Interplay between protease and reverse transcriptase dimerization in a model HIV-1 polyprotein.
Gag‐Pol polyprotein
HIV‐1
darunavir
dimerization
inhibitor
protease
reverse transcriptase
Journal
Protein science : a publication of the Protein Society
ISSN: 1469-896X
Titre abrégé: Protein Sci
Pays: United States
ID NLM: 9211750
Informations de publication
Date de publication:
Jul 2024
Jul 2024
Historique:
revised:
01
05
2024
received:
12
02
2024
accepted:
31
05
2024
medline:
19
6
2024
pubmed:
19
6
2024
entrez:
19
6
2024
Statut:
ppublish
Résumé
The Gag-Pol polyprotein in human immunodeficiency virus type I (HIV-1) encodes enzymes that are essential for virus replication: protease (PR), reverse transcriptase (RT), and integrase (IN). The mature forms of PR, RT and IN are homodimer, heterodimer and tetramer, respectively. The precise mechanism underlying the formation of dimer or tetramer is not yet understood. Here, to gain insight into the dimerization of PR and RT in the precursor, we prepared a model precursor, PR-RT, incorporating an inactivating mutation at the PR active site, D25A, and including two residues in the p6* region, fused to a SUMO-tag, at the N-terminus of the PR region. We also prepared two mutants of PR-RT containing a dimer dissociation mutation either in the PR region, PR(T26A)-RT, or in the RT region, PR-RT(W401A). Size exclusion chromatography showed both monomer and dimer fractions in PR-RT and PR(T26A)-RT, but only monomer in PR-RT(W401A). SEC experiments of PR-RT in the presence of protease inhibitor, darunavir, significantly enhanced the dimerization. Additionally, SEC results suggest an estimated PR-RT dimer dissociation constant that is higher than that of the mature RT heterodimer, p66/p51, but slightly lower than the premature RT homodimer, p66/p66. Reverse transcriptase assays and RT maturation assays were performed as tools to assess the effects of the PR dimer-interface on these functions. Our results consistently indicate that the RT dimer-interface plays a crucial role in the dimerization in PR-RT, whereas the PR dimer-interface has a lesser role.
Substances chimiques
HIV Reverse Transcriptase
EC 2.7.7.49
HIV Protease
EC 3.4.23.-
reverse transcriptase, Human immunodeficiency virus 1
EC 2.7.7.-
p16 protease, Human immunodeficiency virus 1
EC 3.4.23.-
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
e5080Subventions
Organisme : University of Pittsburgh
ID : NIH P50 AI150481
Organisme : University of Pittsburgh
ID : NIH R01 GM135919
Informations de copyright
© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.
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