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HRP2 determines the efficiency and specificity of HIV-1 integration in LEDGF/p75 knockout cells but does not contribute to the antiviral activity of a potent LEDGF/p75-binding site integrase inhibitor.

Wang H, Jurado KA, Wu X, Shun MC, Li X, Ferris AL, Smith SJ, Patel PA, Fuchs JR, Cherepanov P, Kvaratskhelia M, Hughes SH, Engelman A - Nucleic Acids Res. (2012)

Bottom Line: The double-KO cells nevertheless supported residual integration into genes, indicating that IN and/or other host factors contribute to integration specificity in the absence of LEDGF/p75 and HRP2.Psip1 KO significantly increased the potency of an allosteric inhibitor that binds the LEDGF/p75 binding site on IN, a result that was not significantly altered by Hdgfrp2 disruption.These findings help to rule out the host factor-IN interactions as the primary antiviral targets of LEDGF/p75-binding site IN inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA.

ABSTRACT
The binding of integrase (IN) to lens epithelium-derived growth factor (LEDGF)/p75 in large part determines the efficiency and specificity of HIV-1 integration. However, a significant residual preference for integration into active genes persists in Psip1 (the gene that encodes for LEDGF/p75) knockout (KO) cells. One other cellular protein, HRP2, harbors both the PWWP and IN-binding domains that are important for LEDGF/p75 co-factor function. To assess the role of HRP2 in HIV-1 integration, cells generated from Hdgfrp2 (the gene that encodes for HRP2) and Psip1/Hdgfrp2 KO mice were infected alongside matched control cells. HRP2 depleted cells supported normal infection, while disruption of Hdgfrp2 in Psip1 KO cells yielded additional defects in the efficiency and specificity of integration. These deficits were largely restored by ectopic expression of either LEDGF/p75 or HRP2. The double-KO cells nevertheless supported residual integration into genes, indicating that IN and/or other host factors contribute to integration specificity in the absence of LEDGF/p75 and HRP2. Psip1 KO significantly increased the potency of an allosteric inhibitor that binds the LEDGF/p75 binding site on IN, a result that was not significantly altered by Hdgfrp2 disruption. These findings help to rule out the host factor-IN interactions as the primary antiviral targets of LEDGF/p75-binding site IN inhibitors.

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Related in: MedlinePlus

In vitro integration activities of PICs. Strand transfer activities, normalized to the levels of HIV-Luc substrate DNA in the extracts, are plotted as percent activities in WT E9 cell extracts. Error bars represent the variation obtained from duplicate sets of PCR assays; results are representative of those observed in two (cytoplasmic PICs) to three (nuclear samples) independent experiments. **P < 0.01 as determined by one-tailed Student’s t-test; the lack of statistical significance in the cytoplasmic samples is in part due to the extent of variation among duplicate PCR samples.
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gks913-F4: In vitro integration activities of PICs. Strand transfer activities, normalized to the levels of HIV-Luc substrate DNA in the extracts, are plotted as percent activities in WT E9 cell extracts. Error bars represent the variation obtained from duplicate sets of PCR assays; results are representative of those observed in two (cytoplasmic PICs) to three (nuclear samples) independent experiments. **P < 0.01 as determined by one-tailed Student’s t-test; the lack of statistical significance in the cytoplasmic samples is in part due to the extent of variation among duplicate PCR samples.

Mentions: We previously determined that HIV-1 PICs made in Psip1 KO cells are fully competent to integrate the viral reverse transcript in vitro (15). To determine if the remaining HRP2 in these cells might influence PIC function, cytoplasmic and nuclear material extracted from cells 7 h from the start of infection were incubated with plasmid target DNA in the presence of Mg2+ ions. Purified DNAs were examined by nested qPCR to detect the formation of covalent HIV-plasmid DNA junctions, and these values were normalized to the total level of HIV-Luc DNA in the extracts. As expected, PICs extracted from Psip1 KO cells showed levels of integration activity similar to PICs extracted from WT and Hdgfrp2 KO cells (Figure 4). PICs extracted from double-KO cells supported about half this level of activity, linking the drop in chromosomal integration in double-KO cells to a reduction in the strand transfer activity of PIC-associated IN.Figure 4.


