Full-length HIV-1 RNA needs to be exported from the nucleus to carry out its functions including serving as a template for Gag/Gag-Pol translation and as a genome in the newly assembled virion. To study HIV-1 RNA packaging, we have developed a novel assay to directly visualize viral RNA in the particles at single-RNA-molecule sensitivity. This assay reveals that most HIV-1 particles contain viral RNA. Although it is known that retroviral RNAs packaged into particles are dimeric, standard biochemical assays cannot determine the number of RNA molecules in one particle;our results provide evidence to support the long-standing assumption that two RNA molecules (one dimer) are packaged into a particle. To better understand the mechanisms of RNA packaging, we have performed genetic, biochemical, and imaging analyses to show that HIV-1 RNA uses base-pairing of the DIS sequences to select its copackaged RNA partner, and this process occurs in the cytoplasm of the producer cell prior to RNA packaging into particles. We and others have observed that HIV-1 RNA can be manipulated to deviate from using the CRM1-mediated nuclear export pathway and, instead, be exported via the NXF1 pathway, which is used by most cellular mRNAs. However, we found that RNA molecules exported through these two pathways do not copackage efficiently, indicating that the export pathway affects subcellular localization of these RNAs. Our future efforts in this project will be focused on examining the dynamics of HIV-1 RNA export using high-speed, high-resolution microscopy to detect export of a single HIV-1 RNA through the nuclear pore complex. We will investigate the kinetics of export via the CRM1 or NXF1 pathways and explore whether different export pathways use different nuclear pore complexes. We will also investigate the mechanisms that HIV-1 uses to regulate which and how many RNAs are packaged. Additionally, we will utilize an RNA-binding protein to redirect HIV-1 RNA-packaging specificity. These studies will allow us to understand several questions that are fundamentally important to HIV replication, which can be used to generate new strategies to block the spread of HIV.[Corresponds to Hu Project 2 in the October 2011 site visit report of the HIV Drug Resistance Program]

National Institute of Health (NIH)
National Cancer Institute (NCI)
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National Cancer Institute Division of Basic Sciences
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Liu, Yang; Chen, Jianbo; Nikolaitchik, Olga A et al. (2018) The roles of five conserved lentiviral RNA structures in HIV-1 replication. Virology 514:1-8
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
Desimmie, Belete A; Burdick, Ryan C; Izumi, Taisuke et al. (2016) APOBEC3 proteins can copackage and comutate HIV-1 genomes. Nucleic Acids Res :
Chen, Jianbo; Grunwald, David; Sardo, Luca et al. (2014) Cytoplasmic HIV-1 RNA is mainly transported by diffusion in the presence or absence of Gag protein. Proc Natl Acad Sci U S A 111:E5205-13
Kuzembayeva, Malika; Dilley, Kari; Sardo, Luca et al. (2014) Life of psi: how full-length HIV-1 RNAs become packaged genomes in the viral particles. Virology 454-455:362-70
Nikolaitchik, Olga A; Hu, Wei-Shau (2014) Deciphering the role of the Gag-Pol ribosomal frameshift signal in HIV-1 RNA genome packaging. J Virol 88:4040-6
Nikolaitchik, Olga A; Dilley, Kari A; Fu, William et al. (2013) Dimeric RNA recognition regulates HIV-1 genome packaging. PLoS Pathog 9:e1003249
Dilley, Kari A; Ni, Na; Nikolaitchik, Olga A et al. (2011) Determining the frequency and mechanisms of HIV-1 and HIV-2 RNA copackaging by single-virion analysis. J Virol 85:10499-508
Ni, Na; Nikolaitchik, Olga A; Dilley, Kari A et al. (2011) Mechanisms of human immunodeficiency virus type 2 RNA packaging: efficient trans packaging and selection of RNA copackaging partners. J Virol 85:7603-12
Hussein, Islam T M; Ni, Na; Galli, Andrea et al. (2010) Delineation of the preferences and requirements of the human immunodeficiency virus type 1 dimerization initiation signal by using an in vivo cell-based selection approach. J Virol 84:6866-75

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