Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Dec 19;2(12):e1340.
doi: 10.1371/journal.pone.0001340.

Role of PSIP1/LEDGF/p75 in lentiviral infectivity and integration targeting

Heather M Marshall  1 , Keshet Ronen, Charles Berry, Manuel Llano, Heidi Sutherland, Dyana Saenz, Wendy Bickmore, Eric Poeschla, Frederic D Bushman
Affiliations

Role of PSIP1/LEDGF/p75 in lentiviral infectivity and integration targeting

Heather M Marshall et al. PLoS One. .

Abstract

Background: To 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.

Methodology: Here 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/significance: In 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.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Effects of intensified knockdown of LEDGF/p75 in SupT1 cells on the efficiency of lentiviral infection.
A) and B) HIV luc activity was compared for wild-type SupT1 cells, SupT1 containing a control scrambled shRNA (SCRAM), and LEDGF/p75 knockdown (KD) cells. A) High multiplicity of infection (80 ng p24); B) Lower multiplicity of infection (20 ng p24). The designation “p24” indicates the amount of viral stock, measured by the weight of the p24 capsid antigen applied to cells. C) and D) EIAV infectivity was compared in the SupT1 cell set as assayed by quantitative PCR for viral cDNA: C) high multiplicity (100 µl stock), D) lower multiplicity (25 µl stock).
Figure 2
Figure 2. Efficiency of lentiviral infection in control (+/+) and homozygous LEDGF/p75-disrupted (−/−) murine cells, measured by quantitative PCR.
A) HIV infectivity. B) EIAV infectivity.
Figure 3
Figure 3. Integration site consensus at sequences flanking EIAV proviruses in control and LEDGF/p75-knockdown SupT1 cells.
A) Unmodified SupT1 cells. B) Control SCRAM cells. C) LEDGF KD cells. The diagrams were generated using the WebLOGO program (weblogo.berkeley.edu/logo.cgi). The y-axis indicates bits of information–perfect conservation of a base would score as two bits.
Figure 4
Figure 4. EIAV integration site distributions in control and LEDGF/p75-knockdown SupT1 cells.
Integration site distributions are shown relative to A) RefSeq genes, B) CpG islands (plus or minus 1 kb), C) relative G/C content (Integration sites from unmodified and knockdown cells were pooled and divided into 10 equal bins of increasing GC content, and sites in each cell type plotted for each bin), D) gene density, and E) relative gene expression intensity. For each value in A–B) and D), the measured value for the integration site population was divided by that of the matched random control to emphasize the departure of the experimental data from random. P values shown are based on regression analysis (A–C) or Chi Square test for trend (D–E).
Figure 5
Figure 5. Comparison of LEDGF/p75 knockdowns in different human cell types.
The Jurkat and 293T data sets are described in detail in . Integration frequency was compared within RefSeq genes.
Figure 6
Figure 6. Integration site consensus sequence for lentiviral infection of murine control and LEDGF/p75-disrupted cells.
A) HIV in +/+ MEFs. B) HIV integration in −/− MEFs. C) EIAV integration in +/+ MEFs. D) EIAV integration in −/− MEFs. Markings as in Figure 3.
Figure 7
Figure 7. EIAV integration distributions in murine control and LEDGF/p75-disrupted cells.
Integration frequencies are shown relative to A) RefSeq genes, B) CpG islands (1 kb window; note that there were no control sites within <1 kb), C) G/C content, D) Gene density (250 kb window), E) Gene activity. Markings as in Figure 4.
Figure 8
Figure 8. HIV integration distributions in murine control and LEDGF/p75-disrupted cells.
A) RefSeq genes, B) CpG islands (1 kb window), C) G/C content, D) Gene density (250 kb window), E) Gene activity. Markings as in Figure 4.
Figure 9
Figure 9. Correlation between LEDGF/p75 expression and the frequency of HIV integration in genes.
Data is shown for 15 HIV integration site data sets in 10 cell types. The y-axis shows the percentage of integration events within transcription units of the “known gene” set of human genes for each integration site data set. The x-axis shows relative expression values for LEDGF/p75 derived from Affymetrix array data (see methods for details). The R-squared value for the fit is 0.6148 (P<0.0001). The references for the data sets used are as follows: Macrophage 1 is the VSV-G set in ; Macrophage 2 is the CCR5 set in ; SupT1 ; IMR90 1 is the dividing set in ; IMR90 2 is the growth-arrested set in ; CD4 T ; PBMC ; Jurkat 1 is the Mse set in ; Jurkat 2 is the Avr set in ; Jurkat 3 is the initially bright set in ; Jurkat 4 is the initially dark set in ; Jurkat p75 knockdown ; 293T ; 293T Scram ; 293T p75 knockdown .
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Coffin JM, Hughes SH, Varmus HE. Retroviruses. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 1997. - PubMed
    1. Bushman FD. Lateral DNA transfer: Mechanisms and consequences. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2001.
    1. Jordan A, Defechereux P, Verdin E. The site of HIV-1 integration in the human genome determines basal transcriptional activity and response to tat transactivation. EMBO J. 2001;20:1726–1738. - PMC - PubMed
    1. Jordan A, Bisgrove D, Verdin E. HIV reporducibly establishes a latent infection after acute infection of T cells in vitro. EMBO J. 2003;22:1868–1877. - PMC - PubMed
    1. Lewinski M, Bisgrove D, Shinn P, Chen H, Verdin E, et al. Genome-wide analysis of chromosomal features repressing HIV transcription. J Virol. 2005;79:6610–6619. - PMC - PubMed

Publication types

Substances