Defining Mechanisms of HIV-1 Gag:RNA Interactions and Virus Assembly

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

We are studying the trafficking of HIV-1 macromolecules and assembly. Once it has exited the nucleus, HIV-1 RNA needs to travel to various subcellular locations to carry out its functions, including dimerizing with another viral RNA and assembling into a viral particle. Our current and future studies are focused on exploring the initiation of Gag:RNA interaction in the cells, examining RNA trafficking in T cells, and defining the role of the viral RNA genome in particle assembly. We will also determine the kinetics of virus maturation by live-cell imaging and determine the factors that shape RNA structures in the virions. ___BACKGROUND: To generate infectious particles, HIV-1 RNA and proteins traffic to the plasma membrane, the major virus assembly site. The Gag protein drives HIV-1 assembly and interacts with viral RNA and proteins to ensure the packaging of the viral genome and replication machinery. Additionally, Gag interacts with host proteins for virus egress. It has often been suggested that the interactions of HIV-1 RNA and Gag leading to assembly are initiated in the cytoplasm. To better understand the regulation of virus assembly, we are examining cytoplasmic HIV-1 Gag:RNA and RNA:RNA interactions. We are also studying HIV-1 RNA trafficking in T cells and exploring the role of the RNA genome in HIV assembly. ___The studies in this project sought to address several unanswered questions on the trafficking of HIV-1 macromolecules and virus assembly, which are essential processes in viral replication. We visualized HIV-1 RNA and monitored its movement in the cytoplasm by using single-molecule tracking and determined that most of the HIV-1 RNA molecules have diffusive movement. Additionally, we showed that, in the presence of Gag, HIV-1 RNA is transported by diffusion with mobility similar to that of RNAs unable to express functional Gag. These studies have defined a major mechanism important to HIV-1 gene expression. Polarized T cells not only constitute a majority of HIV-1 target cells in vivo but also play a critical role in the spread of HIV-1 via cell-to-cell infection. To determine the distribution of HIV-1 RNA in polarized T cells, we visualized the RNA and found that HIV-1 RNAs were enriched near the uropod plasma membrane in a Gag-dependent manner. These results indicated that HIV-1 RNA is enriched during the process of virus assembly. As the Gag-enriched uropod is more likely to form a virological synapse, such targeting facilitates cell-mediated infection and virus spread in vivo. : We have examined the dynamics of viral RNA and Gag-RNA interactions near the plasma membrane by total internal reflection fluorescence (TIRF) microscopy. We found that in the absence of Gag, most of the HIV-1 RNAs stayed near the plasma membrane transiently. The presence of Gag significantly increased the time RNAs stay near the plasma membrane. We observed that the frequency of HIV-1 RNA packaging was dependent on the Gag expression level. Our results showed that only a small proportion of the HIV-1 RNAs (approximately one tenth to one third) that reached the plasma membrane was incorporated into viral protein complexes. These studies determined the dynamics of HIV-1 RNA on the plasma membrane and obtained the temporal information of RNA-Gag interactions that lead to RNA encapsidation. ___We have studied the role of HIV-1 RNA during virus assembly. We hypothesize that HIV-1 full-length RNA facilitates the formation of viral particles. To test our hypothesis, we examined the efficiencies of particle formation with and without RNA containing HIV-1 packaging signal. We found that, although viral particles can be generated without the presence of RNA genome, the HIV-1 RNA genome facilitates the production of HIV-1 particles. Furthermore, the effects of the RNA genome are dependent on the level of Gag expressed in the cells. These observations are consistent with our hypothesis that packaging a dimeric RNA is the nucleation process of HIV-1 assembly. ACCOMPLISHMENT: Although several nucleocapsid (NC)-binding sites have been identified in the 5' UTR of HIV-1 RNA, whether these sites direct HIV-1 RNA genome packaging has not been fully investigated. It has been shown that HIV-1 NC binds exposed guanosines. Thus, we introduced G-to-A substitution on exposed guanosines in the NC-binding sites and examined the ability of these mutant RNAs to compete for packaging with wild-type RNA. We observed that multiple NC-binding sites affect RNA packaging; of the sites tested, those located at stem-loop 1 of the 5' UTR had the most significant effects. These sites were previously reported as the primary NC-binding sites using a chemical probing reverse-footprinting assay and the major Gag binding sites using an in vitro assay. We found that substituting 3 to 4 guanosines resulted in less than twofold defects in packaging. However, when mutations were combined, severe defects were observed. Furthermore, combining mutations had synergistic effects on RNA packaging defects, suggesting redundancy in Gag:RNA interactions and a requirement for multiple Gag proteins to bind RNA to encapsidate the HIV-1 genome. Additionally, we examined whether exposed guanosines in the 5' untranslated region are important for HIV-2 genome packaging. We found that mutating as few as three guanosines significantly reduce RNA packaging efficiency. However, not all guanosines examined have the same effect; instead, a hierarchical order exists wherein a primary site, a secondary site, and three tertiary sites are identified. Furthermore, there are functional overlaps in these sites and mutations of more than one site can act synergistically to cause genome packaging defects. HIV-1 Gag:RNA interactions mediate genome packaging, but the mechanism remains unclear. We posited that, besides RNA binding, other properties of Gag contribute to genome packaging. To examine features of Gag that are important for genome packaging, we established complementation systems that separate the particle-assembling and RNA-binding functions of Gag: we used a set of Gag proteins to drive particle assembly and an RNA-binding Gag to package HIV-1 RNA. We have developed two types of RNA-binding Gag in which packaging is mediated by the authentic nucleocapsid (NC) domain or by a nonviral RNA-binding domain. We found that in both cases, mutations that affect the multimerization or plasma membrane anchoring properties of Gag reduce or abolish RNA packaging. These mutant Gag can coassemble into particles but cannot package the RNA genome efficiently. Our findings indicate that HIV-1 RNA packaging occurs at the plasma membrane and RNA-binding Gag needs to multimerize on RNA to encapsidate the viral genome.