Abstract
The aim of the present study was to understand the biology of unintegrated HIV-1 DNA and reveal the mechanisms involved in its transcriptional silencing. We found that histones are loaded on HIV-1 DNA after its nuclear import and before its integration in the host genome. Nucleosome positioning analysis along the unintegrated and integrated viral genomes revealed major differences in nucleosome density and position. Indeed, in addition to the well-known nucleosomes Nuc0, Nuc1, and Nuc2 loaded on integrated HIV-1 DNA, we also found NucDHS, a nucleosome that covers the DNase hypersensitive site, in unintegrated viral DNA. In addition, unintegrated viral DNA-associated Nuc0 and Nuc2 were positioned slightly more to the 5' end relative to their position in integrated DNA. The presence of NucDHS in the proximal region of the long terminal repeat (LTR) promoter was associated with the absence of RNAPII and of the active histone marks H3K4me3 and H3ac at the LTR. Conversely, analysis of integrated HIV-1 DNA showed a loss of NucDHS, loading of RNAPII, and enrichment in active histone marks within the LTR. We propose that unintegrated HIV-1 DNA adopts a repressive chromatin structure that competes with the transcription machinery, leading to its silencing.
Highlights
The aim of the present study was to understand the biology of unintegrated HIV-1 DNA and reveal the mechanisms involved in its transcriptional silencing
To investigate histone loading on HIV DNA, we infected Jurkat cells with VSV-G pseudotyped HIV-1 engineered to express RFP under the control of the long terminal repeat (LTR) and GFP under the control of the CMV promoter (CMV-gfp transcriptional unit inserted after nef-rfp)
H3 and H2B were loaded on newly reverse-transcribed viral DNA, because incubation with nevirapin and infection with heat-inactivated HIV-1 resulted in loss of the H3 signal at HIV DNA, with no effect on the chromatin immunoprecipitation (ChIP) signals at genomic loci (Fig. 1H)
Summary
The aim of the present study was to understand the biology of unintegrated HIV-1 DNA and reveal the mechanisms involved in its transcriptional silencing. In the life cycle of HIV, on fusion of the viral and cellular membranes and release of the viral core in the host cell cytoplasm, the viral RNA genome is reverse-transcribed into a double-stranded linear viral DNA (dslvDNA). This dslvDNA, associated with the viral integrase (IN) and various viral and cellular proteins, forms a large nucleoprotein complex, the preintegration complex (PIC) [1, 2]. On integration in the host genome, HIV-1 DNA adopts a chromatin structure with precisely positioned nucleosomes (Nuc0, Nuc, and Nuc2) and a DNase hypersensitive site (DHS) around the HIV long terminal repeat (LTR) [15, 16]. Activation of HIV-1 gene expression is accompanied by chromatin remodeling, at the repressive Nuc positioned immediately downstream of the transcription start site (TSS) [15, 16]; it is Significance
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