HRP2 determines the efficiency and specificity of HIV-1 integration in LEDGF/p75 knockout cells but does not contribute to the antiviral activity of a potent LEDGF/p75-binding site integrase inhibitor.

Wang H, Jurado KA, Wu X, Shun MC, Li X, Ferris AL, Smith SJ, Patel PA, Fuchs JR, Cherepanov P, Kvaratskhelia M, Hughes SH, Engelman A - Nucleic Acids Res. (2012)

In vitro integration activities of PICs. Strand transfer activities, normalized to the levels of HIV-Luc substrate DNA in the extracts, are plotted as percent activities in WT E9 cell extracts. Error bars represent the variation obtained from duplicate sets of PCR assays; results are representative of those observed in two (cytoplasmic PICs) to three (nuclear samples) independent experiments. **P < 0.01 as determined by one-tailed Student’s t-test; the lack of statistical significance in the cytoplasmic samples is in part due to the extent of variation among duplicate PCR samples.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526291&req=5

gks913-F4: In vitro integration activities of PICs. Strand transfer activities, normalized to the levels of HIV-Luc substrate DNA in the extracts, are plotted as percent activities in WT E9 cell extracts. Error bars represent the variation obtained from duplicate sets of PCR assays; results are representative of those observed in two (cytoplasmic PICs) to three (nuclear samples) independent experiments. **P < 0.01 as determined by one-tailed Student’s t-test; the lack of statistical significance in the cytoplasmic samples is in part due to the extent of variation among duplicate PCR samples.
Mentions: We previously determined that HIV-1 PICs made in Psip1 KO cells are fully competent to integrate the viral reverse transcript in vitro (15). To determine if the remaining HRP2 in these cells might influence PIC function, cytoplasmic and nuclear material extracted from cells 7 h from the start of infection were incubated with plasmid target DNA in the presence of Mg2+ ions. Purified DNAs were examined by nested qPCR to detect the formation of covalent HIV-plasmid DNA junctions, and these values were normalized to the total level of HIV-Luc DNA in the extracts. As expected, PICs extracted from Psip1 KO cells showed levels of integration activity similar to PICs extracted from WT and Hdgfrp2 KO cells (Figure 4). PICs extracted from double-KO cells supported about half this level of activity, linking the drop in chromosomal integration in double-KO cells to a reduction in the strand transfer activity of PIC-associated IN.Figure 4.

Bottom Line: The double-KO cells nevertheless supported residual integration into genes, indicating that IN and/or other host factors contribute to integration specificity in the absence of LEDGF/p75 and HRP2.Psip1 KO significantly increased the potency of an allosteric inhibitor that binds the LEDGF/p75 binding site on IN, a result that was not significantly altered by Hdgfrp2 disruption.These findings help to rule out the host factor-IN interactions as the primary antiviral targets of LEDGF/p75-binding site IN inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA.

ABSTRACT
The binding of integrase (IN) to lens epithelium-derived growth factor (LEDGF)/p75 in large part determines the efficiency and specificity of HIV-1 integration. However, a significant residual preference for integration into active genes persists in Psip1 (the gene that encodes for LEDGF/p75) knockout (KO) cells. One other cellular protein, HRP2, harbors both the PWWP and IN-binding domains that are important for LEDGF/p75 co-factor function. To assess the role of HRP2 in HIV-1 integration, cells generated from Hdgfrp2 (the gene that encodes for HRP2) and Psip1/Hdgfrp2 KO mice were infected alongside matched control cells. HRP2 depleted cells supported normal infection, while disruption of Hdgfrp2 in Psip1 KO cells yielded additional defects in the efficiency and specificity of integration. These deficits were largely restored by ectopic expression of either LEDGF/p75 or HRP2. The double-KO cells nevertheless supported residual integration into genes, indicating that IN and/or other host factors contribute to integration specificity in the absence of LEDGF/p75 and HRP2. Psip1 KO significantly increased the potency of an allosteric inhibitor that binds the LEDGF/p75 binding site on IN, a result that was not significantly altered by Hdgfrp2 disruption. These findings help to rule out the host factor-IN interactions as the primary antiviral targets of LEDGF/p75-binding site IN inhibitors.

Show MeSH
Related in: MedlinePlus