HIV-1 gene expression and replication largely depend on the regulatory proteins Tat and Rev, but it is unclear how the intracellular levels of these viral proteins are regulated after infection.Here we report that HIV-1 Rev causes specific degradation of cytoplasmic Tat, which results in inhibition of HIV-1 replication. The nuclear export signal (NES) region of Rev is crucial for this activity but is not involved in direct interactions with Tat. Rev reduces the levels of ubiquitinated forms of Tat, which have previously been reported to be important for its transcriptional properties. Tat is stabilized in the presence of NAD(P)H:quinine oxidoreductase 1 (NQO1), and potent degradation of Tat is induced by dicoumarol, an NQO1 inhibitor. Furthermore, Rev causes specific reduction in the levels of endogenous NQO1. Thus, we propose that Rev is able to induce degradation of Tat indirectly by downregulating NQO1 levels. Our findings have implications in HIV-1 gene expression and latency.

Deubiquitinases (DUBs) are key regulators of complex cellular processes. HIV-1 Tat is synthesized early after infection and is mainly responsible for enhancing viral production. Here, we report that one of the DUBs, USP7, stabilized the HIV-1 Tat protein through its deubiquitination. Treatment with either a general DUB inhibitor (PR-619) or USP7-specific inhibitor (P5091) resulted in Tat protein degradation. The USP7-specific inhibitor reduced virus production in a latently infected T-lymphocytic cell line J1.1, which produces large amounts of HIV-1 upon stimulation. A potent increase in Tat-mediated HIV-1 production was observed with USP7 in a dose-dependent manner. As expected, deletion of the USP7 gene using the CRISPR-Cas9 method reduced the Tat protein and supported less virus production. Interestingly, the levels of endogenous USP7 increased after HIV-1 infection in human T-cells (MOLT-3) and in mammalian cells transfected with HIV-1 proviral DNA. Thus, HIV-1 Tat is stabilized by the host cell deubiquitinase USP7, leading to enhanced viral production, and HIV-1 in turn up-regulates the USP7 protein level.

Murine double minute 2 (Mdm2) is known to enhance the transactivation potential of human immunodeficiency virus (HIV-1) Tat protein by causing its ubiquitination. However, the regulation of Mdm2 during HIV-1 infection and its implications for viral replication have not been well studied. Here, we show that the Mdm2 protein level increases during HIV-1 infection and this effect is mediated by HIV-1 Tat protein. Tat appears to stabilise Mdm2 at the post-translational level by inducing its phosphorylation at serine-166 position through AKT. Although p53 is one of the key players for Mdm2 induction, Tat-mediated stabilisation of Mdm2 appears to be independent of p53. Moreover, the non-phosphorylatable mutant of Mdm2 (S166A) fails to interact with Tat and shows decreased half-life in the presence of Tat compared with wild-type Mdm2. Furthermore, the non-phosphorylatable mutant of Mdm2 (S166A) is unable to support HIV-1 replication. Thus, HIV-1 Tat appears to stabilise Mdm2, which in turn enhances Tat-mediated viral replication. This study highlights the importance of post-translational modifications of host cellular factors in HIV-1 replication and pathogenesis.

In addition to glial cells, HIV-1 infection occurs in multipotent human neural precursor cells (hNPCs) and induces quiescence in NPCs. HIV-1 infection of the brain alters hNPC stemness, leading to perturbed endogenous neurorestoration of the CNS following brain damage by HIV-1, compounding the severity of dementia in adult neuroAIDS cases. In pediatric neuroAIDS cases, HIV-1 infection of neural stem cell can lead to delayed developmental milestones and impaired cognition. Using primary cultures of human fetal brain-derived hNPCs, we gained novel insights into the role of a neural stem cell determinant, tripartite containing motif 32 (TRIM32), in HIV-1 Tat-induced quiescence of NPCs. Acute HIV-1 Tat treatment of hNPCs resulted in proliferation arrest but did not induce differentiation. Cellular localization and levels of TRIM32 are critical regulators of stemness of NPCs. HIV-1 Tat exposure increased nuclear localization and levels of TRIM32 in hNPCs. The in vitro findings were validated by studying TRIM32 localization and levels in frontal cortex of HIV-1-seropositive adult patients collected at post mortem as well as by infection of hNPCs by HIV-1. We observed increased percentage of cells with nuclear localization of TRIM32 in the subventricular zone (SVZ) as compared with age-matched controls. Our quest for probing into the mechanisms revealed that TRIM32 is targeted by miR-155 as downregulation of miR-155 by HIV-1 Tat resulted in upregulation of TRIM32 levels. Furthermore, miR-155 or siRNA against TRIM32 rescued HIV-1 Tat-induced quiescence in NPCs. Our findings suggest a novel molecular cascade involving miR-155 and TRIM32 leading to HIV-1 Tat-induced attenuated proliferation of hNPCs. The study also uncovered an unidentified role for miR-155 in modulating human neural stem cell proliferation, helping in better understanding of hNPCs and diseased brain.

The innate immune response is crucial for defense against viral infections. Cells recognize virus infection through pattern recognition receptors and induce type I interferons as well as proinflammatory cytokines to orchestrate an innate immune response. Herpes simplex virus 1 (HSV-1) triggers both the cyclic GMP-AMP synthase (cGAS)–stimulator of interferon genes (STING) and Toll-like receptor 3 (TLR3) pathways. It is well known that TLR3 uses the adaptor protein Toll/interleukin-1 receptor (IL-1R) domain-containing adaptor-inducing beta interferon (TRIF) for signaling, but we recently reported that STING signaling also requires TRIF. Because STING directly binds to TRIF, we identified the STING-interacting domain of TRIF and generated STING-noninteracting mutants of human and mouse TRIFs. The mutant TRIFs were unable to support STING signaling, although they were fully functional in the TLR3 pathway. These mutants were used to assess the relative contributions of the TLR3 and STING pathways to the attenuation of HSV-1 replication in mouse and human cell lines. For this purpose, the mouse L929 and NB41A3 cell lines and the human HT1080 and HeLa-M cell lines, in which both the TLR3 and the STING pathways are operational, were used. The TRIF gene was disrupted in these lines by CRISPR/Cas9, before reconstituting them with mutant and wild-type TRIF expression vectors. Infection of the reconstituted cells with HSV-1 revealed that both the cGAS-STING and the TLR3 signaling pathways are required for the attenuation of virus replication, but their relative contributions in attenuating HSV-1 replication were found to be different in mouse versus human cell lines. Thus, our study suggests that the relative contributions of the cGAS-STING and the TLR3 pathways in the attenuation of viral infection may be species specific. IMPORTANCE The magnitude of fatal infections caused by all different viruses in human and animal populations justifies a better understanding of the host innate immune response process that attenuates virus replication. In particular, the relative contributions of different signaling pathways which are responsible for the generation of the innate immune response are still largely unknown. In this study, we used STING-noninteracting TRIF mutants to decipher the relative contributions of the TLR3 and cGAS-STING signaling pathways to the attenuation of HSV-1 infection. We show that the relative contributions of the two pathways to the attenuation of viral infection are different in mouse versus human cell lines. Together, our results provide new insights into the relative contributions of two different signaling pathways in the attenuation of viral infection and may lead to the development of new antiviral strategies aimed at blocking viral infection at very early stages.