The HIV-1 capsid-binding host factor CPSF6 is post-transcriptionally regulated by the cellular microRNA miR-125b.

Cleavage and polyadenylation specificity factor 6 (CPSF6) is a cellular protein involved in mRNA processing. Emerging evidence suggests that CPSF6 also plays key roles in HIV-1 infection, specifically during nuclear import and integration targeting. However, the cellular and molecular mechanisms that regulate CPSF6 expression are largely unknown. In this study, we report a post-transcriptional mechanism that regulates CPSF6 via the cellular microRNA miR-125b. An in silico analysis revealed that the 3'UTR of CPSF6 contains a miR-125b-binding site that is conserved across several mammalian species. Because miRNAs repress protein expression, we tested the effects of miR-125b expression on CPSF6 levels in miR-125b knockdown and over-expression experiments, revealing that miR-125b and CPSF6 levels are inversely correlated. To determine whether miR-125b post-transcriptionally regulates CPSF6, we introduced the 3'UTR of CPSF6 mRNA into a luciferase reporter and found that miR-125b negatively regulates CPSF6 3'UTR-driven luciferase activity. Accordingly, mutations in the miR-125b seed sequence abrogated the regulatory effect of the miRNA on the CPSF6 3'UTR. Finally, pulldown experiments demonstrated that miR-125b physically interacts with CPSF6 3'UTR. Interestingly, HIV-1 infection down-regulated miR-125b expression concurrent with up-regulation of CPSF6. Notably, miR-125b down-regulation in infected cells was not due to reduced pri-miRNA or pre-miRNA levels. However, miR-125b down-regulation depended on HIV-1 reverse transcription but not viral DNA integration. These findings establish a post-transcriptional mechanism that controls CPSF6 expression and highlight a novel function of miR-125b during HIV-host interaction.

Bortezomib Sustains T Cell Function by Inducing miR-155-Mediated Downregulation of SOCS1 and SHIP1.

Suppressive mechanisms operating within T cells are linked to immune dysfunction in the tumor microenvironment. We have previously reported using adoptive T cell immunotherapy models that tumor-bearing mice treated with a regimen of proteasome inhibitor, bortezomib - a dipeptidyl boronate, show increased antitumor lymphocyte effector function and survival. Here, we identify a mechanism for the improved antitumor CD8+ T cell function following bortezomib treatment. Intravenous administration of bortezomib at a low dose (1 mg/kg body weight) in wild-type or tumor-bearing mice altered the expression of a number of miRNAs in CD8+ T cells. Specifically, the effect of bortezomib was prominent on miR-155 - a key cellular miRNA involved in T cell function. Importantly, bortezomib-induced upregulation of miR-155 was associated with the downregulation of its targets, the suppressor of cytokine signaling 1 (SOCS1) and inositol polyphosphate-5-phosphatase (SHIP1). Genetic and biochemical analysis confirmed a functional link between miR-155 and these targets. Moreover, activated CD8+ T cells treated with bortezomib exhibited a significant reduction in programmed cell death-1 (PD-1) expressing SHIP1+ phenotype. These data underscore a mechanism of action by which bortezomib induces miR-155-dependent downregulation of SOCS1 and SHIP1 negative regulatory proteins, leading to a suppressed PD-1-mediated T cell exhaustion. Collectively, data provide novel molecular insights into bortezomib-mediated lymphocyte-stimulatory effects that could overcome immunosuppressive actions of tumor on antitumor T cell functions. The findings support the approach that bortezomib combined with other immunotherapies would lead to improved therapeutic outcomes by overcoming T cell exhaustion in the tumor microenvironment.