Peptides Derived from HIV-1 Integrase that Bind Rev Stimulate Viral Genome Integration

Abstract

BackgroundThe 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 FindingsUsing 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/SignificanceThe 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.

Nucleocytoplasmic shuttling of HIV-1 integrase is controlled by the viral Rev protein

“…4A and B) and Rev 53-67 (not shown)], blocked nuclear import of IN without affecting the intracellular localization of Rev. The opposite effect, inhibition of Rev and augmentation of IN nuclear import, was observed following the addition of the Rev-interacting IN-derived peptides 21 INr-1 ( Fig. 4C and D 20 The notion that formation of the Rev-IN complex induces inhibition of IN in virus-infected cells can also be inferred from experiments showing that disruption of these complexes results in stimulation of integration.…”

“…20,21 The HIV-1 Rev is a 116 residues protein, which has been assumed to be expressed after the integration process is complete. 22 It bears a well-defined nuclear localization signal (NLS) and nuclear export signal (NES) sequences that enable its nucleocytoplasmic shuttling.…”

“…28 The in vitro experiments revealed that the interaction with Rev inhibits IN enzymatic activity. 21 Rev-derived peptides (Rev 13-23 and Rev 53-67) also inhibited the enzymatic activity of the HIV-1 IN. 28 These cell-permeable Rev-derived peptides inhibited integration of the viral cDNA in virus-infected cells.…”

“…28 These cell-permeable Rev-derived peptides inhibited integration of the viral cDNA in virus-infected cells. 20,21,28 Inhibition of viral DNA integration was also observed when cells overexpressing Rev were infected with the wild-type (WT) HIV, suggesting an…”

“…4C and D 20 The notion that formation of the Rev-IN complex induces inhibition of IN in virus-infected cells can also be inferred from experiments showing that disruption of these complexes results in stimulation of integration. 20,21 This may be achieved by IN-derived peptides (INrs), which promote dissociation of the Rev-IN complexes in vitro 21 as well as in vivo. 20 Support for the view that the limited number of integration events in HIV-infected cells is due to the presence of an inhibitory Rev was also obtained from experiments showing multiple integration of cDNA following infection by a ∆Rev virus.…”