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. These experiments will provide an understanding of aspects of HIV-1 replication that we currently know very little about and will allow us to better understand how HIV-1 uses the cellular machinery to traffic its macromolecules and generate infectious viruses.__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.__Immature particles need to go through a maturation process to become infectious viruses. During this process, protease (PR) in the Gag-Pol polyprotein is activated and cleaves Gag/Gag-Pol polyproteins to release mature proteins. This process allows the rearrangement of the virion structure including the capsid proteins, which form a conical core. We are studying the timing of the proteolytic cleavage and virus maturation by using live-cell imaging.__During virion maturation, the viral RNA dimer also changes and becomes more thermodynamically stable. Additionally, the dimeric RNA genome needs to be included inside the core along with reverse transcriptase and integrase, which allows the synthesis and integration of the viral DNA when entering the new host cell. We are studying the structures of the virion RNA and factors that influence these structures. These five areas of focus in this project seek to address several unanswered questions on HIV-1 assembly and maturation, which are essential processes in viral replication.__ACCOMPLISHMENTS: HIV-1 RNA must go to specific subcellular compartments to be translated or packaged into viral particles. Proper RNA trafficking is required for the functions of RNA and its encoded proteins. However, little was known about how HIV-1 RNA is transported in the cytoplasm. We visualized HIV-1 RNA and monitored its movement in the cytoplasm by using single-molecule tracking. We observed that most of the HIV-1 RNA molecules moved in a nondirectional, random-walk manner, which did not require intact cytoskeletal structure, and that the mean-squared distance traveled by the RNA increased linearly with time, indicative of diffusive movement. We also observed that a single HIV-1 RNA molecule could move at various speeds when traveling through the cytoplasm, indicating that its movement was strongly affected by the immediate environment. When Gag was expressed, a significant portion of HIV-1 RNA may be transported as Gag-RNA complexes, whose properties could differ greatly from Gag-free RNA. Therefore, we also analyzed the cytoplasmic HIV-1 RNA movement in the presence of sufficient Gag for virion assembly and found that HIV-1 RNA is still 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 by using live-cell microscopy and a Bgl-YFP construct that specifically recognizes stem-loop sequences engineered into the HIV-1 genome. We found that HIV-1 RNAs were enriched near the uropod plasma membrane; furthermore, RNA enrichment was dependent on Gag and its ability to interact with the HIV-1 RNA. 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 than regions at the leading edge, such targeting facilitates cell-mediated infection and virus spread in vivo. The results from these studies provide additional molecular insights into the preferential assembly of HIV-1 virions at the uropod of polarized T cells.__To gain insights into RNA packaging and virus assembly mechanisms, we examined the dynamics of viral RNA and Gag-RNA interactions near the plasma membrane by total internal reflection fluorescence (TIRF) microscopy. HIV-1 RNA was labeled with a photo-convertible Eos protein via a BglG protein that recognizes stem-loop sequences engineered into the viral genome. UV light exposure causes an irreversible structural change in Eos and alters its emitted fluorescence from green to red. The dynamics of HIV-1 RNA were determined by photoconverting Eos near the plasma membrane and by following the population of the photoconverted red-Eos-labeled RNA signals over time. We found that in the absence of Gag, most of the HIV-1 RNAs stayed near the plasma membrane transiently for a few minutes. The presence of Gag significantly increased the time RNAs stay near the plasma membrane; most of the RNAs were detected after 30 minutes. We then quantified the proportion of HIV-1 RNAs near the plasma membrane that was packaged into assembling viral complexes. By tagging Gag with a blue fluorescent protein, 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.__Dimerization of the RNA genome is a key event in HIV-1 virion assembly and has a strong impact in viral replication and evolution. Although its biological importance is appreciated, very little is known about the genome dimerization process. HIV-1 RNA genomes dimerize prior to packaging into virions, and RNA interacts with the viral structural protein Gag in the cytoplasm. Thus, it is often hypothesized that RNAs dimerize in the cytoplasm and the RNA-Gag complex is transported to the plasma membrane for virus assembly. In this study, we tagged HIV-1 RNAs with fluorescent proteins, via interactions of RNA-binding proteins and motifs in the RNA genomes, and studied their behavior at the plasma membrane by using TIRF microscopy. We showed that 1) HIV-1 RNAs dimerize not in the cytoplasm but on the plasma membrane; 2) dynamic interactions occur among HIV-1 RNAs and stabilization of the RNA dimer requires Gag protein; 3) dimerization often occurs at an early stage of the virus assembly process; and 4) the dimerization process is probably mediated by the interactions of two RNA-Gag complexes, rather than two RNAs. This study defines the timing, location, and partners involved in RNA dimerization, an important process for HIV-1 replication.__[Corresponds to Hu Project 2 in the July 2016 site visit report of the HIV Dynamics and Replication Program]