HIV-1 Gag co-localizes with euchromatin histone marks at the nuclear periphery

ABSTRACT The retroviral Gag protein of human immunodeficiency virus type 1 (HIV-1) plays a central role in the selection of unspliced viral genomic RNA (gRNA) for packaging into new virions. Previously, we demonstrated that full-length HIV-1 Gag undergoes nuclear trafficking, where it associates with unspliced viral RNA (USvRNA) at transcription sites. To further examine the kinetics of HIV-1 Gag nuclear localization, we used biochemical and imaging techniques to determine the timing of HIV-1 entry into the nucleus. We also aimed to determine more precisely Gag’s subnuclear distribution to test the hypothesis that Gag associates with euchromatin, the transcriptionally active region of the nucleus. We observed that HIV-1 Gag localized to the nucleus at low expression levels shortly after its synthesis in the cytoplasm, suggesting that nuclear trafficking was not strictly concentration dependent. Furthermore, we found that HIV-1 Gag preferentially localized to the transcriptionally active euchromatin fraction compared to the heterochromatin-rich region in a latently infected T-cell line (J-Lat 10.6) treated with latency-reversal agents. Interestingly, HIV-1 Gag was more closely co-localized with euchromatin-associated histone marks near the nuclear periphery, the preferred location of HIV-1 proviral integration. Although the precise function of Gag’s association with histones in transcriptionally active chromatin regions remains uncertain, together with previous reports, this finding is consistent with a potential role for euchromatin-associated Gag molecules to initiate the selection of newly transcribed USvRNA in the nucleus for incorporation into virions. IMPORTANCE The traditional view of retrovirus assembly posits that packaging of gRNA by HIV-1 Gag occurs in the cytoplasm or at the plasma membrane. However, our previous studies showing that HIV-1 Gag enters the nucleus and binds to USvRNA at transcription sites suggest that gRNA selection may occur in the nucleus. In the present study, we observed that HIV-1 Gag trafficked to the nucleus and co-localized with USvRNA within 8 hours of expression. In infected T cells (J-Lat 10.6) reactivated from latency and in a HeLa cell line stably expressing an inducible Rev-dependent HIV-1 construct, we found that Gag preferentially localized with euchromatin histone marks associated with enhancer and promoter regions near the nuclear periphery, which is the favored site HIV-1 integration. These observations support the innovative hypothesis that HIV-1 Gag associates with euchromatin-associated histones to localize to active transcription sites, promoting capture of newly synthesized gRNA for packaging. The traditional view of retrovirus assembly posits that packaging of gRNA by HIV-1 Gag occurs in the cytoplasm or at the plasma membrane. However, our previous studies showing that HIV-1 Gag enters the nucleus and binds to USvRNA at transcription sites suggest that gRNA selection may occur in the nucleus. In the present study, we observed that HIV-1 Gag trafficked to the nucleus and co-localized with USvRNA within 8 hours of expression. In infected T cells (J-Lat 10.6) reactivated from latency and in a HeLa cell line stably expressing an inducible Rev-dependent HIV-1 construct, we found that Gag preferentially localized with euchromatin histone marks associated with enhancer and promoter regions near the nuclear periphery, which is the favored site HIV-1 integration. These observations support the innovative hypothesis that HIV-1 Gag associates with euchromatin-associated histones to localize to active transcription sites, promoting capture of newly synthesized gRNA for packaging.