentiviruses. Retroviruses can interact with each other through complementation. Proteins and RNAs from two different viruses can comingle and interact in doubly infected cells. An estimated one million people are dually infected with HIV-1 and HIV-2, providing the basis for possible interactions between these two viruses in human populations. Using two different systems, we examined whether heterologous Gag proteins can coassemble and complement each others functions. We have observed that MLV and SNV Gag can interact but their genomes must encode a homologous capsid (CA) domain in Gag to rescue virus replication. In contrast, the Gag proteins of HIV-1 and HIV-2 can coassemble and rescue virus replication via functional complementation. We will study the properties of particles containing both HIV-1 and HIV-2 Gag, and further define the extent of interactions and complementation of Gag proteins from different retroviruses. We will quantify the functional nucleocapsid (NC) and late-domain motif needed for virus replication and will determine the requirement of various viral protein functions in different steps of viral replication. With our newly developed imaging systems, we will study the Gag-Gag and Gag-RNA interactions in living cells. We will also explore the link between formation of the condensed core and a stable RNA dimer. Results from these studies will provide insights into retroviral interactions via complementation and will further our understanding of the process of virus assembly and the functions of Gag proteins in various stages of viral replication. [Corresponds to Hu Project 3 in the April 2007 site visit report of the HIV Drug Resistance Program]