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Identification of HIV Tat and NF-kB binding proteins associated with semen derived extracellular vesicles. In this study, we used orthogonal pull-downs, mobility shift, and electrophoretic protein binding assays coupled with proteomic and secretomic analyses to highlights the previously unappreciated SEV-associated transcriptional repressors that may target HIV Tat to suppress viral transcription.

ABSTRACT: Semen-derived extracellular vesicles (SEVs) have been shown to inhibit the human immunodeficiency virus type 1 (HIV-1) LTR transactivation through the blockade of the interaction of transactivator of transcription (Tat) and host transcription factors NF-kB and Sp1 (Tat•NF-ᴋB p65 and Tat•Sp1). The ability of SEVs to regulate the activities of transcription factors suggests that SEVs may contain transcription activators and repressors. In this present study, we used orthogonal SEVs-based screens, including pull-downs, mobility shift assays, and electrophoretic protein binding assays coupled with mass spectrometry (MS) to identify host proteins in SEVs that interact with the different transcription factors (SEVs•Tat and SEVs•NF-ᴋB p65 binding proteins). Protein network analysis performed on SEVs•Tat and SEVs•NF-ᴋB p65 complexes revealed interactome networks that associate with 7SK snRNA binding, mRNA Processing, P-TEFb complex, NELF complex. Additionally, Pathway enrichment analyses of SEVs•Tat and SEVs•NF-ᴋB p65 complexes identifies biological functions involved in initiation of transcription and translation elongation at the HIV LTR, formation of HIV and RNA Pol II elongation complexes, protein, nucleic acid, and chromatin binding, as well as chromatin assembly and disassembly. Finally, integrative analysis of SEVs•Tat and SEVs•NF-ᴋB p65 binding proteins identifies four proteins (AKAP9, ARHGEF28, INTS1, BRD2) to be common to both complexes. Immunoprecipitation assays suggest that SEVs-associated BRD2 and NELFB bind HIV Tat. The identification of extracellular interactome of Tat and NF-κB p65 in HIV transcription paves the way for the rational design of novel inhibitors of HIV transcription, with potential for therapeutic targeting.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Semen, Extracellular Vesicle

DISEASE(S): Human Immunodeficiency Virus Infectious Disease

SUBMITTER: Wasifa Naushad

LAB HEAD: Chioma M

PROVIDER: PXD062816 | Pride | 2026-06-15

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	Hussein_12-2018_SG_Hum_NFkB_complex.RAW	Raw
	Hussein_12-2018_SG_Hum_NFkB_complex.RAW.mzxml	Mzxml
	Hussein_12-2018_SG_Hum_NFkB_complex.mgf	Mgf
	db.mzidentml_1_1_0.xml	Mzid
	db.pep.xml	Pepxml

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Publications

Identification of proteins in semen-derived extracellular vesicles that bind to Tat and NF-κB and that may impair HIV replication.

Okeoma Bryson C BC Kaddour Hussein H Naushad Wasifa W Paromov Victor V Chaudhary Ashok A Noghero Alessio A Stapleton Jack T JT Okeoma Chioma M CM

Science signaling 20250909 903

Replication of HIV-1 requires the coordinated action of host and viral transcription factors, most critically the viral transactivator Tat and the host nuclear factor κB (NF-κB). This activity is disrupted in infected cells that are cultured with extracellular vesicles (EVs) present in human semen, suggesting that they contain factors that could inform the development of new therapeutics. Here, we explored the contents of semen-derived EVs (SEVs) from uninfected donors and individuals with HIV-1 ...[more]

PMID: 40924817

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Project description:Our previous work demonstrated that HIV-1 infection progressively reduces TCR/CD3 expression due to a defect in CD3g gene transcripts. We further found that knocking down expression of the viral tat and/or nef genes was correlated with CD3g transcript and TCR/CD3 surface receptor levels on HIV-1 infected cells. This study was undertaken to investigate the direct effect of HIV-1 Tat expression on the TCR/CD3 machinery. Progressive downregulation from TCR/CD3hi to TCR/CD3lo to TCR/CD3â was observed on Tat expressing cells in a manner that emulated HIV-1 infection, with a lack of CD3g transcripts again responsible for the defect. When Tat cell cultures containing a mixture of TCR/CD3 surface densities were separated into TCR/CD3hi and TCR/CD3lo/â populations, they quickly reverted to a mixed CD3 phenotype. Thus, the progression TCR/CD3hi to TCR/CD3lo to TCR/CD3â is an active, reversible process with receptor levels fluctuating in response to intracellular dynamics. Examination of tat mutants found that the regions involved in Tat-mediated transactivation and TAR binding are required for TCR/CD3 downregulation while the lysine at position 28 and Tat exon 2 are dispensable. Global gene expression, assessed in association with TCR/CD3 downregulation in HIV-1 infected and Tat expressing cells, detected broad suppression of TCR/CD3 signaling, co-stimulation and negative regulatory genes along with target transcription factors, ligands and receptors. A significant subset of the genes altered in HIV-1 infected cells was specifically targeted by Tat in association with TCR/CD3 loss. Our finding that Tat negatively regulates many facets of the TCR/CD3 machinery has important implications for disease pathogenesis. We used microarrays to investigate changes in CD4+ T cell gene expression induced by expression of the HIV-1 Tat protein. Examine changes in gene expression in HIV-1 Tat transduced cells compared with cells transduced with an antisense Tat sequence used as a control.

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Publications

Identification of proteins in semen-derived extracellular vesicles that bind to Tat and NF-κB and that may impair HIV replication.

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