Human herpesvirus-encoded MicroRNA in host-pathogen interaction.

Human herpesviruses (HHV) are ubiquitous, linear dsDNA viruses that establish lifelong latency, disrupted by sporadic reactivation. HHV have evolved diverse ingenious mechanisms to evade robust host defenses. Incorporation of unique stem loop sequences that generate viral microRNAs (v-miRs) exemplifies one such evolutionary adaptation in HHV. These noncoding RNAs can control cellular and viral transcriptomes highlighting their ability in shaping host-HHV interactions. We summarize recent developments in functional characterization of HHV-encoded miRNAs in shaping the outcome of host-pathogen interaction. Non-immunogenic dissemination of v-miRs through exosomes confer added advantage to HHV in incessant modulation of host microenvironment. This review delineates the mechanistic role of v-miRs in facilitating viral persistence and tropism by targeting genes associated with cellular (apoptosis, angiogenesis, cell migration, etc.) and viral life cycle (latency, lytic and reactivation). Burgeoning evidences indicate plausible association of v-miRs in various immune-mediated diseases (nasopharyngeal carcinoma, neurological disorders, periodontal diseases, etc.) and herpesvirus-related malignancies indicating their broad-spectrum impact on host cellular pathways. We propose to exploit tisssue and systemic levels of v-miRs as diagnostic and prognostic markers for cancers and immune-mediated diseases. Therapeutic targeting of v-miRs will advance the promising outcomes of preclinical discoveries to bedside application.

Herpesviruses and MicroRNAs: New Pathogenesis Factors in Oral Infection and Disease?

The oral cavity incessantly encounters a plethora of microorganisms. Effective and efficient oral innate and adaptive immune responses are incumbent to maintain healthy mucosa. A higher prevalence of Human Herpesviruses (HHV), a family of large enveloped DNA viruses, has been reported in multiple oral inflammatory diseases suggesting their involvement in disease progression. However, the viral components contributing to oral disease remain obscure. MicroRNAs (miRNA) are non-protein coding, single stranded ribonucleic acid (RNA) molecules that post-transcriptionally regulate diverse messenger RNAs. Thus, miRNAs can control large repertoire of biological processes. Changes in miRNA expression are associated with various oral infections and diseases. Cellular miRNAs can act as pro- or anti-viral factors and dysregulation of host miRNA expression occurs during herpesviruses infection. This strongly suggest a critical role of cellular miRNAs in host-herpesvirus interaction. Interestingly, HHV also encode multiple miRNAs (called viral miRNAs) that may play key role in host-pathogen interaction by modulating both host biological pathways and controlling viral life cycle. Recent studies from our laboratory have identified viral miRNAs (v-miRs) in diseased oral tissue biopsies and demonstrate their immunomodulatory roles. This review discusses the association of miRNAs (both host and viral) and herpesviruses in the pathogenesis of oral inflammatory diseases.

Viral miRNAs Alter Host Cell miRNA Profiles and Modulate Innate Immune Responses.

Prevalence of the members of herpesvirus family in oral inflammatory diseases is increasingly acknowledged suggesting their likely role as an etiological factor. However, the underlying mechanisms remain obscure. In our recent miRNA profiling of healthy and diseased human tooth pulps, elevated expression of human herpesvirus encoded viral microRNAs (v-miRs) were identified. Based on the fold induction and significance values, we selected three v-miRs namely miR-K12-3-3p [Kaposi sarcoma-associated virus (KSHV)], miR-H1 [herpes simplex virus 1 (HSV1)], and miR-UL-70-3p [human cytomegalovirus (HCMV)] to further examine their impact on host cellular functions. We examined their impact on cellular miRNA profiles of primary human oral keratinocytes (HOK). Our results show differential expression of several host miRNAs in v-miR-transfected HOK. High levels of v-miRs were detected in exosomes derived from v-miR transfected HOK as well as the KSHV-infected cell lines. We show that HOK-derived exosomes release their contents into macrophages (Mφ) and alter expression of endogenous miRNAs. Concurrent expression analysis of precursor (pre)-miRNA and mature miRNA suggest transcriptional or posttranscriptional impact of v-miRs on the cellular miRNAs. Employing bioinformatics, we predicted several pathways targeted by deregulated cellular miRNAs that include cytoskeletal organization, endocytosis, and cellular signaling. We validated three novel targets of miR-K12-3-3p and miR-H1 that are involved in endocytic and intracellular trafficking pathways. To evaluate the functional consequence of this regulation, we performed phagocytic uptake of labeled bacteria and noticed significant attenuation in miR-H1 and miR-K12-3-3p but not miR-UL70-3p transfected primary human Mφ. Multiple cytokine analysis of E. coli challenged Mφ revealed marked reduction of secreted cytokine levels with important roles in innate and adaptive immune responses suggesting a role of v-miRs in immune subversion. Our findings reveal that oral disease associated v-miRs can dysregulate functions of key host cells that shape oral mucosal immunity thus exacerbating disease severity and progression.

