Browsing by Subject "HCV"

RNA is a versatile and tractable biomolecule that serves as a critical component of life, whether as a script for protein production, a carrier of genetic information, a scaffold, or an enzyme. The fate and function of RNA can be influenced by chemical modifications such as N6-methyladenosine (m6A). Here we sought to identify the role of m6A during infection by positive-sense RNA viruses in the Flaviviridae family.

First, we investigated the role of m6A on viral RNA. We mapped m6A on the viral RNA genomes of hepatitis C virus (HCV), dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), and yellow fever virus (YFV). We then studied HCV as a model RNA and virus to understand the function of m6A on Flaviviridae RNA genomes. We found that the m6A methyltransferases METTL3 and METTL14 reduced HCV infectious particle production without affection viral RNA replication, while the m6A demethylase FTO had the opposite effect. Similarly, the m6A-binding YTHDF1-3 proteins also inhibited HCV particle production. Furthermore, the YTHDF proteins relocalized to cytoplasmic lipid droplets, the sites of HCV particle assembly, during infection. We then identified that m6A in a specific region of viral RNA was responsible for the role of m6A in viral particle production. Abrogation of m6A modification increased viral RNA binding to the capsid protein Core, an important step of HCV assembly, and also increased HCV particle production. These data suggest that m6A inhibits HCV particle production and that m6A modification of viral RNA can have a functional consequence for infection.

We then investigated how m6A on cellular mRNA can impact Flaviviridae infection. Working in collaboration with Dr. Chris Mason’s lab, we developed stringent analytical tools for detecting m6A changes. When we applied these tools, we found that a subset of cellular transcripts had altered m6A modification following infection by DENV, ZIKV, WNV, and HCV. We identified that innate immune signaling and ER stress, cellular pathways which are activated during Flaviviridae infection, contribute to altered m6A modification of two model transcripts RIOK3 and CIRBP. The gain of m6A on RIOK3 promotes the translation of this transcript, while loss of m6A on CIRBP influences its alternative splicing. Importantly, the RIOK3, CIRBP, and other transcripts with altered m6A modification can promote or inhibit Flaviviridae infection. Taken together, these results highlight the important role of m6A on both viral and cellular RNA in regulating infection.

Hepatitis C virus (HCV) evasion of the host immune system is largely mediated by the actions of the HCV NS3-NS4A protease complex, which consists of the serine protease and RNA helicase NS3 and its membrane targeting co-factor NS4A. NS3-NS4A has multiple functions in the HCV life cycle, with roles in both HCV replication and regulation of innate immune signaling. To regulate innate immune signaling, NS3-NS4A inactivates multiple signaling proteins, including MAVS, an adaptor protein in the RIG-I antiviral signaling pathway, and Riplet, an E3 ubiquitin ligase that activates RIG-I. Inactivation of these host proteins results in an inhibition of downstream signaling through the transcription factor IRF3 and inhibition of the subsequent induction of IFN-β. What directs the multiple functions of NS3-NS4A throughout the HCV life cycle is largely undetermined. Here, we identify a tyrosine residue within the transmembrane domain of NS4A that uncouples the various function of NS3-NS4A.

First, to uncouple the roles of NS3-NS4A in replication and immune evasion, I focused on the NS4A transmembrane domain and generated an NS4A mutant (Y16F) in a full-length HCV infectious clone, a subgenomic replicon, and an over-expression construct. I then assessed viral replication of HCV wild-type (WT) and Y16F viruses by measuring replication of a subgenomic HCV replicon in two related liver hepatoma cell lines: Huh7, which have functional RIG-I signaling, and Huh-7.5 cells, which lack functional RIG-I signaling. The HCV Y16F virus replicated to similar levels as WT HCV in Huh-7.5 cells. However, in Huh7 cells, replication of HCV Y16F was decreased compared to the HCV WT. I used CRISPR-Cas9 gene editing to delete proteins in the RIG-I pathway, including RIG-I, MAVS, and IRF3, in Huh7 cells, infected these cells with HCV WT or Y16F viruses, and then measured virus replication. I found that Y16F viral replication was not restored to the levels of WT in Huh7-RIG-I KO cells or the Huh7-MAVS KO cells, but it was restored to the levels of WT in the Huh7-IRF3 KO cells. I also found that the HCV NS3-NS4A Y16F mutation reduced the ability of over-expressed NS3-NS4A to block IRF3 activation, as measured through nuclear translocation via immunofluorescence microscopy. Further the NS3-NS4A Y16F mutation also had a reduced ability to block the induction of interferon-stimulated genes during both HCV replication and infection. This reveals that HCV NS4A Y16 can regulate a RIG-I-independent, yet IRF3-dependent, signaling pathway that limits viral replication.

Second, to further characterize this RIG-I-independent, IRF3-dependent signaling pathway, I examined the interactions of HCV NS3-NS4A with two of its known host substrates, MAVS and Riplet. To test whether the Y16F mutation prevented NS3-NS4A cleavage of MAVS, I performed a MAVS cleavage assay during both overexpression of NS3-NS4A and MAVS and also during infection with HCV WT and Y16F viruses. NS3-NS4A Y16F was able to cleave MAVS just like WT during both conditions. Next, I found that over-expression of NS3-NS4A WT changed Riplet intracellular localization and that NS4A interacted with Riplet. However, the NS4A Y16F mutation prevented NS4A-Riplet interactions in both of these contexts. Interestingly, I found that Huh-7.5 cells express lower levels of Riplet protein and mRNA compared to Huh7 cells. When full-length Riplet was added exogenously to Huh-7.5 cells, HCV Y16F virus replication was reduced compared to WT. However, when a Riplet construct missing the RING domain, which is essential for Riplet signaling, was added exogenously to Huh-7.5, both WT and Y16F viruses now replicated similarly. Taken together, these data identify NS3-NS4A Y16 as important for regulating a previously uncharacterized Riplet-mediated signaling pathway that limits HCV infection.