Interaction between Nef and INI1/SMARCB1 augments replicability of HIV-1 in resting human peripheral blood mononuclear cells

Abstract

A central feature of HIV-1 infection is the inability of entering virus to integrate into chromosomes of resting T lymphocytes unless they are mitogenically activated. In contrast, SIVpbj1.9 replicates in initially resting T lymphocytes by activating infected cells. Previous reports have shown that a difference in Nef-mediated T cell activation between HIV-1 and SIVpbj1.9 plays a critical role in the differing abilities of these viruses to replicate in resting lymphocytes. However, the molecular details of these differences are still unclear. Here, we show that infection with a chimeric virus, HSIVnef, which harbors the 5′ 308 nucleotides of SIVpbj1.9 nef in place of the 5′ 221 nucleotides of HIV-1 nef in the HIV-1 proviral backbone, resulted in integration of the provirus into host chromosomes without mitogenic activation and thereby replication in resting human PBMCs (hPBMCs). These results indicate that Nef is an essential viral determinant for the integration of provirus into host chromosomes in resting T cells. Using the yeast two-hybrid system, we identified integrase interactor-1 (INI1/SMARCB1) as a cellular factor that is involved in the integration process via interaction with Nef. Although INI1 interacted with both SIVpbj1.9 and HIV-1 Nefs, SIVpbj1.9 Nef, but not HIV-1 Nef, enhanced proviral integration into host DNA. Furthermore, mutational analysis revealed that the basic-amino-acid-rich amino-terminal domain in SIVpbj1.9 Nef is crucial for interaction with INI1 and virus replication in resting hPBMCs. Taken together, these data indicate that Nef is a critical viral protein for incorporating nascent proviral DNA into host chromosomes in resting PBMCs and that this occurs through interaction with INI1. This elucidates the basis for replication of the integrated provirus when the host cell is in a resting state.

Function of ubiquitin (Ub) specific protease 15 (USP15) in HIV-1 replication and viral protein degradation

“…Alternatively, USP15 could activate other cellular protein(s) in the USP15/Nef complex, and the activated protein(s) in turn degrades Nef protein. We accordingly screened for a potential cellular component involved in the UPS-mediated protein degradation by interacting with Nef, using the yeast- followed by the mammalian-two-hybrid assays (Kalpana et al, 1994; Pyeon and Park, 2015). We found that Nef binds to ubiquitin-protein ligase E3A (UBE3A/E6AP) which induces protein degradation by attaching Ub to substrates (Bernassola et al, 2008; Vande Pol and Klingelhutz, 2013), i.e.…”

“…For RT assay, virions in the supernatant were pelleted by centrifugation at 12,000 g for 1 h, and the RT activity was determined, as described (Pyeon and Park, 2015). For virion preparation for WB, cells and cell debris in the culture supernatant were removed by centrifugation at 800 g for 100 min, and the cleared supernatant was passed through a 0.22 μm filter (Corning, NY) to ensure complete removal of smaller cell debris.…”

“…For virion preparation for WB, cells and cell debris in the culture supernatant were removed by centrifugation at 800 g for 100 min, and the cleared supernatant was passed through a 0.22 μm filter (Corning, NY) to ensure complete removal of smaller cell debris. Virions in the filtrate were precipitated in 20% sucrose in PBS by centrifugation at 238,000 g for 90 min, as described (Pyeon and Park, 2015), and virion proteins were analyzed by WB, as described above.…”

Comparative molecular genetic analysis of simian and human HIV-1 integrase interactor INI1/SMARCB1/SNF5

“…His-tagged HIV-1 INT was purified using Ni-NTA agarose beads (QIAGEN, Valencia, CA) from overnight culture of BL21 cells transformed with pINSD.His.Sol, obtained from Dr. Robert Craigie through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. The in vitro DNA strand transfer assay was then performed, using purified INT and INI1 as described previously [4, 8]. Briefly, the HIV-1 INT substrate U5.5 (5′-GGATCCGGAAAA TCTCTAGCA) was labeled with 50 μCi of [γ- 32 P]ATP (NEN) by incubating with 10 U of T4 polynucleotide kinase (New England Biolabs, Beverly, MA).…”

“…Our recent studies have revealed that the Nef protein of simian immunodeficiency virus PBj1.9 enhanced proviral DNA integration into the host chromosome in association with INT and human INI1 [8]. To verify that simian INI1 also has the same function in proviral DNA integration, we sequenced simian INI1 cDNA and compared its function with that of its human counterpart.…”