DHX9/RHA Binding to the PBS-Segment of the Genomic RNA during HIV-1 Assembly Bolsters Virion Infectivity

Cellular RNA-binding proteins incorporated into virions during human immunodeficiency virus type 1 (HIV-1) assembly promote the replication efficiency of progeny virions. Despite its critical role in bolstering virion infectivity, the molecular basis for the incorporation of DHX9/RNA helicase A (RHA...

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Veröffentlicht in:Journal of molecular biology 2016-06, Vol.428 (11), p.2418-2429
Hauptverfasser: Boeras, Ioana, Song, Zhenwei, Moran, Andrew, Franklin, Jarryd, Brown, William Clay, Johnson, Marc, Boris-Lawrie, Kathleen, Heng, Xiao
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container_end_page 2429
container_issue 11
container_start_page 2418
container_title Journal of molecular biology
container_volume 428
creator Boeras, Ioana
Song, Zhenwei
Moran, Andrew
Franklin, Jarryd
Brown, William Clay
Johnson, Marc
Boris-Lawrie, Kathleen
Heng, Xiao
description Cellular RNA-binding proteins incorporated into virions during human immunodeficiency virus type 1 (HIV-1) assembly promote the replication efficiency of progeny virions. Despite its critical role in bolstering virion infectivity, the molecular basis for the incorporation of DHX9/RNA helicase A (RHA) to virions remains unclear. Here, cell-based experiments demonstrate that the truncation of segments of the HIV-1 5′-untranslated region (5′-UTR) distinct from the core encapsidation sequence eliminated virion incorporation of RHA, indicating that RHA recruitment is mediated by specific interactions with the HIV-1 5′-UTR. In agreement with biological data, isothermal titration calorimetry determined that the dimer conformation of the 5′-UTR binds one RHA molecule per RNA strand, and the interaction is independent of nucleocapsid protein binding. NMR spectra employing a deuterium-labeling approach enabled resolution of the dimeric 5′-UTR in complex with the RHA N-terminal domain. The structure of the large molecular mass complex was dependent on RHA binding to a double-stranded region of the primer binding site (PBS)-segment of the 5′-UTR. A single A-to-C substitution was sufficient to disrupt biophysical conformation and attenuate virion infectivity in cell-based assays. Taken together, our studies demonstrate the structural basis for HIV-1 genomic RNA to recruit beneficial cellular cofactor to virions. The support of progeny virion infectivity by RHA is attributable to structure-dependent binding at the PBS‐segment of the HIV-1 5′-UTR during virus assembly. [Display omitted] •Host DHX9/RHA is recruited in HIV-1 virions to support infectivity.•Cell-based assays identified the 5′-UTR necessary to RHA recruitment to virions.•NMR data of the complex (290kDa) pinpointed PBS as the RHA-binding site.•A PBS point mutation that precluded RHA binding attenuated virion infectivity.•Our work sheds light on the RHA involvement in the early viral replication events.
doi_str_mv 10.1016/j.jmb.2016.04.011
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Despite its critical role in bolstering virion infectivity, the molecular basis for the incorporation of DHX9/RNA helicase A (RHA) to virions remains unclear. Here, cell-based experiments demonstrate that the truncation of segments of the HIV-1 5′-untranslated region (5′-UTR) distinct from the core encapsidation sequence eliminated virion incorporation of RHA, indicating that RHA recruitment is mediated by specific interactions with the HIV-1 5′-UTR. In agreement with biological data, isothermal titration calorimetry determined that the dimer conformation of the 5′-UTR binds one RHA molecule per RNA strand, and the interaction is independent of nucleocapsid protein binding. NMR spectra employing a deuterium-labeling approach enabled resolution of the dimeric 5′-UTR in complex with the RHA N-terminal domain. The structure of the large molecular mass complex was dependent on RHA binding to a double-stranded region of the primer binding site (PBS)-segment of the 5′-UTR. A single A-to-C substitution was sufficient to disrupt biophysical conformation and attenuate virion infectivity in cell-based assays. Taken together, our studies demonstrate the structural basis for HIV-1 genomic RNA to recruit beneficial cellular cofactor to virions. The support of progeny virion infectivity by RHA is attributable to structure-dependent binding at the PBS‐segment of the HIV-1 5′-UTR during virus assembly. [Display omitted] •Host DHX9/RHA is recruited in HIV-1 virions to support infectivity.•Cell-based assays identified the 5′-UTR necessary to RHA recruitment to virions.•NMR data of the complex (290kDa) pinpointed PBS as the RHA-binding site.•A PBS point mutation that precluded RHA binding attenuated virion infectivity.•Our work sheds light on the RHA involvement in the early viral replication events.</description><identifier>ISSN: 0022-2836</identifier><identifier>EISSN: 1089-8638</identifier><identifier>DOI: 10.1016/j.jmb.2016.04.011</identifier><identifier>PMID: 27107641</identifier><language>eng</language><publisher>England: Elsevier Ltd</publisher><subject>5' Untranslated Regions - genetics ; Binding Sites - genetics ; DEAD-box RNA Helicases - metabolism ; dimeric 5′-UTR ; DNA Primers - genetics ; Genomics ; HIV Infections - genetics ; HIV Infections - virology ; HIV-1 - genetics ; Human immunodeficiency virus 1 ; Humans ; Lentivirus ; Neoplasm Proteins - metabolism ; NMR ; PBS-segment ; RNA, Viral - genetics ; RNA-binding domain ; Virion - genetics ; Virus Assembly - genetics ; virus infectivity ; Virus Replication - genetics</subject><ispartof>Journal of molecular biology, 2016-06, Vol.428 (11), p.2418-2429</ispartof><rights>2016 Elsevier Ltd</rights><rights>Copyright © 2016 Elsevier Ltd. 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subjects 5' Untranslated Regions - genetics
Binding Sites - genetics
DEAD-box RNA Helicases - metabolism
dimeric 5′-UTR
DNA Primers - genetics
Genomics
HIV Infections - genetics
HIV Infections - virology
HIV-1 - genetics
Human immunodeficiency virus 1
Humans
Lentivirus
Neoplasm Proteins - metabolism
NMR
PBS-segment
RNA, Viral - genetics
RNA-binding domain
Virion - genetics
Virus Assembly - genetics
virus infectivity
Virus Replication - genetics
title DHX9/RHA Binding to the PBS-Segment of the Genomic RNA during HIV-1 Assembly Bolsters Virion Infectivity
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