Unintegrated HIV-1 DNAs are loaded with core and linker histones and transcriptionally silenced

Abstract

Significance Retroviruses are characterized by the reverse transcription of the viral RNA genome into DNA and the integration of that DNA to form the provirus. However, little is known about the nature of unintegrated HIV-1 DNAs early upon delivery into the nucleus. Using chromatin immunoprecipitation assays, we found that both core and H1 linker histones are deposited onto unintegrated HIV-1 DNAs. We also confirmed transcriptional silencing of unintegrated HIV-1 DNAs and determined the presence of posttranslational histone modifications characteristic of inactive chromatin. Our results will help to increase the efficiency of expression from nonintegrating HIV-1–based vectors and after transient transfections with DNA. Upon delivery into the nucleus of the host cell, linear double-stranded retroviral DNAs are either integrated into the host genome to form the provirus or act as a target of the DNA damage response and become circularized. Little is known about the chromatinization status of the unintegrated retroviral DNAs of the human immunodeficiency virus type 1 (HIV-1). In this study, we used chromatin immunoprecipitation to investigate the nature of unintegrated HIV-1 DNAs and discovered that core histones, the histone variant H3.3, and H1 linker histones are all deposited onto extrachromosomal HIV-1 DNA. We performed a time-course analysis and determined that the loading of core and linker histones occurred early after virus application. H3.3 and H1 linker histones were also found to be loaded onto unintegrated DNAs of the Moloney murine leukemia virus. The unintegrated retroviral DNAs are potently silenced, and we provide evidence that the suppression of extrachromosomal HIV-1 DNA is histone-related. Unintegrated DNAs were marked by posttranslational histone modifications characteristic of transcriptionally inactive genes: high levels of H3K9 trimethylation and low levels of H3 acetylation. These findings reveal insights into the nature of unintegrated retroviral DNAs.