Expression Of Viral microRNAs Research Articles

Human and Viral microRNA Expression in Acute and Chronic HIV Infections.

Human and viral microRNAs (miRNAs) are involved in the regulation of gene transcription, and the establishment of their profiles in acute (AHI) and chronic (CHI) HIV infections may shed light on the pathogenetic events related to different phases of HIV disease. Next-generation sequencing (NGS) of miRNA libraries was performed, and the reads were used to analyze miRNA differential expression in the plasma with AHI and CHI. Functional analysis was then undertaken to investigate the biological processes characterizing the two phases of HIV infection. Except for hsa-miR-122-5p, which was found in 3.39% AHI vs. 0.18% CHI, the most represented human miRNAs were similarly represented in AHI and CHI. However, when considering the overall detected miRNAs in AHI and CHI, 15 displayed differential expression (FDR p < 0.05). Functional analysis identified 163 target mRNAs involved in promoting angiogenesis activation in AHI versus CHI through the action of hsa-miR10b-5p, hsa-miR1290, hsa-miR1-3p, and hsa-miR296-5p. The viral miRNAs detected, all belonging to herpesviruses, accounted for only 0.014% of total reads. The present data suggest that AHI patients exhibit strong innate immune activation through the upregulation of hsa-miR-122-5p and early activation of angiogenesis. More specific investigations are needed to study the role of viral miRNAs in HIV pathogenesis.

Human cytomegalovirus infection perturbs neural progenitor cell fate via the expression of viral microRNAs.

Human cytomegalovirus (HCMV) preferentially targets neural progenitor cells (NPCs) in congenitally infected fetal brains, inducing neurodevelopmental disorders. While HCMV expresses several microRNAs (miRNAs) during infection, their roles in NPC infection are unclear. Here, we characterized expression of cellular and viral miRNAs in HCMV-infected NPCs during early infection by microarray and identified seven differentially expressed cellular miRNAs and six significantly upregulated HCMV miRNAs. Deep learning approaches were used to identify potential targets of significantly upregulated HCMV miRNAs against differentially expressed cellular mRNAs, and the associations with miRNA-mRNA expression changes were observed. Gene ontology enrichment analysis indicated cellular gene targets were significantly enriched in pathways involved in neurodevelopment and cell-cycle processes. Viral modulation of selected miRNAs and cellular gene targets involved in neurodevelopmental processes were further validated by qRT-PCR. Finally, a predicted 3' UTR target site of hcmv-miR-US25-1 in Jag1, a factor important for neurogenesis, was confirmed by mutagenesis. Reduction of Jag1 RNA and protein levels in NPCs was observed in response to transient expression of hcmv-miR-US25-1. A hcmv-miR-US25-1 mutant virus (ΔmiR-US25) displayed limited ability to downregulate Jag1 mRNA levels and protein levels during the early infection stage compared with the wild type virus. Our collective experimental and computational investigation of miRNAs and cellular mRNAs expression in HCMV-infected NPCs yields new insights into the roles of viral miRNAs in regulating NPC fate and their contributions to HCMV neuropathogenesis. This article is protected by copyright. All rights reserved.

Expression of Viral microRNAs in Serum and White Blood Cells of Cows Exposed to Bovine Leukemia Virus.

