Studies on exosomal microRNAs and proteins associated with Hepatitis C and Dengue virus induced pathogenesis

Abstract

Studies on the exosomal microRNAs and proteins associated with Hepatitis C and Dengue virus-induced pathogenesis.

Hepatitis C virus and Dengue virus are the group of positive-strand RNA viruses belonging to the family Flaviviridae which causes hepatitis and dengue fever, respectively. Various host factors including small non-coding RNAs, micro RNAs (miRNAs) and proteins are the major regulators of the cellular pathways that are involved in virus induced pathogenesis.

Exosomes are the small extracellular vesicles, secreted out by the cells and have significant role in cell to cell signaling. It comprises of various host factors like miRNA, lncRNAs, mRNAs and cellular proteins. As the disease progresses from an early stage to severe form of infection, the composition of the exosome changes which might directly correlate to the pathogenesis. Study of the exosomal miRNAs and proteins can provide an insight into its role in disease progression, which has been highly under explored. The current study focuses on the differential expression of the exosomal miRNAs and proteins upon two distinctly different viruses with diverse phenotype viz. Hepatitis C virus (HCV) causes chronic infection and Dengue virus causes acute infection.

Part I: Elucidation of the role of differentially expressed exosomal miRNAs in HCV infection and pathogenesis.

Hepatitis C virus (HCV) is one of the most common causes of liver disease. In some infected patients, the virus gets cleared by the host immune system, but others develop chronic disease, cirrhosis and often lead to hepatocellular carcinoma (HCC). In this part, the role of exosome-associated microRNAs (miRNAs) in HCV-induced disease pathology has been investigated. RNA-sequencing was performed to identify miRNAs that are differentially regulated in the exosomes isolated from patient sera at different stages of HCV infection viz. cirrhosis and hepatocellular carcinoma. Among the differentially expressed miRNAs, miR-375 was shortlisted for further studies. miR-375 was found to be significantly upregulated in the exosomes isolated from patients with both cirrhosis and HCC. A similar upregulation pattern was observed in the intracellular and extracellular/exosomal levels of miR-375 in HCV-JFH1 RNA transfected Huh7.5 cells. The depletion of miR-375 in infected cells inhibited the HCV-induced cell migration and proliferation without affecting viral replication or release, suggesting a supportive role for miR-375 in HCV induced pathogenesis. Naïve Huh 7.5 cells would uptake miR-375 that is secreted through exosomes derived from HCV-infected cells that will lead to an increase in cell proliferation and migration in the recipient cells. Further, IGFBP4 was identified as a potential target of miR-375, which was validated using luciferase reporter constructs. Since IGFBP4 is involved in cell growth and malignancy, it is possible that miR-375 exerts its effect on cell proliferation through IGFBP4. Taken together, the results demonstrate the critical involvement of exosome-associated miR-375 in HCV-induced pathogenesis.

Part II: To study the exosomal miRNAs signatures during disease progression in dengue virus infection.

Dengue virus is a causative agent of the dengue fever, a most common mosquito transmitted disease. As an epidemic disease, dengue has put almost half of the world’s population at risk. With the lack of specific treatment and effective vaccine, it is most important to diagnose and prognose the disease as early as possible. Based on its severity, dengue infection has been categorized into 3 group plans viz. WHO Plan A (dengue without warning), WHO Plan B (dengue with warning) and WHO Plan C (Dengue haemorrhagic fever). In this part, we examined the exosome associated micro-RNA in human patient’s serum sample at different stages of dengue infection using RNA-sequencing. We could identify 50 differentially expressed microRNAs that were either significantly up regulated or down regulated in dengue infection. After extensive validation, we observed that miR-96-5p was significantly upregulated in all stages of dengue infection whereas miR-146a-5p was found to be significantly downregulated in all stages of dengue infection. Further study on miR-146a-5p showed that it regulates the expression of most of the proteins involved in immune response viz. IL-8, NF-kappa, HuR, indicating its role in regulating the immune response during dengue infection. Further, we could identify a few potential miRNAs that are upregulated in the patients that progress to severe form of dengue infection.

Part III: Studying the exosomal proteins differentially expressed in HCV and Dengue infection: Possible implications

Protein composition of the exosomes are also known to change during the viral infection. In the final part of the study, the exosomal proteins expressed during different stages of HCV and Dengue virus infection were evaluated using LC-MS and 2D gel analysis respectively. Interestingly, in case of HCV infection, certain proteins, such as Apo B100, Tetranectin and Gelsolin were found to be gradually upregulated with the disease progression from chronic to cirrhosis to HCC. Whereas Transthyretin and Hsp90 co-chaperon showed differential expression at different stages (Plan A, Plan B and Plan C) of dengue virus infection. Interestingly, Haptoglobin was found to be upregulated in both the viral infections, indicating some common role. Results suggest possible involvement of some common and different exosomal proteins in mediating cellular pathogenesis in the context of two different viruses with diverse phenotype.

Overall, the study reveals the potential role of exosomal miRNA and proteins, as part of host response and viral strategies, which could collectively contribute to disease progression, underpinning novel targets for possible therapeutic intervention.