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Peptides derived from HIV-1 integrase that bind Rev stimulate viral genome integration.

Levin A, Hayouka Z, Helfer M, Brack-Werner R, Friedler A, Loyter A - PLoS ONE (2009)

Bottom Line: Both INr-1 and INr-2 were found to be cell-permeable and nontoxic, allowing a study of their effect in HIV-1-infected cultured cells.The results of the present study raise the possibility that in HIV-infected cells, the Rev protein may be involved in the integration of proviral DNA by controlling/regulating the activity of the integrase.Release from such inhibition leads to stimulation of IN activity and multiple viral DNA integration events.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, Israel.

ABSTRACT

Background: The human immunodeficiency virus type 1 (HIV-1) integrase protein (IN), catalyzes the integration of viral DNA into the host cell genome. IN catalyzes the first step of the integration process, namely the 3'-end processing in which IN removes a pGT dinucleotide from the 3' end of each viral long terminal repeat (LTR). Following nuclear import of the viral preintegration complex, the host chromosomal DNA becomes accessible to the viral cDNA and the second step of the integration process, namely the strand-transfer step takes place. This ordered sequence of events, centered on integration, is mandatory for HIV replication.

Methodology/principal findings: Using an integrase peptide library, we selected two peptides, designated INr-1 and INr-2, which interact with the Rev protein and probably mediate the Rev-integrase interaction. Using an in-vitro assay system, we show that INr-1 and INr-2 are able to abrogate the inhibitory effects exerted by Rev and Rev-derived peptides on integrase activity. Both INr-1 and INr-2 were found to be cell-permeable and nontoxic, allowing a study of their effect in HIV-1-infected cultured cells. Interestingly, both INr peptides stimulated virus infectivity as estimated by production of the viral P24 protein, as well as by determination of the appearance of newly formed virus particles. Furthermore, kinetics studies revealed that the cell-permeable INr peptides enhance the integration process, as was indeed confirmed by direct determination of viral DNA integration by real-time PCR.

Conclusions/significance: The results of the present study raise the possibility that in HIV-infected cells, the Rev protein may be involved in the integration of proviral DNA by controlling/regulating the activity of the integrase. Release from such inhibition leads to stimulation of IN activity and multiple viral DNA integration events.

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Related in: MedlinePlus

Inhibition of IN enzymatic activity by Rev and its abrogation by the INr peptides.(A) Rev-GFP (dark green) or GFP (turquoise) were incubated at different molar ratios with IN (390 nM) and the IN enzymatic activity was determined as described previously [22] and in Materials and Methods. (B) Rev-GFP was preincubated with the indicated INr peptides at a molar ratio of 5∶1 (peptide∶Rev-GFP) and the resultant mixture was added to a solution containing IN to give a molar ratio of 1∶200 and 1∶400 (IN∶Rev-GFP). Following an incubation period, IN activity was estimated as described previously [22]. All other experimental conditions are described in Materials and Methods.
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pone-0004155-g002: Inhibition of IN enzymatic activity by Rev and its abrogation by the INr peptides.(A) Rev-GFP (dark green) or GFP (turquoise) were incubated at different molar ratios with IN (390 nM) and the IN enzymatic activity was determined as described previously [22] and in Materials and Methods. (B) Rev-GFP was preincubated with the indicated INr peptides at a molar ratio of 5∶1 (peptide∶Rev-GFP) and the resultant mixture was added to a solution containing IN to give a molar ratio of 1∶200 and 1∶400 (IN∶Rev-GFP). Following an incubation period, IN activity was estimated as described previously [22]. All other experimental conditions are described in Materials and Methods.

Mentions: The results depicted in Fig 2A clearly show that the enzymatic activity of IN is inhibited by Rev-GFP due to specific interaction with the Rev protein itself and not with the GFP (Fig 2A). At a Rev-GFP∶IN (mole/mole) ratio of about 100, approx. 30% inhibition was already observed, reaching up to 70% inhibition at a ratio of 400 (Fig 2A). Interestingly, the INr peptides abrogated the inhibitory activities of Rev-GFP (Fig 2B). The results in Fig 3A confirm previously described results [22] showing that the Rev-derived peptides (Rev 53–67 and Rev 13–23 blocked IN enzymatic activity, reaching 60 to 70% inhibition at a peptide∶IN (mol/mol) ratio of about 150 (Fig 3A and B). It is also evident that the INr peptides were able to abrogate the inhibitory effect of the Rev derived peptides on integrase activity. Integrase activity was fully restored in the presence of both INr peptides at a molar ratio of Rev∶INr peptides of 1∶5–1∶10 (Fig 3A and B)). The fact that no abrogation was observed when the Rev∶INr peptides ratios was 1∶1 can be explained by the affinity differences towards their binding partners ([22] and table 2). This appeared to be due to the ability of the INrs to specifically interact with Rev-GFP and the Rev-derived peptides, since incubation with a scrambled peptide did not restore the integrase activity (Fig. 3B).