Bovine leukemia virus (BLV) affects the health and productivity of cattle. The virus causes abnormal immune function and immunosuppression. MicroRNAs (miRNAs) are involved in gene expression, having been associated with stress and immune response, tumor growth, and viral infection. The objective of this study was to determine the expression of circulating miRNAs produced by BLV in animals exposed to the virus. Sera from 14 animals were collected to establish IgG reactivity to BLV by ELISA, where seven animals were seropositive and seven were seronegative for BLV exposure. White blood cells (WBC) from each animal were also collected and miRNAs were identified by sequencing from sera and WBC. The seropositive group had higher counts of BLV miRNAs when compared to seronegative group in sera and WBC. Blv-miR-1-3p, blv-miR-B2-5p, blv-miR-B4-3p, and blv-miR-B5-5p were statistically significant (P < 0.00001) in serum with an average of 7 log2 fold difference between seropositive and seronegative groups. Blv-miR-B1-3p, blv-miR-B1-5p, blv-miR-B3, blv-miR-B4-3p, blv-miR-B4-5p, blv-miR-B5-5p were statistically significant (P < 1.08e−9) in WBC with an average of 7 log2 fold difference between the seropositive and the seronegative groups. Blv-miR-B2-3p and blv-miR-B2-5p were also statistically significant in WBC (P < 2.79e-17), with an average of 27 log2 fold difference between the seropositive and the seronegative groups. There were 18 genes identified as being potential targets for blv-miR-B1-5p, and 3 genes for blv-miR-B4-5p. Gene ontology analysis indicated that the target genes are mainly involved in the response to stress and in the immune system process. Several of the identified genes have been associated with leukemia development in humans and cattle. Differential expression of genes targeted by BLV miRNAs should be evaluated to determine their effect in BLV replication.

Impact of Natural Occurring ERAP1 Single Nucleotide Polymorphisms within miRNA-Binding Sites on HCMV Infection.

Human cytomegalovirus (HCMV) is a β-herpesvirus that causes serious problems in people with a compromised immune system, whereas it coexists asymptomatically within the host with a healthy immune system. Like other viruses, HCMV has adopted multiples strategies to manipulate the host’s immune responses. Among them, expression of viral microRNAs (miRNAs) is one of the most intriguing. HCMV miR-UL112-5p and miR-US4-1 have been found to contribute to immune evasion by targeting the endoplasmic reticulum aminopeptidase 1 (ERAP1), a highly polymorphic key component of antigen processing. The current incomplete picture on the interplay between viral miRNAs and host immunity implies the need to better characterize the host genetic determinants. Naturally occurring single nucleotide polymorphisms (SNPs) within the miRNA binding sites of target genes may affect miRNA–target interactions. In this review, we focus on the relevance of 3′ untranslated region (3′UTR) ERAP1 SNPs within miRNA binding sites in modulating miRNA–mRNA interactions and the possible consequent individual susceptibility to HCMV infection. Moreover, we performed an in silico analysis using different bioinformatic algorithms to predict ERAP1 variants with a putative powerful biological function. This evidence provides a basis to deepen the knowledge on how 3′UTR ERAP1 variants may alter the mechanism of action of HCMV miRNAs, in order to develop targeted antiviral therapies.

BK polyomavirus microRNA expression and sequence variation in polyomavirus-associated nephropathy

BackgroundBK polyomavirus (BKPyV) infection is a common asymptomatic viral infection in the general population. Severe complications are seen in immunocompromised individuals, such as polyomavirus-associated nephropathy (PyVAN) in renal transplant recipients. Information on BKPyV microRNA expressions is scarce, although polyomavirus-encoded microRNAs have been shown to control viral replication and assist in immune evasion. Whereas the pathogenic role of rearrangements in JC polyomavirus has been well established, little is known about BKPyV rearrangements in PyVAN. ObjectivesTo assess viral microRNA expression and transcriptional control region (TCR) sequence variation in PyVAN patients. Study designbkv-miR-B1-3p and bkv-miR-B1-5p microRNA expression was quantified in 55 plasma samples from 9 PyVAN patients and 2 controls using specific miRNA assays. TCR architectures among the viral populations in each patient were characterized by massive parallel sequencing. Resultsbkv-miR-B1-3p and bkv-miR-B1-5p miRNA expression was established in 85.5% and 98.2% of samples, respectively. On average, an 8.9-fold (bkv-miR-B1-3p) and 8.7-fold (bkv-miR-B1-5p) higher expression levels were detected in PyVAN patients as compared to controls. Rearranged BKPyV strains with duplications and deletions were detected in 7/9 PyVAN patients, but 77.6–99.9% of all sequence reads in all samples represented archetype strains. ConclusionsThe frequent detection and increased expression of miRNAs suggest involvement in PyVAN pathogenesis. Despite the predominance of archetype BKPyV strains, the frequent detection of minor rearranged viral populations urges further study on their role in severe kidney disease. Our results suggest that miRNA expression is increased in PyVAN patients, as well as in the presence of rearranged viral strains.

