A novel role for gag as a cis-acting element regulating RNA structure, dimerization and packaging in HIV-1 lentiviral vectors.

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cam.issuedOnline2021-12-20
dc.contributor.authorVamva, Eirini
dc.contributor.authorGriffiths, Alex
dc.contributor.authorVink, Conrad A
dc.contributor.authorLever, Andrew ML
dc.contributor.authorKenyon, Julia C
dc.contributor.orcidVink, Conrad A [0000-0001-6739-6669]
dc.contributor.orcidKenyon, Julia C [0000-0001-6055-7052]
dc.date.accessioned2022-01-28T16:47:43Z
dc.date.available2022-01-28T16:47:43Z
dc.date.issued2022-01-11
dc.date.updated2022-01-28T16:47:43Z
dc.descriptionFunder: Philosophical Society of Cambridge
dc.descriptionFunder: Darwin College
dc.descriptionFunder: Homerton College
dc.descriptionFunder: University of Cambridge
dc.descriptionFunder: Clinical Academic Reserve
dc.description.abstractClinical usage of lentiviral vectors is now established and increasing but remains constrained by vector titer with RNA packaging being a limiting factor. Lentiviral vector RNA is packaged through specific recognition of the packaging signal on the RNA by the viral structural protein Gag. We investigated structurally informed modifications of the 5' leader and gag RNA sequences in which the extended packaging signal lies, to attempt to enhance the packaging process by facilitating vector RNA dimerization, a process closely linked to packaging. We used in-gel SHAPE to study the structures of these mutants in an attempt to derive structure-function correlations that could inform optimized vector RNA design. In-gel SHAPE of both dimeric and monomeric species of RNA revealed a previously unreported direct interaction between the U5 region of the HIV-1 leader and the downstream gag sequences. Our data suggest a structural equilibrium exists in the dimeric viral RNA between a metastable structure that includes a U5-gag interaction and a more stable structure with a U5-AUG duplex. Our data provide clarification for the previously unexplained requirement for the 5' region of gag in enhancing genomic RNA packaging and provide a basis for design of optimized HIV-1 based vectors.
dc.description.sponsorshipEV was supported by a grant from the BBSRC (BB/N503708/1 to AMLL and CV) and received personal support from Darwin College, the University of Cambridge trust and the Philosophical society of Cambridge. JCK received personal support from Homerton College. AMLL is supported by the Clinical Academic Reserve and his laboratory by the NIHR Cambridge BRC (Grant RCAG/18).
dc.identifier.doi10.17863/CAM.80747
dc.identifier.eissn1362-4962
dc.identifier.issn0305-1048
dc.identifier.other34928383
dc.identifier.otherPMC8754630
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/333324
dc.languageeng
dc.publisherOxford University Press (OUP)
dc.publisher.urlhttp://dx.doi.org/10.1093/nar/gkab1206
dc.rightsAttribution 4.0 International
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.sourcenlmid: 0411011
dc.sourceessn: 1362-4962
dc.subjectGenetic Vectors
dc.subjectHEK293 Cells
dc.subjectHIV-1
dc.subjectHumans
dc.subjectNucleic Acid Conformation
dc.subjectRNA, Viral
dc.subjectRegulatory Sequences, Nucleic Acid
dc.subjectVirus Assembly
dc.titleA novel role for gag as a cis-acting element regulating RNA structure, dimerization and packaging in HIV-1 lentiviral vectors.
dc.typeArticle
dcterms.dateAccepted2021-11-30
prism.endingPage448
prism.issueIdentifier1
prism.publicationNameNucleic Acids Res
prism.startingPage430
prism.volume50
pubs.funder-project-idBiotechnology and Biological Sciences Research Council (BB/N503708/1)
rioxxterms.licenseref.urihttps://creativecommons.org/licenses/by/4.0/
rioxxterms.versionVoR
rioxxterms.versionofrecord10.1093/nar/gkab1206
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