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Ovine herpesvirus-2-encoded microRNAs target virus genes involved in virus latency.

ABSTRACT: Herpesviruses encode microRNAs (miRNAs) that target both virus and host genes; however, their role in herpesvirus biology is understood poorly. We identified previously eight miRNAs encoded by ovine herpesvirus-2 (OvHV-2), the causative agent of malignant catarrhal fever (MCF), and have now investigated the role of these miRNAs in regulating expression of OvHV-2 genes that play important roles in virus biology. ORF20 (cell cycle inhibition), ORF50 (reactivation) and ORF73 (latency maintenance) each contain predicted targets for several OvHV-2 miRNAs. Co-transfection of miRNA mimics with luciferase reporter constructs containing the predicted targets showed the 5' UTRs of ORF20 and ORF73 contain functional targets for ovhv-miR-2 and ovhv2-miR-8, respectively, and the 3' UTR of ORF50 contains a functional target for ovhv2-miR-5. Transfection of BJ1035 cells (an OvHV-2-infected bovine T-cell line) with the relevant miRNA mimic resulted in a significant decrease in ORF50 and a smaller but non-significant decrease in ORF20. However, we were unable to demonstrate a decrease in ORF73. MCF is a disease of dysregulated lymphocyte proliferation; miRNA inhibition of ORF20 expression may play a role in this aberrant lymphocyte proliferation. The proteins encoded by ORF50 and ORF73 play opposing roles in latency. It has been hypothesized that miRNA-induced inhibition of virus genes acts to ensure that fluctuations in virus mRNA levels do not result in reactivation under conditions that are unfavourable for viral replication and our data supported this hypothesis.

SUBMITTER: Riaz A

PROVIDER: S-EPMC4820034 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Project description:UnlabelledGammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi sarcoma-associated herpesvirus (KSHV, or HHV-8), and murine gammaherpesvirus 68 (MHV68, γHV68, or MuHV-4), are B cell-tropic pathogens that each encode at least 12 microRNAs (miRNAs). It is predicted that these regulatory RNAs facilitate infection by suppressing host target genes involved in a wide range of key cellular pathways. However, the precise contribution that gammaherpesvirus miRNAs make to viral life cycle and pathogenesis in vivo is unknown. MHV68 infection of mice provides a highly useful system to dissect the function of specific viral elements in the context of both asymptomatic infection and disease. Here, we report (i) analysis of in vitro and in vivo MHV68 miRNA expression, (ii) generation of an MHV68 miRNA mutant with reduced expression of all 14 pre-miRNA stem-loops, and (iii) comprehensive phenotypic characterization of the miRNA mutant virus in vivo. The profile of MHV68 miRNAs detected in infected cell lines varied with cell type and did not fully recapitulate the profile from cells latently infected in vivo. The miRNA mutant virus, MHV68.Zt6, underwent normal lytic replication in vitro and in vivo, demonstrating that the MHV68 miRNAs are dispensable for acute replication. During chronic infection, MHV68.Zt6 was attenuated for latency establishment, including a specific defect in memory B cells. Finally, MHV68.Zt6 displayed a striking attenuation in the development of lethal pneumonia in mice deficient in IFN-γ. These data indicate that the MHV68 miRNAs may facilitate virus-driven maturation of infected B cells and implicate the miRNAs as a critical determinant of gammaherpesvirus-associated disease.ImportanceGammaherpesviruses such as EBV and KSHV are widespread pathogens that establish lifelong infections and are associated with the development of numerous types of diseases, including cancer. Gammaherpesviruses encode many small noncoding RNAs called microRNAs (miRNAs). It is predicted that gammaherpesvirus miRNAs facilitate infection and disease by suppressing host target transcripts involved in a wide range of key cellular pathways; however, the precise contribution that these regulatory RNAs make to in vivo virus infection and pathogenesis is unknown. Here, we generated a mutated form of murine gammaherpesvirus (MHV68) to dissect the function of gammaherpesvirus miRNAs in vivo. We demonstrate that the MHV68 miRNAs were dispensable for short-term virus replication but were important for establishment of lifelong infection in the key virus reservoir of memory B cells. Moreover, the MHV68 miRNAs were essential for the development of virus-associated pneumonia, implicating them as a critical component of gammaherpesvirus-associated disease.

Project description:Background: Human cytomegalovirus (HCMV) is a beta-hersvirinae that has a high latent infection rate worldwide and can cause serious consequences in immunocompromised patients when reactivation; however, the mechanism of how HCMV convert from latent to reactivation has rarely been investigated. In the present study, we aimed to perform a comprehensive analysis of the HCMV-encoded microRNA (miRNA) profile in serum of patients upon HCMV reactivation from latency and to further evaluate its clinical significance for the disease monitoring and preventing usefulness.Methods: Serum samples from 59 viremia patients and 60 age-gender matched controls were enrolled in this study for screening and validation of different expression of HCMV miRNAs. Serum concentrations of 22 known HCMV miRNAs were determined by a hydrolysis probe-based stem-loop quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay. HCMV DNA was measured by quantitative real-time PCR (qPCR) with the whole blood sample. Serum HCMV IgG and IgM were assessed using enzyme linked immunosorbent assay (ELISA). Another 47 samples from 5 patients at different time points were collected to evaluate the monitoring effectiveness and disease prediction ability of differential expression HCMV-miRNAs during the antiviral treatment.Results: The RT-qPCR analysis revealed that the serum levels of 16 of the 22 examined HCMV miRNAs were elevated in HCMV viremia patients compared with controls, and a profile of 8 HCMV miRNAs including hcmv-miR-US25-2-3p, hcmv-miR-US4-5p, hcmv-miR-US25-2-5p, hcmv-miR-US25-1-3p, hcmv-miR-US25-1, hcmv-miR-UL36, hcmv-miR-UL148D, hcmv-miR-US29-3p were markedly elevated (fold change?>?2, P?<?0.01). Receiver operating characteristic curve (ROC) analysis were performed on the selected HCMV-miRNAs in all of the patients and controls that enrolled in this study, and which ranged from 0.72 to 0.80 in the autoimmune patients. In addition, hcmv-miR-US25-1-3p levels were significantly correlated with HCMV DNA load (r?=?0.349, P?=?0.007), and were obviously higher in the reactivation set than the latency set in the autoimmune patients, which could be a predictor for the monitoring of the antiviral treatment.Conclusions: HCMV miRNAs profile showed markedly shift-switch from latency to reactivation in circulation from HCMV infected patients and hcmv-miR-US25-1-3p may be served as a predictor for the switch upon reactivation from latency in patients suffered with autoimmune diseases.

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Ovine herpesvirus-2-encoded microRNAs target virus genes involved in virus latency.

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