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

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

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. 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. We have studied the assembly of HIV-1 Gag containing heterologous NC domain that does not recognize HIV-1 RNA and the adaptation of HIV-1 to improve Gag:RNA interactions. These studies revealed the minor changes in the NC domain can alter Gag:RNA interactions and improve RNA packaging and viral replication. 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. 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 will continue these live-cell imaging studies to determine the kinetics of HIV-1 and nonviral RNA on the plasma membrane and examine the factors that affect their retention on the plasma membrane. 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. We will continue these studies to examine the role of viral RNA in facilitating HIV particle assembly.