Peptides derived from HIV-1 integrase that bind Rev stimulate viral genome integration.

Levin A, Hayouka Z, Helfer M, Brack-Werner R, Friedler A, Loyter A - PLoS ONE (2009)

Inhibition of IN enzymatic activity by Rev and its abrogation by the INr peptides.(A) Rev-GFP (dark green) or GFP (turquoise) were incubated at different molar ratios with IN (390 nM) and the IN enzymatic activity was determined as described previously [22] and in Materials and Methods. (B) Rev-GFP was preincubated with the indicated INr peptides at a molar ratio of 5∶1 (peptide∶Rev-GFP) and the resultant mixture was added to a solution containing IN to give a molar ratio of 1∶200 and 1∶400 (IN∶Rev-GFP). Following an incubation period, IN activity was estimated as described previously [22]. All other experimental conditions are described in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2607543&req=5

pone-0004155-g002: Inhibition of IN enzymatic activity by Rev and its abrogation by the INr peptides.(A) Rev-GFP (dark green) or GFP (turquoise) were incubated at different molar ratios with IN (390 nM) and the IN enzymatic activity was determined as described previously [22] and in Materials and Methods. (B) Rev-GFP was preincubated with the indicated INr peptides at a molar ratio of 5∶1 (peptide∶Rev-GFP) and the resultant mixture was added to a solution containing IN to give a molar ratio of 1∶200 and 1∶400 (IN∶Rev-GFP). Following an incubation period, IN activity was estimated as described previously [22]. All other experimental conditions are described in Materials and Methods.
Mentions: The results depicted in Fig 2A clearly show that the enzymatic activity of IN is inhibited by Rev-GFP due to specific interaction with the Rev protein itself and not with the GFP (Fig 2A). At a Rev-GFP∶IN (mole/mole) ratio of about 100, approx. 30% inhibition was already observed, reaching up to 70% inhibition at a ratio of 400 (Fig 2A). Interestingly, the INr peptides abrogated the inhibitory activities of Rev-GFP (Fig 2B). The results in Fig 3A confirm previously described results [22] showing that the Rev-derived peptides (Rev 53–67 and Rev 13–23 blocked IN enzymatic activity, reaching 60 to 70% inhibition at a peptide∶IN (mol/mol) ratio of about 150 (Fig 3A and B). It is also evident that the INr peptides were able to abrogate the inhibitory effect of the Rev derived peptides on integrase activity. Integrase activity was fully restored in the presence of both INr peptides at a molar ratio of Rev∶INr peptides of 1∶5–1∶10 (Fig 3A and B)). The fact that no abrogation was observed when the Rev∶INr peptides ratios was 1∶1 can be explained by the affinity differences towards their binding partners ([22] and table 2). This appeared to be due to the ability of the INrs to specifically interact with Rev-GFP and the Rev-derived peptides, since incubation with a scrambled peptide did not restore the integrase activity (Fig. 3B).

Bottom Line: Both INr-1 and INr-2 were found to be cell-permeable and nontoxic, allowing a study of their effect in HIV-1-infected cultured cells.The results of the present study raise the possibility that in HIV-infected cells, the Rev protein may be involved in the integration of proviral DNA by controlling/regulating the activity of the integrase.Release from such inhibition leads to stimulation of IN activity and multiple viral DNA integration events.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, Israel.

ABSTRACT

Background: The human immunodeficiency virus type 1 (HIV-1) integrase protein (IN), catalyzes the integration of viral DNA into the host cell genome. IN catalyzes the first step of the integration process, namely the 3'-end processing in which IN removes a pGT dinucleotide from the 3' end of each viral long terminal repeat (LTR). Following nuclear import of the viral preintegration complex, the host chromosomal DNA becomes accessible to the viral cDNA and the second step of the integration process, namely the strand-transfer step takes place. This ordered sequence of events, centered on integration, is mandatory for HIV replication.

Methodology/principal findings: Using an integrase peptide library, we selected two peptides, designated INr-1 and INr-2, which interact with the Rev protein and probably mediate the Rev-integrase interaction. Using an in-vitro assay system, we show that INr-1 and INr-2 are able to abrogate the inhibitory effects exerted by Rev and Rev-derived peptides on integrase activity. Both INr-1 and INr-2 were found to be cell-permeable and nontoxic, allowing a study of their effect in HIV-1-infected cultured cells. Interestingly, both INr peptides stimulated virus infectivity as estimated by production of the viral P24 protein, as well as by determination of the appearance of newly formed virus particles. Furthermore, kinetics studies revealed that the cell-permeable INr peptides enhance the integration process, as was indeed confirmed by direct determination of viral DNA integration by real-time PCR.

Conclusions/significance: The results of the present study raise the possibility that in HIV-infected cells, the Rev protein may be involved in the integration of proviral DNA by controlling/regulating the activity of the integrase. Release from such inhibition leads to stimulation of IN activity and multiple viral DNA integration events.

Show MeSH
Related in: MedlinePlus