Abstract
The present work describes a novel interaction between the human immunodeficiency virus type 1 (HIV-1) Rev protein and the cellular lens epithelium-derived growth factor p75 (LEDGF/p75) protein in vitro and in virus-infected cells. Here we show, for the first time, that formation of an Rev-LEDGF/p75 complex is a crucial step in regulating viral cDNA integration. Coimmunoprecipitation experiments at various times after virus infection revealed that, first, an integrase enzyme (IN)-LEDGF/p75 complex is formed, which is then replaced by a Rev-LEDGF/p75 and Rev-IN complexes. This was supported by in vitro experiments showing that Rev promotes dissociation of the IN-LEDGF/p75 complex. Combination of the viral IN and the cellular LEDGF/p75 is required for proper integration of the viral cDNA into the host chromosomal DNA. Our findings demonstrate that integration of HIV-1 cDNA is regulated by an interplay between viral Rev and the host-cell LEDGF/p75 proteins.
Highlights
Integration of the HIV genome into the host cell chromosome is a central event in the viral replication cycle [1]
Using an enzyme-linked immunosorbent assay (ELISA)-based system, a Rev-LEDGF/ p75 interaction was observed with an apparent Kd in the low nanomolar range, similar to that observed for the integrase enzyme (IN)-LEDGF/p75 and Rev-IN interactions (Figure 1A, B)
The fact that restoration of fluorescence demonstrates specific protein-protein interaction is strengthened by the control experiments that show no fluorescence was restored when cells were transformed with the linker plasmids containing only the half green florescent protein (GFP) (N-termini half of GFP [GN] or C-termini half of GFP [GC]) or when such linkers were coexpressed with Rev, IN or LEDGF/p75 conjugated to the second half of the GFP
Summary
Integration of the HIV genome into the host cell chromosome is a central event in the viral replication cycle [1]. In addition to the IN, the cellular protein lens epithelium-derived growth factor (LEDGF/p75), was shown to be required for promoting integration of the viral DNA [3,4]. The contribution of LEDGF/p75 to the tethering of IN to the host chromatin, and to the whole integration process, has been demonstrated by the dramatic decrease in HIV-1 replication in siRNA LEDGF/p75-knockdown cells [5,6,7,8]. In addition to the stimulatory LEDGF/p75 protein, the integration process is subjected to inhibition by another regulatory factor. Our recent data suggest that this factor may be the viral Rev protein [11,12]
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