In silico prediction of cellular gene targets of herpesvirus encoded microRNAs.

Herpesviruses have evolved to encode multiple microRNAs [viral miRNAs (v-miRs)], a unique feature of this family of double stranded DNA (dsDNA) viruses. However, functional role of these v-miRs in host-pathogen interaction remains poorly studied. In this data, we examined the impact of oral disease associated v-miRs viz., miR-H1 [encoded by herpes simplex virus 1 (HSV1)] and miR-K12-3 [encoded by Kaposi sarcoma-associated herpesvirus (KSHV)] by identifying putative targets of viral miRNAs. We used our published microarray data (GSE107005) to identify the transcripts downregulated by the v-miRs. The 3' untranslated region (UTR) of these genes were extracted using BioMart tool on Ensembl and subjected to RNA:RNA interaction employing RNA Hybrid. We obtained hundreds of potential and novel miR-H1 and miR-K12-3 binding sites on the 3'UTR of the genes downregulated by these v-miRs. The information can provide likely regulatory mechanisms of the candidate v-miRs through which they can exert biological impact during herpesvirus infection and pathogenesis.

Herpesvirus-encoded microRNAs detected in human gingiva alter host cell transcriptome and regulate viral infection.

MicroRNAs (miRNAs) are small, non-coding RNAs of ~18-25 nucleotides that have gained extensive attention as critical regulators in complex gene networks including immune cell lineage commitment, differentiation, maturation, and maintenance of immune homeostasis and function. Many viruses encode miRNAs that directly downregulate the expression of factors of the innate immune system, which includes proteins involved in promoting apoptosis and recruitment. In this study, we examined the expression profiles of three previously identified viral miRNAs (v-miRs) from the human herpesvirus (HHV) family, HSV-1 (miR-H1), KSHV (miR-K12-3-3p), and HCMV (miR-US4) in healthy and diseased periodontal tissues and observed increased levels of v-miRs in diseased tissues. To understand the significance of this increase, we overexpressed v-miRs in human oral keratinocytes (HOK), a common target for various HHV, and analyzed the impact of miR-H1 and miR-K12-3-3p on the host transcriptome. More than 1300 genes were altered in HOK overexpressing miR-H1 and miR-K12-3-3p. Global pathway analysis of deregulated genes identified several key cellular pathways that may favor viral persistence. Using bioinformatic analysis, we predicted hundreds of potential v-miR binding sites on genes downregulated by miR-H1 and miR-K12-3-3p and validated three novel target v-miR sites suggesting widespread direct and indirect modulation of numerous host genes/pathways by a single v-miR. Finally, in vitro HSV-1 infection assays showed that miR-H1 can regulate viral entry and infection in human oral keratinocytes (HOK). Overall, our results demonstrate clinical and functional relevance of pathogenic viral molecules viz., v-miRs that regulate both host and viral functions and may contribute to the pathogenesis of inflammatory oral diseases.

Radiation Exposure from Diagnostic Imaging in a Cohort of Pediatric Transplant Recipients.

Recipients of solid organ transplants (SOT) have extensive diagnostic imaging (DI). The purpose of this study was to quantify this exposure. Children from northern Alberta with SOTs at Stollery Children's Hospital, Edmonton, Alberta January 1, 2006, to July 31, 2012, were included. Effective doses of radiation were estimated using published norms for DI performed post-transplant up to October 16, 2014. The 54 eligible children had 6215 DI studies (5628 plain films, 293 computerized tomography (CT) scans, 149 positron emission topography (PET) -CT scans, 47 nuclear medicine scans and 98 cardiac catheterizations). Children less than 5 years of age underwent more DI studies than did older children (median (IQR) 140 (66-210) vs 49 (19-105), p = 0.010). Children with post-transplant lymphoproliferative disorder (N = 8) had more CT scans (median (IQR) 13 (5.5-36) vs 1 (0-5), p<0.001) and PET-CT scans (median (IQR) 3.5 (1.5-8) vs 0 (0-0), p<0.001) than did other children. The estimated cumulative effective dose attributed to DI studies post-transplant was median (range) 78 (4.1-400) millisievert (mSv), and 19 of 54 children (35%; 95% confidence interval 24-49%) had a dose >100 mSv. In conclusion, a significant proportion of pediatric transplant recipients have sufficient radiation exposure post-transplant for DI to be at potential risk for radiation-induced malignancies.