Loquacious modulates flaviviral RNA replication in mosquito cells.

Arthropod-borne viruses infect both mosquito and mammalian hosts. While much is known about virus-host interactions that modulate viral gene expression in their mammalian host, much less is known about the interactions that involve inhibition, subversion or avoidance strategies in the mosquito host. A novel RNA-Protein interaction detection assay was used to detect proteins that directly or indirectly bind to dengue viral genomes in infected mosquito cells. Membrane-associated mosquito proteins Sec61A1 and Loquacious (Loqs) were found to be in complex with the viral RNA. Depletion analysis demonstrated that both Sec61A1 and Loqs have pro-viral functions in the dengue viral infectious cycle. Co-localization and pull-down assays showed that Loqs interacts with viral protein NS3 and both full-length and subgenomic viral RNAs. While Loqs coats the entire positive-stranded viral RNA, it binds selectively to the 3' end of the negative-strand of the viral genome. In-depth analyses showed that the absence of Loqs did not affect translation or turnover of the viral RNA but modulated viral replication. Loqs also displayed pro-viral functions for several flaviviruses in infected mosquito cells, suggesting a conserved role for Loqs in flavivirus-infected mosquito cells.

Exosome-associated microRNA-375 induces cell proliferation by regulating IGFBP4 upon hepatitis C virus infection.

Hepatitis C virus (HCV) infection is one of the most common causes of liver cancer. HCV infection causes chronic disease followed by cirrhosis, which often leads to hepatocellular carcinoma (HCC). In this study, we investigated the roles of exosome-associated miRNAs in HCV-induced disease pathology. Small RNA sequencing was performed to identify miRNAs that are differentially regulated in exosomes isolated from patient sera at two different stages of HCV infection: cirrhosis and hepatocellular carcinoma. Among the differentially expressed miRNAs, miR-375 was found to be significantly upregulated in exosomes isolated from patients with cirrhosis and HCC. A similar upregulation was observed in intracellular and extracellular/exosomal levels of miR-375 in HCV-JFH1 infected Huh7.5 cells. The depletion of miR-375 in infected cells inhibited HCV-induced cell migration and proliferation, suggesting a supportive role for miR-375 in HCV pathogenesis. miR-375, secreted through exosomes derived from HCV-infected cells, could also be transferred to naïve Huh7.5 cells, resulting in an increase in cell proliferation and migration in the recipient cells. Furthermore, we identified Insulin growth factor binding protein 4 (IGFBP4), a gene involved in cell growth and malignancy, as a novel target of miR-375. Our results demonstrate the critical involvement of exosome-associated miR-375 in HCV-induced disease progression.

CRISPR Screen Reveals PACT as a Pro-Viral Factor for Dengue Viral Replication.

The dengue virus is a single-stranded, positive-sense RNA virus that infects ~400 million people worldwide. Currently, there are no approved antivirals available. CRISPR-based screening methods have greatly accelerated the discovery of host factors that are essential for DENV infection and that can be targeted in host-directed antiviral interventions. In the present study, we performed a focused CRISPR (Clustered Regularly Interspaced Palindromic Repeats) library screen to discover the key host factors that are essential for DENV infection in human Huh7 cells and identified the Protein Activator of Interferon-Induced Protein Kinase (PACT) as a novel pro-viral factor for DENV. PACT is a double-stranded RNA-binding protein generally known to activate antiviral responses in virus-infected cells and block viral replication. However, in our studies, we observed that PACT plays a pro-viral role in DENV infection and specifically promotes viral RNA replication. Knockout of PACT resulted in a significant decrease in DENV RNA and protein abundances in infected cells, which was rescued upon ectopic expression of full-length PACT. An analysis of global gene expression changes indicated that several ER-associated pro-viral genes such as ERN1, DDIT3, HERPUD1, and EIF2AK3 are not upregulated in DENV-infected PACT knockout cells as compared to infected wildtype cells. Thus, our study demonstrates a novel role for PACT in promoting DENV replication, possibly through modulating the expression of ER-associated pro-viral genes.

Cross-species microRNA transmission modulates flavivirus growth in mosquitoes.

Mosquitoes can be infected with a variety of RNA viruses. Recently,Zhu et al. demonstrated that human microRNA hsa-miR-150-5p is acquired by mosquitoes during blood meals and protects the Dengue virus by downregulation of chymotrypsin AaCT-1 mRNA. This finding suggests the use of microRNA antagomirs as an antiviral approach in mosquitoes.

The tale of two flaviviruses: subversion of host pathways by RNA shapes in dengue and hepatitis C viral RNA genomes.

Pathogenic RNA viruses continue to emerge owing to their rapid evolutionary rates. The family of the Flaviviridae contains enveloped, single-stranded, positive-sense RNA viruses that include mosquito borne viruses such as dengue virus and the blood-borne hepatitis C virus. Upon infection, the genomic viral RNA needs to first compete with a sea of host mRNAs for host ribosomes that synthesize the viral proteins. Then, the positive-sense template needs to be amplified and packaged into newly assembled virions. To accomplish these tasks, the virus subverts several biochemical machineries from the host. The participation of specific structures in the viral RNA mediates specific RNA-RNA and RNA-protein interactions that dictate many viral subversion strategies. In this review, we shall focus on the various mechanisms by which RNA elements in the dengue virus and hepatitis C virus untranslated regions aid the viral infectious cycle and contribute to viral fitness.