Exploring histone loading on HIV DNA reveals a dynamic nucleosome positioning between unintegrated and integrated viral genome

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.

Epigenetic Mechanisms of HIV-1 Persistence

“…More specifically, HIV-1 episomes have been found to be enriched in histone modifications typical of a repressive heterochromatin architecture, such as the trimethylation of histone 3 lysine 9 (H3K9me3) [ 42 ]. A recent report has confirmed this observation and shown that histones are loaded soon after the synthesis of HIV-1 DNA and before its integration in the host chromatin [ 43 ]. In particular, nucleosome positioning was found to dynamically vary at the 5′LTR between unintegrated and integrated forms of HIV-1 DNA [ 43 ].…”

“…A recent report has confirmed this observation and shown that histones are loaded soon after the synthesis of HIV-1 DNA and before its integration in the host chromatin [ 43 ]. In particular, nucleosome positioning was found to dynamically vary at the 5′LTR between unintegrated and integrated forms of HIV-1 DNA [ 43 ]. The more closed nucleosome lattice in the unintegrated viral promoter, in turn, prevented the transcription of viral genes [ 43 ].…”

“…In particular, nucleosome positioning was found to dynamically vary at the 5′LTR between unintegrated and integrated forms of HIV-1 DNA [ 43 ]. The more closed nucleosome lattice in the unintegrated viral promoter, in turn, prevented the transcription of viral genes [ 43 ]. However, the mechanisms by which epigenetic silencing is exerted on unintegrated HIV-1 episomes remains to be elucidated.…”

Silencing of Unintegrated Retroviral DNAs

“…For these reasons, it has been of interest to determine the properties of newly synthesized DNA formed by HIV-1 and compare them with those of the MLVs. Unintegrated HIV-1 DNAs formed by integration-defective virions, or formed in the presence of integrase inhibitors, were found to be heavily silenced, much like those of the MLVs [65][66][67]. Examination of the presence of histones on the unintegrated HIV-1 DNA by ChIP revealed the rapid loading of histones H3 and H2B, as seen for the MLVs [65,66].…”

“…Unintegrated HIV-1 DNAs formed by integration-defective virions, or formed in the presence of integrase inhibitors, were found to be heavily silenced, much like those of the MLVs [65][66][67]. Examination of the presence of histones on the unintegrated HIV-1 DNA by ChIP revealed the rapid loading of histones H3 and H2B, as seen for the MLVs [65,66]. Moreover, analysis of the histones by ChIP again revealed the presence of marks associated with silencing: histones bound to the HIV-1 viral DNA were heavily marked by H3K9me3, and were very low in H3 acetylation.…”