Role of PSIP1/LEDGF/p75 in Lentiviral Infectivity and Integration Targeting

Heather M Marshall,Keshet Ronen,Manuel Llano,Heidi Sutherland,Wendy Bickmore,Eric Poeschla,Frederic D Bushman,Dyana Saenz,Charles Berry,Debbie Fox

doi:10.1371/journal.pone.0001340

Abstract

BackgroundTo replicate, lentiviruses such as HIV must integrate DNA copies of their RNA genomes into host cell chromosomes. Lentiviral integration is favored in active transcription units, which allows efficient viral gene expression after integration, but the mechanisms directing integration targeting are incompletely understood. A cellular protein, PSIP1/LEDGF/p75, binds tightly to the lentiviral-encoded integrase protein (IN), and has been reported to be important for HIV infectivity and integration targeting.MethodologyHere we report studies of lentiviral integration targeting in 1) human cells with intensified RNAi knockdowns of PSIP1/LEDGF/p75, and 2) murine cells with homozygous gene trap mutations in the PSIP1/LEDGF/p75 locus. Infections with vectors derived from equine infections anemia virus (EIAV) and HIV were compared. Integration acceptor sites were analyzed by DNA bar coding and pyrosequencing.Conclusions/SignificanceIn both PSIP1/LEDGF/p75-depleted cell lines, reductions were seen in lentiviral infectivity compared to controls. For the human cells, integration was reduced in transcription units in the knockdowns, and this reduction was greater than in our previous studies of human cells less completely depleted for PSIP1/LEDGF/p75. For the homozygous mutant mouse cells, similar reductions in integration in transcription units were seen, paralleling a previous study of a different mutant mouse line. Integration did not become random, however–integration in transcription units in both cell types was still favored, though to a reduced degree. New trends also appeared, including favored integration near CpG islands. In addition, we carried out a bioinformatic study of 15 HIV integration site data sets in different cell types, which showed that the frequency of integration in transcription units was correlated with the cell-type specific levels of PSIP1/LEDGF/p75 expression.

Highlights

Steps of retroviral replication involve reverse transcription to generate a DNA copy of the viral RNA genome, and integration, which results in the covalent connection of the viral DNA to host cell DNA
Like HIV IN, equine infectious anemia virus (EIAV) IN is known to bind LEDGF/p75 [20], and EIAV is known to integrate in active transcription units [31], so EIAV is a suitable model for analysis of the influence of LEDGF/ p75 on lentivirus infection
Extensive characterization has shown that these cells have stronger knockdowns than those studied previously (e. g. [12,40]), providing an improved model for the role of LEDGF/p75 in lentiviral integration targeting in human T-cells

Summary

Introduction

Steps of retroviral replication involve reverse transcription to generate a DNA copy of the viral RNA genome, and integration, which results in the covalent connection of the viral DNA to host cell DNA (for reviews see [1,2]). We present data on the role of a host-cell encoded protein, PSIP1/LEDGF/p75, that guides integration site selection by lentiviruses, the viral genus including HIV ( we use ‘‘LEDGF/p75’’ because this name is widely used in the HIV field). To replicate, lentiviruses such as HIV must integrate DNA copies of their RNA genomes into host cell chromosomes. We carried out a bioinformatic study of 15 HIV integration site data sets in different cell types, which showed that the frequency of integration in transcription units was correlated with the cell-type specific levels of PSIP1/LEDGF/p75 expression

Methods

Results

Conclusion