SUMMARY MicroRNAs (miRNAs), ~22-nucleotide noncoding RNAs, assemble into RNA-induced silencing complexes (RISC) and localize to cytoplasmic substructures called P-bodies. Dictated by base-pair complementarity between miRNA and a target mRNA, miRNAs specifically repress posttranscriptional expression of several mRNAs. Here, we report that HIV-1 mRNA interacts with RISC proteins and that disrupting P-body structures enhances viral production and infectivity. In HIV-1-infected human T lymphocytes, we identified a highly abundant miRNA, miR-29a, which specifically targets the HIV-1 3’-UTR region. Inhibiting miR-29a enhanced HIV-1 viral production and infectivity, whereas expressing a miR-29 mimic suppressed viral replication. We also found that specific miR-29a-HIV-1 mRNA interactions enhance viral mRNA association with RISC and P-body proteins. Thus, we provide an example of a single host miRNA regulating HIV-1 production and infectivity. These studies highlight the significance of miRNAs and P-bodies in modulating host cell interactions with HIV-1 and possibly other viruses.
The SF1 helicase MOV10 is an antiviral factor that is incorporated into human immunodeficiency virus type 1 (HIV-1) virions. We now report that HIV-1 virions also incorporate UPF1, which belongs to the same SF1 helicase subfamily as MOV10 and functions in the nonsense-mediated decay (NMD) pathway. Unlike ectopic MOV10, the overexpression of UPF1 does not impair the infectivity of HIV-1 progeny virions. However, UPF1 becomes a potent inhibitor of HIV-1 progeny virion infectivity when residues required for its helicase activity are mutated. In contrast, equivalent mutations abolish the antiviral activity of MOV10. Importantly, cells depleted of endogenous UPF1, but not of another NMD core component, produce HIV-1 virions of substantially lower specific infectivity. The defect is at the level of reverse transcription, the same stage of the HIV-1 life cycle inhibited by ectopic MOV10. Thus, whereas ectopic MOV10 restricts HIV-1 replication, the related UPF1 helicase functions as a cofactor at an early postentry step.
Kaposi sarcoma (KS) herpesvirus (KSHV), also known as human herpesvirus-8, is the causative agent for KS and a plasmablastic form of multicentric Castleman disease (MCD; KSHV-MCD). 1 KSHV-MCD is a relapsing and remitting B-cell lymphoproliferative disorder that occurs primarily in people living with HIV. 2,3 Patients present with anemia, thrombocytopenia, and inflammatory symptoms associated with elevated cytokines including interleukin-6 (IL-6) and IL-10, elevated levels of C-reactive protein (CRP), and elevated KSHV viral loads (KSHV-VLs). KSHV encodes for viral IL-6 (vIL-6), an analog of human IL-6 (hIL-6), which contributes to KSHV-MCD pathogenesis, in part by driving production of hIL-6. [4][5][6] Treatment options include rituximab (a humanized monoclonal antibody against CD20), rituximab with liposomal doxorubicin or etoposide, or virus-activated cytotoxic therapy with high-dose zidovudine (AZT) and valganciclovir (VGC). 7-13 However, some patients do not respond to rituximab, and, when used alone, it is associated with worsening or development of KS in 35% to 67%. 8,9
From various screens, we found that Kaposi’s sarcoma-associated herpesvirus (KSHV) viral microRNAs (miRNAs) target several enzymes in the mevalonate/cholesterol pathway. 3-Hydroxy-3-methylglutaryl-coenzyme A (CoA) synthase 1 (HMGCS1), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR [a rate-limiting step in the mevalonate pathway]), and farnesyl-diphosphate farnesyltransferase 1 (FDFT1 [a committed step in the cholesterol branch]) are repressed by multiple KSHV miRNAs. Transfection of viral miRNA mimics in primary endothelial cells (human umbilical vein endothelial cells [HUVECs]) is sufficient to reduce intracellular cholesterol levels; however, small interfering RNAs (siRNAs) targeting only HMGCS1 did not reduce cholesterol levels. This suggests that multiple targets are needed to perturb this tightly regulated pathway. We also report here that cholesterol levels were decreased in de novo-infected HUVECs after 7 days. This reduction is at least partially due to viral miRNAs, since the mutant form of KSHV lacking 10 of the 12 miRNA genes had increased cholesterol compared to wild-type infections. We hypothesized that KSHV is downregulating cholesterol to suppress the antiviral response by a modified form of cholesterol, 25-hydroxycholesterol (25HC). We found that the cholesterol 25-hydroxylase (CH25H) gene, which is responsible for generating 25HC, had increased expression in de novo-infected HUVECs but was strongly suppressed in long-term latently infected cell lines. We found that 25HC inhibits KSHV infection when added exogenously prior to de novo infection. In conclusion, we found that multiple KSHV viral miRNAs target enzymes in the mevalonate pathway to modulate cholesterol in infected cells during latency. This repression of cholesterol levels could potentially be beneficial to viral infection by decreasing the levels of 25HC.
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