Interleukin-6-dependent growth in a newly established plasmablastic lymphoma cell line and its therapeutic targets

Plasmablastic lymphoma (PBL) is a rare, highly aggressive subtype of non-Hodgkin lymphoma with plasma-cell differentiation occurring typically in immune-suppressed patients such as those with AIDS. This study reports the establishment and characterization of a new cell line, PBL-1, derived from a patient with AIDS-associated PBL. Morphological assessment of PBL-1 indicated plasma-cell differentiation with a CD20(−) CD38(+) CD138(+) immunophenotype and IgH/c-myc translocation. The cell line harbours Epstein-Barr virus, but a 52.7-kbp length defect was identified in its genome, resulting in no expression of viral microRNAs encoded in the BamHI-A Rightward Transcript region. Importantly, supplementation of culture medium with >5 ng/mL of interleukin-6 (IL-6) was required for PBL-1 growth. Starvation of IL-6 or addition of tocilizumab, an inhibitory antibody for the IL-6 receptor, induced apoptosis of PBL-1. Transduction of IL-6 into PBL-1 by lentivirus vector induced autologous growth without IL-6 supplementation of culture medium. These data indicate the IL-6 dependency of PBL-1 for proliferation and survival. mTOR inhibitors induced cell death effectively, suggesting mTOR in the IL-6 signalling pathway is a potential therapeutic target for PBL. This established PBL cell line will be a useful tool to further understand the pathophysiology of PBL and aid the future development of PBL treatment.

Gastric adenocarcinoma microRNA profiles in fixed tissue and in plasma reveal cancer-associated and Epstein-Barr virus-related expression patterns

MicroRNA expression in formalin-fixed paraffin-embedded tissue (FFPE) or plasma may add value for cancer management. The GastroGenus miR Panel was developed to measure 55 cancer-specific human microRNAs, Epstein-Barr virus (EBV)-encoded microRNAs, and controls. This Q-rtPCR panel was applied to 100 FFPEs enriched for adenocarcinoma or adjacent non-malignant mucosa, and to plasma of 31 patients. In FFPE, microRNAs upregulated in malignant versus adjacent benign gastric mucosa were hsa-miR-21, -155, -196a, -196b, -185, and -let-7i. Hsa-miR-18a, 34a, 187, -200a, -423-3p, -484, and -744 were downregulated. Plasma of cancer versus non-cancer controls had upregulated hsa-miR-23a, -103, and -221 and downregulated hsa-miR-378, -346, -486-5p, -200b, -196a, -141, and -484. EBV-infected versus uninfected cancers expressed multiple EBV-encoded microRNAs, and concomitant dysregulation of four human microRNAs suggests that viral infection may alter cellular biochemical pathways. Human microRNAs were dysregulated between malignant and benign gastric mucosa and between plasma of cancer patients and non-cancer controls. Strong association of EBV microRNA expression with known EBV status underscores the ability of microRNA technology to reflect disease biology. Expression of viral microRNAs in concert with unique human microRNAs provides novel insights into viral oncogenesis and reinforces the potential for microRNA profiles to aid in classifying gastric cancer subtypes. Pilot studies of plasma suggest the potential for a noninvasive addition to cancer diagnostics.

Virus-encoded microRNA contributes to the molecular profile of EBV-positive Burkitt lymphomas.

