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Global changes in the RNA binding specificity of HIV-1 gag regulate virion genesis.

ABSTRACT: The HIV-1 Gag protein orchestrates all steps of virion genesis, including membrane targeting and RNA recruitment into virions. Using crosslinking-immunoprecipitation (CLIP) sequencing, we uncover several dramatic changes in the RNA-binding properties of Gag that occur during virion genesis, coincident with membrane binding, multimerization, and proteolytic maturation. Prior to assembly, and after virion assembly and maturation, the nucleocapsid domain of Gag preferentially binds to psi and Rev Response elements in the viral genome, and GU-rich mRNA sequences. However, during virion genesis, this specificity transiently changes in a manner that facilitates genome packaging; nucleocapsid binds to many sites on the HIV-1 genome and to mRNA sequences with a HIV-1-like, A-rich nucleotide composition. Additionally, we find that the matrix domain of Gag binds almost exclusively to specific tRNAs in the cytosol, and this association regulates Gag binding to cellular membranes.

SUBMITTER: Kutluay SB

PROVIDER: S-EPMC4247003 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Global changes in the RNA binding specificity of HIV-1 gag regulate virion genesis.

Kutluay Sebla B SB Zang Trinity T Blanco-Melo Daniel D Powell Chelsea C Jannain David D Errando Manel M Bieniasz Paul D PD

Cell 20141106 5

The HIV-1 Gag protein orchestrates all steps of virion genesis, including membrane targeting and RNA recruitment into virions. Using crosslinking-immunoprecipitation (CLIP) sequencing, we uncover several dramatic changes in the RNA-binding properties of Gag that occur during virion genesis, coincident with membrane binding, multimerization, and proteolytic maturation. Prior to assembly, and after virion assembly and maturation, the nucleocapsid domain of Gag preferentially binds to psi and Rev R ...[more]

PMID: 25416948

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Project description: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 in trans In contrast, artificially tethering full-length viral RNAs or surrogate gag-pol mRNAs 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 two cis-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 the gag-pol mRNA actively compartmentalize Gag to preferred sites within the cytoplasm and/or PM.IMPORTANCE The 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-pol mRNAs) 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-1 gag-pol mRNA 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.

Dataset Information

Global changes in the RNA binding specificity of HIV-1 gag regulate virion genesis.

Publications

Global changes in the RNA binding specificity of HIV-1 gag regulate virion genesis.

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