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

• Published in: Journal of molecular biology 2016-06, Vol.428 (11), p.2418-2429
• Main Authors: Boeras, Ioana, Song, Zhenwei, Moran, Andrew, Franklin, Jarryd, Brown, William Clay, Johnson, Marc, Boris-Lawrie, Kathleen, Heng, Xiao
• Format: Article
• Language: English
• 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
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2016.04.011

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.