Burkitt lymphoma (BL) is an aggressive neoplasm characterized by consistent morphology and phenotype, typical clinical behavior and distinctive molecular profile. The latter is mostly driven by the MYC over-expression associated with the characteristic translocation (8;14) (q24; q32) or with variant lesions. Additional genetic events can contribute to Burkitt Lymphoma pathobiology and retain clinical significance. A pathogenetic role for Epstein-Barr virus infection in Burkitt lymphomagenesis has been suggested; however, the exact function of the virus is largely unknown.In this study, we investigated the molecular profiles (genes and microRNAs) of Epstein-Barr virus-positive and -negative BL, to identify specific patterns relying on the differential expression and role of Epstein-Barr virus-encoded microRNAs.First, we found significant differences in the expression of viral microRNAs and in selected target genes. Among others, we identified LIN28B, CGNL1, GCET2, MRAS, PLCD4, SEL1L, SXX1, and the tyrosine kinases encoding STK10/STK33, all provided with potential pathogenetic significance. GCET2, also validated by immunohistochemistry, appeared to be a useful marker for distinguishing EBV-positive and EBV-negative cases. Further, we provided solid evidences that the EBV-encoded microRNAs (e.g. BART6) significantly mold the transcriptional landscape of Burkitt Lymphoma clones.In conclusion, our data indicated significant differences in the transcriptional profiles of EBV-positive and EBV-negative BL and highlight the role of virus encoded miRNA.

Hcmv-miR-UL22A-5p: A Biomarker in Transplantation With Broad Impact on Host Gene Expression and Potential Immunological Implications

Cytomegalovirus (CMV) encodes multiple microRNAs. While these have been partially characterized in vitro, their relevance to clinical CMV infection has not been evaluated. We analyzed samples from a cohort of solid organ transplant patients with CMV disease (n = 245) for viral microRNA expression. Several CMV microRNAs were readily detectable in patients with CMV disease in variable relative abundance. Expression level generally correlated with DNA viral load and the absence of viral microRNA was associated with faster viral clearance. Detection of hcmv-miR-UL22A-5p at baseline independently predicted the recurrence of CMV viremia upon discontinuation of antiviral therapy (OR 3.024, 95% CI: 1.35-6.8; p = 0.007). A combination of direct mRNA targeting by the microRNA and indirect modulation of gene expression involving isoforms of the transcriptional regulator C-MYC may be responsible for the broad effects seen in the association of gene transcripts with the RNA-induced silencing complex and in global protein expression upon hcmv-miR-UL22A-5p transfection. This novel study of in vivo viral microRNA expression profiles provides unique insight into the complexity of clinical CMV infection following transplantation. We provide evidence that viral microRNAs may have complex effects on gene expression and be associated with specific virologic and clinical outcomes, and thus could be further evaluated as biomarkers.

The JCPYV DNA load inversely correlates with the viral microrna expression in blood and cerebrospinal fluid of patients at risk of PML

BackgroundIn light of their regulatory role, changes in the expression of Polyomavirus JC (JCPyV) microRNAs may be relevant for virus reactivation and the development of progressive multifocal leukoencephalopathy (PML). ObjectivesTo investigate the presence of JCPyV-DNA and JCPyV microRNA expression in clinical specimens of patients at risk for PML. Study designThe JCPyV-DNA and microRNA status was assessed in peripheral blood mononuclear cells (PBMCs) and plasma from 100 HIV patients, in serum and cerebrospinal fluid (CSF) from 14 HIV PML patients and in PBMCs and plasma from 50 healthy controls using Multiplex real-time PCR and JCPyV miRNA-J1-3p and −5p stem-loop RT-PCR. The JCPyV-DNA microRNA-expressing region was also sequenced. ResultsA positive JCPyV-DNA status was more prevalent in HIV patients (67%, 67/100) compared to healthy controls (18%, 9/50). Among these, 46% and 42% of the HIV patients and 18% and 0% of the healthy controls were positive based on PBMC and plasma determinations, respectively. PBMC JCPyV microRNA positivity was observed in 22 out of 46 (48%) JCPyV+ HIV patients and in 3 out of 9 (33%) JCPyV+ healthy controls. Moreover, JCPyV microRNAs in exosomes were found in 6 out of 100 (6%) HIV plasma samples, in 12 out of 50 (24%) healthy samples, in 6 out of 14 (43%) serum samples, and in 3 out of 5 (60%) HIV PML CSF samples. Of note, the JCPyV-DNA load was inversely correlated with expression of the viral microRNA. The JCPyV microRNA genomic expression region showed a different combination of three mutations. ConclusionsThe low levels of JCPyV microRNA expression in HIV patients with high JCPyV-DNA prevalence observed in this study highlight the potential clinical relevance of JCPyV microRNAs in PML risk assessment.

