1. MicroRNAs and Viral Infections
Viruses have been demonstrated to alter the expression of human microRNAs, cause specific degradation of microRNAs, and even encode their own, virally-derived microRNAs. These can all aid in maintaining viral latency, evasion of immune responses, and ultimately can dictate the outcomes of viral infections. Conversely, human microRNAs can alter the immune system and directly regulate responses to viral infections. Numerous broadly antiviral microRNAs have now been identified; however, their targets and roles in viral infections remain mysterious. What are the target genes of these microRNAs? How do they influence the viral life cycle? What is their role in modulating the immune response and disease pathogenesis? Can they be targeted for antiviral therapy? The answers to these questions will provide a broader understanding of the role of microRNAs in viral infections, immune responses and disease pathogenesis.
2. Hepatitis C Virus (HCV) and miR-122
Hepatitis C virus (HCV) is a global health problem, affecting approximately 3% of the world population, including more than 268,000 Canadians (Sagan SM et al. Can J Gastro Hepatol 2016). Currently, there is no vaccine available and current treatment protocols have limited efficacy. Thus, there is a pressing need for novel anti-HCV strategies.
MicroRNA-122 (miR-122) is a highly abundant liver-specific microRNA that was demonstrated to have a genetic interaction with two sites within the 5’ non-coding region (NCR) of the HCV genome. This is an unusual microRNA interaction as it promotes viral RNA accumulation both in cell culture and in vivo. Curiously, miR-122 has a minimal effect on viral translation and the rate of RNA synthesis. This suggests that miR-122 plays a protective role in preventing the degradation of the viral RNA. We have demonstrated that miR-122 has more extensive interactions with the HCV genome beyond the seed sequences, involving the 5’ terminus of the viral genome (Machlin ES, Sarnow P, and Sagan SM, PNAS 2011). Binding to the HCV RNA genome in this way creates a 3’ overhang which suggests that miR-122 may prevent recognition of the HCV RNA 5’ terminus by nucleases or enzymes that induce innate immune responses. Interestingly, we have demonstrated that a similar interaction occurs between miR-122 and a related virus, termed GB virus B (GBV-B) in cultured liver cells (Sagan SM, Sarnow P, and Wilson JA, J Virol. 2013). We are currently using novel pull-down and RNA analysis strategies to analyze miR-122 complexes in HCV-infected cells, to determine the host and viral factors involved in, and the mechanism of, this unusual microRNA-target RNA interaction. These studies will help to identify novel host-virus interactions, defining new targets for therapeutic intervention.
3. Dynamic Structure of Viral RNAs
The viral RNA genome of positive-sense RNA viruses is highly complex, as it must serve as a template for translation, replication as well as packaging of the viral genome. As such, viral RNA is a dynamic structure associated with numerous host and viral RNA-binding proteins. Using structural probing strategies, such as Selective 2' Hydroxyl Acylation analyzed by Primer Extension (SHAPE), we are currently analyzing viral RNA structure, in vitro and in vivo in numerous contexts. Combined with pull-down strategies we hope to learn more about RNA binding proteins and RNA structures important for viral RNA translation, replication and packaging.
4. Host and viral determinants of Zika virus pathogenesis
Zika virus (ZIKV) is an emerging mosquito-borne pathogen of tremendous public health concern. The World Health Organization estimates that as many as 2.2 billion people are at risk of ZIKV infection. Currently, there is are no vaccines or antiviral therapies to treat ZIKV infection and our understanding of the virus and its pathogenesis is limited.
Although historically a benign infectious agent, recent introduction of ZIKV to the Western Hemisphere has resulted in an explosive ongoing epidemic in South America associated with novel pathogenicity, including Guillain-Barré syndrome and fetal microcephaly. While genomic analyses have revealed that ZIKV has undergone evolution since its discovery, little is known regarding the impact of these polymorphisms on ZIKV pathogenesis. As such, we hypothesize that the contemporary strain(s) of ZIKV have evolved ways to increase viral fitness, counter the host immune response, and promote fetal pathogenesis. To investigate this, we are currently working with several ZIKV isolates, including the African reference strain (ZIKV-AF), and two Asian lineage isolates (an isolate from a ZIKV-infected returning Canadian traveller, ZIKV-CDN and a Brazilian isolate, ZIKV-BR). In addition, together with Dr. Martin Richer (McGill University) we have developed an immunocompetent mouse model of infection (Pardy RD, Rajah MM, Condotta SA, Taylor NG, Sagan, SM and Richer MJ, PLoS Path 2017). Our lab is using comparative analyses, chimeric viruses, and site-directed mutagenesis to determine the impact of viral evolution on ZIKV fitness, infectivity, replication, and pathogenesis. Our preliminary data also indicates that ZIKV is able to induce a robust immune response in our immunocompetent mouse model and we are characterizing the immune response to infection with both the African and Asian lineage isolates. Together, these new tools will allow us to directly address the impact of viral polymorphisms, and will elucidate host and viral determinants of ZIKV pathogenesis.