Dendritic cells maturated by co-culturing with HIV-1 latently infected Jurkat T cells or stimulating with AIDS-associated pathogens secrete TNF-α to reactivate HIV-1 from latency.

Elucidation of mechanisms underlying the establishment, maintenance of and reactivation from HIV-1 latency is essential for the development of therapeutic strategies aimed at eliminating HIV-1 reservoirs. Microbial translocation, as a consequence of HIV-1-induced deterioration of host immune system, is known to result in a systemic immune activation and transient outbursts of HIV-1 viremia in chronic HIV-1 infection. How these microbes cause the robust HIV-1 reactivation remains elusive. Dendritic cells (DCs) have previously been shown to reactivate HIV-1 from latency; however, the precise role of DCs in reactivating HIV-1 from latently infected T-cell remains obscure. In this study, by using HIV-1 latently infected Jurkat T cells, we demonstrated that AIDS-associated pathogens as represented by Mycobacterium bovis (M. bovis) Bacillus Calmette-Guérin (BCG) and bacterial component lipopolysaccharide (LPS) were unable to directly reactivate HIV-1 from Jurkat T cells; instead, they mature DCs to secrete TNF-α to accomplish this goal. Moreover, we found that HIV-1 latently infected Jurkat T cells could also mature DCs and enhance their TNF-α production during co-culture in a CD40-CD40L-signaling-dependent manner. This in turn led to viral reactivation from Jurkat T cells. Our results reveal how DCs help AIDS-associated pathogens to trigger HIV-1 reactivation from latency.

Amino acids at positions 3, 168, and 169 are associated with the ability of Nef proteins from HIV-1 CRF01_AE to downmodulate CD4.

Several HIV-1 subtypes are co-circulating among various high-risk groups in China, and an increasing prevalence of CRF01_AE was observed among MSM (men who have sex with men) within recent years. Patients infected with CRF01_AE may experience a more rapid disease progression than patients infected with non-CRF01_AE; however, the underlying mechanisms remains elusive. HIV-1 Nef is a multifunctional protein and plays critical roles in viral pathogenesis. Nef downregulates CD4 and human leukocyte antigen (HLA) to promote viral transmission and escape from the host immune response. In this study, we investigated the CD4 downmodulation activity of Nef proteins isolated from HIV-1 CRF01_AE and analyzed a potential relationship of Nef's capacity to downregulate CD4 with disease progression. We found that the majority of these Nefs from HIV-1 CRF01_AE efficiently downregulated CD4; Nefs with weaker CD4 downmodulation activity tended to be associated with higher CD4 levels and lower viral loads. Further elucidation revealed that amino acid residues at positions 3, 168, and 169 of CRF01_AE Nefs were associated with the capacity to downregulate CD4. Our data suggest that the capacity of Nef-mediated CD4 downregulation is not the only determinant for controlling disease progression, and other host and viral factors should be considered to explain the rapid disease progression of patients infected with HIV-1 CRF01_AE.

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

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.

Toll-interacting protein inhibits HIV-1 infection and regulates viral latency.

HIV-1 latency is mainly characterized by a reversible silencing of long-terminal repeat (LTR)-driven transcription of provirus. The existing of repressive factors has been described to contribute to transcription silencing of HIV-1. Toll-interacting protein (Tollip) has been identified as a repressor of Toll like receptors (TLR)-mediated signaling. Our previous study has found that Tollip inhibited NF-κB-dependent HIV-1 promoter LTR-driven transcription, indicating the potential role of Tollip in governing viral latency. In this study, by using HIV-1 latently infected Jurkat T-cell and central memory CD4(+) T-cells, we demonstrate the role of Tollip in regulating HIV-1 latency, as the knock-down of Tollip promoted HIV-1 reactivation from both HIV-1 latently infected Jurkat CD4(+) T cells and primary central memory T cells (TCM). Moreover, we found that the activities of LTRs derived from multiple HIV-1 subtypes could be repressed by Tollip; Knock-down of Tollip promoted HIV-1 transcription and infection in CD4(+) T cells. Our data indicate a key role of Tollip in suppressing HIV-1 infection and regulating viral latency, which provides a potential host target for combating HIV-1 infection and latency.

Toll-Interacting Protein Suppresses HIV-1 Long-Terminal-Repeat-Driven Gene Expression and Silences the Post-Integrational Transcription of Viral Proviral DNA.

Toll-interacting protein (Tollip) is a host adaptor protein for negatively regulating Toll-like receptor 2-, 4-, and IL-1R (interleukin-1 receptor)-mediated signaling. We found that Tollip expression could be induced in MDDCs (monocyte-derived dendritic cells) by HIV-1 particles and recombinant gp120 glycoprotein. Hence, we investigated the role of Tollip in modulating HIV-1 infection. We found that Tollip expression suppressed NF-κB-dependent HIV-1 long terminal repeat (LTR)-driven transcription and thus inhibited HIV-1 infection. Our protein truncation experiments proved that the intact C-terminus of Tollip was required for inhibition of both NF-κB activity and HIV-1 LTR-driven gene expression. Intriguingly, Tollip silenced the post-integrational transcription of HIV-1 proviral DNA, indicating the potential role of Tollip in maintaining viral persistence. Our results reveal the novel role of host factor Tollip in modulating HIV-1 infection, and may suggest the hijacking of Tollip as the negative regulator of the TLR pathway and even the downstream signaling, by HIV-1 for maintaining persistent infection. Further elucidation of the mechanisms by which HIV-1 induces Tollip expression and identification of the role of Tollip in modulating HIV-1 latency will facilitate the understanding of host regulation in viral replication and benefit the exploration of novel strategies for combating HIV-1 infection.