Small RNA cloning and sequencing strategy affects host and viral microRNA expression signatures

The establishment of the microRNA (miRNA) expression signatures is the basic element to investigate the role played by these regulatory molecules in the biology of an organism. Marek's disease virus 1 (MDV-1) is an avian herpesvirus that naturally infects chicken and induces T cells lymphomas. During latency, MDV-1, like other herpesviruses, expresses a limited subset of transcripts. These include three miRNA clusters. Several studies identified the expression of virus and host encoded miRNAs from MDV-1 infected cell cultures and chickens. But a high discrepancy was observed when miRNA cloning frequencies obtained from different cloning and sequencing protocols were compared. Thus, we analyzed the effect of small RNA library preparation and sequencing on the miRNA frequencies obtained from the same RNA samples collected during MDV-1 infection of chicken at different steps of the oncoviral pathogenesis. Qualitative and quantitative variations were found in the data, depending on the strategy used. One of the mature miRNA derived from the latency-associated-transcript (LAT), mdv1-miR-M7-5p, showed the highest variation. Its cloning frequency was 50% of the viral miRNA counts when a small scale sequencing approach was used. Its frequency was 100 times less abundant when determined through the deep sequencing approach. Northern blot analysis showed a better correlation with the miRNA frequencies found by the small scale sequencing approach. By analyzing the cellular miRNA repertoire, we also found a gap between the two sequencing approaches. Collectively, our study indicates that next-generation sequencing data considered alone are limited for assessing the absolute copy number of transcripts. Thus, the quantification of small RNA should be addressed by compiling data obtained by using different techniques such as microarrays, qRT-PCR and NB analysis in support of high throughput sequencing data. These observations should be considered when miRNA variations are studied prior addressing functional studies.

Analysis of viral microRNA expression by elephant endotheliotropic herpesvirus 1

Elephant endotheliotropic herpesvirus 1 (EEHV1), a member of the Betaherpesvirinae subfamily, has recently emerged as an important viral pathogen of Asian elephants that can cause a severe, often fatal, hemorrhagic disease. EEHV1 does not replicate in culture and little is currently known about the molecular biology of this emerging pathogen, with the notable exception of its genomic DNA sequence. Here, we have used small RNA deep sequencing to determine whether EEHV1, like other human and murine betaherpesviruses, expresses viral microRNAs in infected tissues in vivo. Our data provide evidence supporting the existence of at least three novel viral microRNAs encoded by EEHV1 and one of these, miR-E3-5p, is shown to repress target mRNA expression. Moreover, miR-E3-5p expression was readily detectable in tissue samples derived from two infected elephants, including in whole blood. These data shed new light on the biology of EEHV1 and identify small RNAs that have the potential to be useful in the diagnosis of sub-clinical infections in captive Asian and African elephants.

Expression of Herpes Simplex Virus 1 MicroRNAs in Cell Culture Models of Quiescent and Latent Infection

To facilitate studies of herpes simplex virus 1 latency, cell culture models of quiescent or latent infection have been developed. Using deep sequencing, we analyzed the expression of viral microRNAs (miRNAs) in two models employing human fibroblasts and one using rat neurons. In all cases, the expression patterns differed from that in productively infected cells, with the rat neuron pattern most closely resembling that found in latently infected human or mouse ganglia in vivo.

Expression of Viral MicroRNAs in Epstein-Barr Virus-Associated Gastric Carcinoma

Epstein-Barr virus (EBV) is associated with about 6 to 16% of gastric carcinoma cases worldwide. Expression of the EBV microRNAs (miRNAs) was observed in B cells and nasopharyngeal carcinoma cells infected with EBV. However, it is not clear if the EBV miRNAs are expressed in EBV-associated gastric carcinomas (EBVaGCs). We found that BART miRNAs but not BHRF1 miRNAs were expressed in EBV-infected gastric carcinoma cell lines and the tumor tissues from patients as well as the animal model. The expression of viral miRNAs in EBVaGCs suggests that these EBV miRNAs may play important roles in the tumorigenesis of EBVaGCs.