Characterizing the viral factors and host determinants that control Dengue virus infection and pathogenesis

Abstract

Background – Dengue virus is a mosquito-borne flavivirus that is the causative agent for dengue fever, which progresses to dengue shock syndrome in many patients. It is estimated that around 390 million dengue infections occur worldwide annually. But the molecular mechanisms governing the adaptation to the dual hosts and the disease progression from mild to severe form are not fully understood. Virus infection and pathogenesis are determined by both viral and host factors, and a better understanding of these determinants is important in the development of therapeutics and better disease management.

To decipher the role of viral miRNAs in dengue-induced pathogenesis.

Dengue virus 3’UTR is highly structured. It has two pseudoknots (PK), PK1 and PK2, which are duplicated structures. It has also been shown that the Dengue Virus UTRs are known to produce small miRNAs called V miRs (Viral miRNAs). The production site of one such miRNA, V miR 5, was found to be in the PK1 structure using mutational studies. Viral miRNAs such as VmiR 5 and VmiR 6 were also found to be enriched in the exosomes (extracellular vesicles) secreted by the infected cells, by which they may play a role in disease pathogenesis. Further, we showed that the production of these VmiRs is dependent on host XRN1 and Dicer proteins, and they are actively loaded into the Ago2-RISC complex. These results indicate a novel mechanism by which the virus produces miRNAs, which could suppress host immune response and contribute to virus pathogenesis.

To identify the role of RNA binding proteins in dengue virus life cycle and pathogenesis.

Dengue virus genome is flanked by 5’UTR (95nt) and 3’UTR (450nt-600nt). During the life cycle, these UTRs are known to interact with various host proteins and miRNAs. We investigated the role of these RBPs in the Detergent-resistant membranes (DRMs) in which the virus replicates upon infection. DRMs were isolated using sub-cellular fractionation followed by mass-spectrometry, which led to the identification of important RNA binding protein Human Antigen R (HuR) in the replication complex. Using in-vitro and ex-vivo assays, it was identified that the HuR protein has a binding site at the Dengue 3’UTR. HuR was further found to play a negative role in Dengue virus replication using silencing and CRISPR knock down studies. In-silico analysis showed that the binding site of HuR and PTB protein were overlapping at Dengue 3’UTR, and UV crosslinking competition assays showed that PTB (positive regulator of virus life cycle) can compete out HuR (negative regulator). The interplay between these proteins thereby regulates the virus's life cycle. Interestingly, HuR was also found to play a positive role in cap-independent translation at a later stage in the virus life cycle. Overall, our results indicated that HuR is a key player in the virus life cycle and virus-induced pathogenesis.

To understand the Role of secretory mRNAs in Disease progression and pathogenesis

Dengue fever has been classified into different plans based on the disease severity, i.e., PLAN A, PLAN B, and PLAN C, with PLAN C representing the patients with Dengue Shock Syndrome (DSS). The molecular mechanisms underlying this transition to a severe form of the disease are not fully understood. Also, the pathogenic role of secretory mRNAs post Dengue Virus infection has not been reported. RNA sequencing from the total RNA isolated from patient serum samples in different PLANs was done to identify the total secretory RNAs in dengue virus infection. Pathway analysis of the mRNAs showed them to play roles in Platelet Degranulation, T cell migration, and other pathways that have previously been shown to play important roles in the transition to Dengue Shock Syndrome. Post validation in patient serum samples, it was found that mRNAs such as Vinculin, TAGLN2, and ITGA2B were specifically upregulated in the serum of patients in PLAN B, which are known to play an important role in the platelet degranulation pathway. This may help us understand the mechanism by which massive thrombocytopenia occurs in severe dengue patients.

Overall, the results demonstrate how dengue viruses exploit specific host factors, adopting various strategies to modulate the viral life cycle and pathogenesis.