title: Subcellular Localization of HIV-1gag-polmRNAs Regulates Sites of Virion Assembly.
abstract: Full-length unspliced human immunodeficiency virus type 1 (HIV-1) RNAs serve dual roles in the cytoplasm as mRNAs encoding the Gag and Gag-Pol capsid proteins as well as genomic RNAs (gRNAs) packaged by Gag into virions undergoing assembly at the plasma membrane (PM). Because Gag is sufficient to drive the assembly of virus-like particles even in the absence of gRNA binding, whether viral RNA trafficking plays an active role in the native assembly pathway is unknown. In this study, we tested the effects of modulating the cytoplasmic abundance or distribution of full-length viral RNAs on Gag trafficking and assembly in the context of single cells. Increasing full-length viral RNA abundance or distribution had little-to-no net effect on Gag assembly competency when provided intransIn contrast, artificially tethering full-length viral RNAs or surrogategag-polmRNAs competent for Gag synthesis to non-PM membranes or the actin cytoskeleton severely reduced net virus particle production. These effects were explained, in large part, by RNA-directed changes to Gag's distribution in the cytoplasm, yielding aberrant subcellular sites of virion assembly. Interestingly, RNA-dependent disruption of Gag trafficking required either of twocis-acting RNA regulatory elements: the 5' packaging signal (Psi) bound by Gag during genome encapsidation or, unexpectedly, the Rev response element (RRE), which regulates the nuclear export of gRNAs and other intron-retaining viral RNAs. Taken together, these data support a model for native infection wherein structural features of thegag-polmRNA actively compartmentalize Gag to preferred sites within the cytoplasm and/or PM.IMPORTANCEThe spatial distribution of viral mRNAs within the cytoplasm can be a crucial determinant of efficient translation and successful virion production. Here we provide direct evidence that mRNA subcellular trafficking plays an important role in regulating the assembly of human immunodeficiency virus type 1 (HIV-1) virus particles at the plasma membrane (PM). Artificially tethering viral mRNAs encoding Gag capsid proteins (gag-polmRNAs) to distinct non-PM subcellular locales, such as cytoplasmic vesicles or the actin cytoskeleton, markedly alters Gag subcellular distribution, relocates sites of assembly, and reduces net virus particle production. These observations support a model for native HIV-1 assembly wherein HIV-1gag-polmRNA localization helps to confine interactions between Gag, viral RNAs, and host determinants in order to ensure virion production at the right place and right time. Direct perturbation of HIV-1 mRNA subcellular localization may represent a novel antiviral strategy.
authors.affiliation: McArdle Laboratory for Cancer Research, Institute for Molecular Virology, and Carbone Cancer Center, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Jordan T
authors.initials: JT
authors.lastname: Becker
authors.affiliation: McArdle Laboratory for Cancer Research, Institute for Molecular Virology, and Carbone Cancer Center, University of Wisconsin-Madison, Madison, Wisconsin, USA nsherer@wisc.edu.
authors.forename: Nathan M
authors.initials: NM
authors.lastname: Sherer
mesh_terms: D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry*; D003593:Cytoplasm / Q000737:chemistry*; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D012333:RNA, Messenger / Q000032:analysis; D012367:RNA, Viral / Q000032:analysis*; D014771:Virion / Q000378:metabolism*; D019065:Virus Assembly; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger; D012367:RNA, Viral; D054301:gag Gene Products, Human Immunodeficiency Virus
keywords: Gag; MS2; Psi; RNA trafficking; RRE; genomes; human immunodeficiency virus; packaging; translation; virus assembly