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) 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.
Department of Veterinary and Biomedical Sciences, University of Minnesota, Saint Paul, MN 55108, USA.
Ioana
I
Boeras
Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA.
Zhenwei
Z
Song
Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA.
Andrew
A
Moran
Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA.
Jarryd
J
Franklin
Center for Structural Biology, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA.
William Clay
WC
Brown
Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO 65211, USA.
Marc
M
Johnson
Department of Veterinary and Biomedical Sciences, University of Minnesota, Saint Paul, MN 55108, USA. Electronic address: kbl@umn.edu.
Kathleen
K
Boris-Lawrie
Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA. Electronic address: hengx@missouri.edu.
Xiao
X
Heng
D020121:5' Untranslated Regions / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics*; D023281:Genomics; D015658:HIV Infections / Q000235:genetics* / Q000821:virology; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D009363:Neoplasm Proteins / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics*; D014771:Virion / Q000235:genetics*; D019065:Virus Assembly / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics
D020121:5' Untranslated Regions; D017931:DNA Primers; D009363:Neoplasm Proteins; D012367:RNA, Viral; C098487:DHX9 protein, human; D053487:DEAD-box RNA Helicases
NMR; PBS-segment; RNA-binding domain; dimeric 5′-UTR; virus infectivity