HIV-1 Gag colocalizes 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 for packaging into new virions. Previously, we demonstrated that full-length HIV-1 Gag undergoes nuclear trafficking where it associates with unspliced viral RNA (vRNA) at transcription sites. To further explore the kinetics of HIV-1 Gag nuclear localization, we used biochemical and imaging techniques to examine 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 would be associated with euchromatin, the transcriptionally active region of the nucleus. We observed that HIV-1 Gag localized to the nucleus 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 CD4+ T cell line (J-Lat 10.6) treated with latency-reversal agents. Interestingly, HIV-1 Gag was more closely associated with transcriptionally-active histone markers near the nuclear periphery, where the HIV-1 provirus was previously shown to integrate. Although the precise function of Gag's association with histones in transcriptionally-active chromatin remains uncertain, together with previous reports, this finding is consistent with a potential role for euchromatin-associated Gag molecules to select newly transcribed unspliced vRNA during the initial stage of virion assembly.

Comparative analysis of retroviral Gag-host cell interactions: focus on the nuclear interactome

“…When the HIV-1 Gag interactomes identified by other laboratories were compared to the interacting proteins identified by us in this report, 190 common proteins were found by at least two independent laboratory groups. Further experimentation will be needed to validate each of these factors to determine whether they play important roles in retrovirus replication or pathogenesis (Fig5).Our past and present cell fractionation experiments demonstrated that, in addition to being present in the nucleoplasm, both the RSV and HIV-1 Gag proteins can be extracted from euchromatin and heterochromatin fractions, complementing our recently published report indicating that HIV-1 Gag localizes with euchromatin marks at the nuclear periphery(9,10). These results suggest that HIV-1 Gag may be specifically targeted to a chromatin-associated compartment through interactions with host nuclear binding partners.…”

“…Tables 3 and 4 list the proteins involved in transcription and splicing, respectively, for the RSV pulldowns. Similarly, Tuffy et al and Chang et al (9, 10) both demonstrated that HIV-1 Gag localizes to transcriptionally active regions in HeLa cells and T cells reactivated from latency. Given these findings, it is feasible that HIV-1 Gag interacts with host nuclear factors involved in transcription, RNA processing, and chromatin remodeling.…”

“…Prior experiments using microscopy revealed that both RSV and HIV-1 Gag proteins localize to the perichromatin compartment of the nucleus where they associate with their cognate USvRNAs at active transcription sites (3, 9, 11). In addition, HIV-1 Gag associates preferentially with marks associated with transcriptionally-active euchromatin compared to heterochromatin (10). To further define the nuclear subcompartments where RSV Gag is localized, cells expressing wild-type or mutant Gag proteins were separated into cytoplasmic, nucleoplasmic, and chromatin-associated protein fractions ( Fig 1 ).…”

“…HIV-1 Gag forms focal RNP complexes with nascent USvRNA in the nucleus, and traffics to the major viral RNA transcription site in T cells reactivated from latency (9). Nuclear localization of HIV-1 Gag occurs in a concentra-tion-independent manner shortly after Gag synthesis begins, and Gag colocalizes with transcriptionally-active euchromatin near the nuclear periphery (10). The function(s) of these nuclear RNPs has yet to be thoroughly investigated, although these data demonstrate that both RSV and HIV-1 Gag proteins traffic to transcription sites and associate with their cognate USvRNAs.…”

Comparative analysis of retroviral Gag-host cell interactions: focus on the nuclear interactome

“…HIV-1 Gag forms focal RNP complexes with nascent USvRNA in the nucleus, and traffics to the major viral RNA transcription site in T cells reactivated from latency [ 9 ]. Nuclear localization of HIV-1 Gag occurs in a concentration-independent manner shortly after Gag synthesis begins, and Gag colocalizes with transcriptionally-active euchromatin near the nuclear periphery [ 10 ]. The function(s) of these nuclear RNPs has yet to be thoroughly investigated, although these data demonstrate that both RSV and HIV-1 Gag proteins traffic to transcription sites and associate with their cognate USvRNAs.…”