Naf1 Regulates HIV-1 Latency by Suppressing Viral Promoter-Driven Gene Expression in Primary CD4 ⁺ T Cells

Abstract

HIV-1 latency is characterized by reversible silencing of viral transcription driven by the long terminal repeat (LTR) promoter of HIV-1. Cellular and viral factors regulating LTR activity contribute to HIV-1 latency, and certain repressive cellular factors modulate viral transcription silencing. Nef-associated factor 1 (Naf1) is a host nucleocytoplasmic shuttling protein that regulates multiple cellular signaling pathways and HIV-1 production. We recently reported that nuclear Naf1 promoted nuclear export of unspliced HIV-1 gag mRNA, leading to increased Gag production. Here we demonstrate new functions of Naf1 in regulating HIV-1 persistence. We found that Naf1 contributes to the maintenance of HIV-1 latency by inhibiting LTR-driven HIV-1 gene transcription in a nuclear factor kappa B-dependent manner. Interestingly, Naf1 knockdown significantly enhanced viral reactivation in both latently HIV-1-infected Jurkat T cells and primary central memory CD4+ T cells. Furthermore, Naf1 knockdown in resting CD4+ T cells from HIV-1-infected individuals treated with antiretroviral therapy significantly increased viral reactivation upon T-cell activation, suggesting an important role of Naf1 in modulating HIV-1 latency in vivo. Our findings provide new insights for a better understanding of HIV-1 latency and suggest that inhibition of Naf1 activity to activate latently HIV-1-infected cells may be a potential therapeutic strategy. IMPORTANCE HIV-1 latency is characterized mainly by a reversible silencing of LTR promoter-driven transcription of an integrated provirus. Cellular and viral proteins regulating LTR activity contribute to the modulation of HIV-1 latency. In this study, we found that the host protein Naf1 inhibited HIV-1 LTR-driven transcription of HIV genes and contributed to the maintenance of HIV-1 latency. Our findings provide new insights into the effects of host modulation on HIV-1 latency, which may lead to a potential therapeutic strategy for HIV persistence by targeting the Naf1 protein.

SAMHD1 Impairs HIV-1 Gene Expression and Reactivation of Viral Latency in CD4+ T-cells

“…7A. Using a SAMHD1 -specific shRNA and established method (54, 55), we knocked down 40-50% of endogenous SAMHD1 expression in latently infected T CM derived from naïve CD4 + T-cells isolated from three healthy donors (Fig. 7B).…”

“…As suppression of the HIV-1 LTR is a common mechanism contributing to latency (52), we aimed to determine whether SAMHD1 affects latency reversal in cells. We utilized two HIV-1 latency cell models, the J-Lat cell line and primary T CM cells (40, 45, 54, 55, 67). In both models, SAMHD1 expression resulted in a suppression of latency reactivation.…”

“…The HTLV-1-LTR luciferase reporter plasmid and pcTax were provided by Patrick Green (The Ohio State University) (71). The HIV-1 FF-luc (pGL3-LTR-luc) was provided by Jian-Hua Wang (Pasteur Institute of Shanghai) (55). The pRenilla-TK plasmid was provided by Kathleen Boris-Lawrie (University of Minnesota).…”

“…We utilized the primary T CM model of latency as described (54, 55). In brief, naïve CD4 + T-cells cells were stimulated for 72 h with anti-CD3/CD28-antibody coated magnetic beads (Dynabeads).…”

Scaffold attachment factor B suppresses HIV-1 infection of CD4+ T cells by preventing binding of RNA polymerase II to HIV-1's long terminal repeat

“…Human peripheral blood mononuclear cells from healthy donors were purchased from the Blood Center of Shanghai (Shanghai, China). Resting CD4 ϩ T cells were isolated from peripheral blood mononuclear cells by using anti-CD4 antibody-coated magnetic beads (Miltenyi Biotec) as described previously (44). CD4 ϩ T cells were cultured in the presence of 20 international units/ml recombinant IL-2 (R&D Systems) and activated with 5 g/ml phytohemagglutinin-P (PHA-P) (Sigma-Aldrich) for 3 days.…”