Contribution of the C-terminal region within the catalytic core domain of HIV-1 integrase to yeast lethality, chromatin binding and viral replication

Zaikun Xu,Yingfeng Zheng,Martin Clement,Pierre Belhumeur,Ganjam V Kalpana,Éric A Cohen,Zhujun Ao,Xiaojian Yao,Andrew J Mouland

doi:10.1186/1742-4690-5-102

Abstract

BackgroundHIV-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.ResultsOur 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.ConclusionOverall, 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.

Highlights

HIV-1 integrase (IN) is a key viral enzymatic molecule required for the integration of the viral cDNA into the genome
Effects of IN mutations on the lethal activity in HP16 yeast cells Caumont et al first demonstrated that HIV-1 IN induced a lethal phenotype in some yeast strains, including the JSC 302, W839-5C and AB2 strains, but not in the W303-1 strain (Rad52+) [26]
We tested whether HIV-1 IN could induce the lethal phenotype in the S. cerevisiae strain HP16 (MATa ura3-52; his3Δ1; leu2; trp1Δ63; prb1-1122; pep4-3 prc1-407)

Summary

Introduction

HIV-1 integrase (IN) is a key viral enzymatic molecule required for the integration of the viral cDNA into the genome. HIV-1 belongs to the Lentiviridae genus of retroviruses and its replication depends on the integration of the reversetranscribed viral genome into the host chromosome. This viral integration step is essential for HIV-1 productive replication, and critical for the re-activation of HIV-1 latent infection. Integration of the viral DNA into the host genome is not random but rather favors active transcription units This is driven by cellular proteins which tether the lentiviral preintegration complexes to specific sites on the host chromosomes. Several cellular proteins, including LEDGF/p75, integrase interactor-1 (Ini1) and barrier-to-autointegration (BAF), have been identified that interact with IN and contribute to its activities during integration and/or other early steps of the HIV-1 life cycle (reviewed in [3])

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