Contribution of the C-terminal region within the catalytic core domain of HIV-1 integrase to yeast lethality, chromatin binding and viral replication.
BACKGROUND
HIV-1 integrase (IN) is a key viral enzymatic molecule required for the integration of the viral cDNA into the genome. Additionally, HIV-1 IN has been shown to play important roles in several other steps during the viral life cycle, including reverse transcription, nuclear import and chromatin targeting. Interestingly, previous studies have demonstrated that the expression of HIV-1 IN induces the lethal phenotype in some strains of Saccharomyces cerevisiae. In this study, we performed mutagenic analyses of the C-terminal region of the catalytic core domain of HIV-1 IN in order to delineate the critical amino acid(s) and/or motif(s) required for the induction of the lethal phenotype in the yeast strain HP16, and to further elucidate the molecular mechanism which causes this phenotype.


RESULTS
Our study identified three HIV-1 IN mutants, V165A, A179P and KR186,7AA, located in the C-terminal region of the catalytic core domain of IN that do not induce the lethal phenotype in yeast. Chromatin binding assays in yeast and mammalian cells demonstrated that these IN mutants were impaired for the ability to bind chromatin. Additionally, we determined that while these IN mutants failed to interact with LEDGF/p75, they retained the ability to bind Integrase interactor 1. Furthermore, we observed that VSV-G-pseudotyped HIV-1 containing these IN mutants was unable to replicate in the C8166 T cell line and this defect was partially rescued by complementation with the catalytically inactive D64E IN mutant.


CONCLUSIONS
Overall, this study demonstrates that three mutations located in the C-terminal region of the catalytic core domain of HIV-1 IN inhibit the IN-induced lethal phenotype in yeast by inhibiting the binding of IN to the host chromatin. These results demonstrate that the C-terminal region of the catalytic core domain of HIV-1 IN is important for binding to host chromatin and is crucial for both viral replication and the promotion of the IN-induced lethal phenotype in yeast.
Xu
Zaikun
Z
Laboratory of Molecular Human Retrovirology, Department of Medical Microbiology, University of Manitoba, 508-730 William Avenue, Winnipeg, R3E 0W3, Canada. xuzaikun@hotmail.com
Zheng
Yingfeng
Y
Ao
Zhujun
Z
Clement
Martin
M
Mouland
Andrew J
AJ
Kalpana
Ganjam V
GV
Belhumeur
Pierre
P
Cohen
Eric A
EA
Yao
Xiaojian
X
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D002522:Chlorocebus aethiops; D002843:Chromatin / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism; D005816:Genetic Complementation Test; D019427:HIV Integrase / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D050296:Microbial Viability; D016296:Mutagenesis; D020125:Mutation, Missense; D000071796:SMARCB1 Protein; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism; D014779:Virus Replication
D048868:Adaptor Proteins, Signal Transducing; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; C504649:PSIP1 protein, human; D000071796:SMARCB1 Protein; C513266:SMARCB1 protein, human; D014157:Transcription Factors; D019427:HIV Integrase; C514801:p31 integrase protein, Human immunodeficiency virus 1