Intracellular Transport of Human Immunodeficiency Virus Type 1 Genomic RNA and Viral Production Are Dependent on Dynein Motor Function and Late Endosome Positioning

Abstract

Our earlier work indicated that the human immunodeficiency virus type 1 (HIV-1) genomic RNA (vRNA) is trafficked to the microtubule-organizing center (MTOC) when heterogeneous nuclear ribonucleoprotein A2/B1 is depleted from cells. Also, Rab7-interacting lysosomal protein promoted dynein motor complex, late endosome and vRNA clustering at the MTOC suggesting that the dynein motor and late endosomes were involved in vRNA trafficking. To investigate the role of the dynein motor in vRNA trafficking, dynein motor function was disrupted by small interference RNA-mediated depletion of the dynein heavy chain or by p50/dynamitin overexpression. These treatments led to a marked relocalization of vRNA and viral structural protein Gag to the cell periphery with late endosomes and a severalfold increase in HIV-1 production. In contrast, rerouting vRNA to the MTOC reduced virus production. vRNA localization depended on Gag membrane association as shown using both myristoylation and Gag nucleocapsid domain proviral mutants. Furthermore, the cytoplasmic localization of vRNA and Gag was not attributable to intracellular or internalized endocytosed virus particles. Our results demonstrate that dynein motor function is important for regulating Gag and vRNA egress on endosomal membranes in the cytoplasm to directly impact on viral production. HIV-12 gene transcription generates a 9-kb primary transcript, the genomic RNA (termed vRNA herein). vRNA can remain unspliced, but it is also singly or multiply spliced to generate over 30 additional mRNAs (1). vRNA encodes 55-and 160-kDa precursor proteins pr55Gag (termed Gag herein) and pr160Gag/Pol that are cleaved following virus assembly during virus maturation to yield both structural proteins and enzymes, which are critical for virus replication. The singly spliced, 4-kb mRNAs encode the auxiliary proteins Vpr, Vpu, and Vif, factors that enhance HIV-1 pathogenesis (2-4) as well as the integral membrane protein envelope (Env) that is found at the surface of virus particles. The multiply spliced, 2-kb mRNAs encode the regulatory proteins Tat, Rev, and Nef, proteins that act at both transcriptional and post-transcriptional levels. Although transcripts that are processed incorrectly are retained and degraded within the nucleus (5, 6), vRNA and the 4-kb mRNAs that both contain intronic sequences are exported from the nucleus to the cytoplasm via Rev and the chromosome region maintenance protein 1 nuclear export pathway (7). The well characterized Rev-mediated RNA transport is accomplished by the concerted activities of host cell factors in the nucleus, nuclear membrane, and in the cytosol (8 -11).It has been largely assumed that following transport of HIV-1 RNAs to the cytoplasm, the RNAs either diffuse to the polysomes for translation and/or diffuse to the plasma membrane for assembly. In fact, the cell cytosol is a cluttered environment requiring regulated and energy-dependent transport processes (Ref. 12, and recently reviewed for HIV-1 (13)). Host cell proteins such as human Rev-intera...

Human Immunodeficiency Virus Type 1 (HIV-1) Induces the Cytoplasmic Retention of Heterogeneous Nuclear Ribonucleoprotein A1 by Disrupting Nuclear Import

“…IF, FISH, Microscopy, and Quantitative Measurements-Immunofluorescence (IF) and fluorescence in situ hybridization (FISH) analyses have been described elsewhere (22,23,40). Microscopy was performed on a Zeiss LSM5 Pascal laser-scanning confocal microscope (Carl-Zeiss) or on a WaveFX/Leica spinning disk confocal microscope equipped with Plan-Apochromat 63ϫ oil immersion objective and an argon/krypton laser.…”

“…Microscopy was performed on a Zeiss LSM5 Pascal laser-scanning confocal microscope (Carl-Zeiss) or on a WaveFX/Leica spinning disk confocal microscope equipped with Plan-Apochromat 63ϫ oil immersion objective and an argon/krypton laser. Details on filter sets employed on the Zeiss LSM5 Pascal are described elsewhere (23,40). Using the WaveFX/Leica spinning disk confocal microscope, AlexaFluor-conjugated secondary antibody emissions were sequentially captured with 500 -550, 570 -620, 665-715, or 435-485 nm (for 4Ј,6-diamidino-2-phenylindole staining) bandpass filters.…”

HIV-1 RRE RNA acts as an RNA silencing suppressor by competing with TRBP-bound siRNAs

“…This result suggests that the difference between a lentiviral vector and HIV-1 to suppress RNAi activity may be related to the presence of viral proteins that could bind to TAR and RRE and mask their function as RSSs. To test this idea, we used the let7-EGFP reporter with overexpressed Rev, which binds RRE, or GagPol, which binds HIV-1 RNA through the nucleocapsid (NC) in the cytoplasm 58,59 to determine if they prevent the RSS activity of pHIV7-tdTomato (Fig. 5B).…”

Properties and functions of the nucleocapsid protein in virus assembly

“…84 Gag and the genomic RNA undergo trafficking through endosomes that appear to be implicated in viral egress. [107][108][109] Indeed, deleting both NC zinc fingers alters the location of HIV-1 Gag in late endosomes, potential sites for viral assembly. 83 The mechanism by which these Gag(NC)-RNA ribonucleic protein complexes traffic and lead to assembly will need future characterizations.…”