HIV-1 must travel through the cytoplasm to reach the nuclear envelope (NE) of an infected cell, transport through a nuclear pore to enter the nucleus, and integrate its genome into the chromosomal DNA of the host cell. During this process, HIV-1 must reverse transcribe its RNA genome into double-strand DNA, protect its genome and proteins from host degradative enzymes, and avoid triggering host defenses that would inhibit its replication. These post-entry events are difficult to study biochemically because there are very few viral complexes in each cell; in addition, it has been difficult to study these events microscopically because methods to efficiently label and track viral complexes have not been available. __We previously observed that HIV-1 viral complexes that enter the nucleus remain at the nuclear periphery and are not randomly distributed throughout the nucleus. We sought to determine whether viral complexes integrate at sites near the nuclear periphery similar to the location of the viral complexes. We developed in situ RNA and DNA fluorescence in situ hybridization (FISH) to identify the nuclear locations of transcriptionally active proviruses. We found that the distribution of transcriptionally active proviruses 24 hours after infection was very similar to that of unintegrated viral complexes, indicating that the viral complexes integrate at sites near the nuclear periphery. We also characterized the nuclear locations of transcriptionally active sites 5 days after infection and found that transcriptionally active proviruses were randomly distributed. These results indicate that HIV-1 integrates at sites near the nuclear periphery; however, the locations of the integration sites become random after a few cell divisions. These results imply that different genes are present at the nuclear periphery in a population of target cells such that essentially the entire genome is available to HIV-1. In addition, the results imply that locations of genes in cells are not fixed and differ from cell to cell. __After fusion, the complex of HIV-1 viral capsid core, reverse transcriptase, and RNA carry out reverse transcription. The viral core must disassemble (uncoat) before integration of the viral DNA into the host genome, but where and when viral core uncoating occurs are not well understood. Studies of uncoating have been hampered by the inability to directly label viral capsid protein (CA) and quantify the amount of CA that remains associated with viral complexes at various stages after infection. We have developed a new method to directly label CA with green fluorescent protein (GFP) in infectious viral capsids with minimal loss of infectivity. We have characterized this labeling method and observed that when only 5-10% of the CA proteins are GFP labeled, the viral complexes retain most of their infectivity, have normal core stability, and bind to cyclophilin A. This robust GFP-CA-labeling method will facilitate future studies of the early stage of HIV-1 replication. ___We have used the direct GFP-CA-labeling method to gain insights into viral core uncoating in infected cells by live-cell microscopy. We observed that, contrary to current models of cytoplasmic or NE-associated viral core uncoating, most viral CA remains associated with nuclear viral complexes. The nuclear viral complexes undergo a rapid nuclear uncoating event just prior to integration. We also found that the nuclear viral complexes remain sensitive to capsid inhibitor PF74, indicating that the nuclear complexes retain CA hexamers. These groundbreaking studies have transformed our view of the early stage of viral replication and viral core uncoating. ___Our previous studies showed that viral complexes exhibit long NE residence times, indicating that translocation of the viral complexes through the nuclear pore is a difficult and time-consuming step during viral replication. However, very little is known about the molecular interactions between viral complexes and host proteins that are essential for nuclear import. To gain insights into this essential yet poorly understood step in viral replication, we have identified CA mutants that exhibit increased NE residence time and a delay in nuclear import. Previous studies have indicated that viral complexes that are not bound to host factor cyclophilin A use a different nuclear import pathway compared with normal viral complexes. Our results indicate that the CA mutants that exhibit a delay in nuclear import involve cyclophilin A binding to the viral complexes. These studies provide novel insights into the mechanism of viral complex translocation into the nucleus.
| Liu, Yang; Nikolaitchik, Olga A; Rahman, Sheikh Abdul et al. (2017) HIV-1 Sequence Necessary and Sufficient to Package Non-viral RNAs into HIV-1 Particles. J Mol Biol 429:2542-2555 |
| Nikolaitchik, Olga; Keele, Brandon; Gorelick, Robert et al. (2015) High recombination potential of subtype A HIV-1. Virology 484:334-40 |
| Delviks-Frankenberry, Krista; Paprotka, Tobias; Cingöz, Oya et al. (2013) Generation of multiple replication-competent retroviruses through recombination between PreXMRV-1 and PreXMRV-2. J Virol 87:11525-37 |
| Delviks-Frankenberry, Krista A; Lengruber, Renan B; Santos, Andre F et al. (2013) Connection subdomain mutations in HIV-1 subtype-C treatment-experienced patients enhance NRTI and NNRTI drug resistance. Virology 435:433-41 |
| Hatch, Steven C; Sardo, Luca; Chen, Jianbo et al. (2013) Gag-dependent enrichment of HIV-1 RNA near the uropod membrane of polarized T cells. J Virol 87:11912-5 |
| Murgai, Meera; Thomas, James; Cherepanova, Olga et al. (2013) Xenotropic MLV envelope proteins induce tumor cells to secrete factors that promote the formation of immature blood vessels. Retrovirology 10:34 |
| Nikolaitchik, Olga A; Dilley, Kari A; Fu, William et al. (2013) Dimeric RNA recognition regulates HIV-1 genome packaging. PLoS Pathog 9:e1003249 |
| Del Prete, Gregory Q; Kearney, Mary F; Spindler, Jon et al. (2012) Restricted replication of xenotropic murine leukemia virus-related virus in pigtailed macaques. J Virol 86:3152-66 |
| Delviks-Frankenberry, Krista; Cingöz, Oya; Coffin, John M et al. (2012) Recombinant origin, contamination, and de-discovery of XMRV. Curr Opin Virol 2:499-507 |
| Cingöz, Oya; Paprotka, Tobias; Delviks-Frankenberry, Krista A et al. (2012) Characterization, mapping, and distribution of the two XMRV parental proviruses. J Virol 86:328-38 |
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