Project Report

Being awarded an East Asia and Pacific Summer Institute (EAPSI) fellowship allowed me to travel to Japan where I had the opportunity to work with Dr. Takemasa Sakaguchi at Hiroshima University. Dr. Sakaguchi is a paramyxovirus researcher with expertise in host immune responses against Sendai virus (SeV), a mouse virus and prototypic member of the paramyxovirus family. Studying SeV is important because it is related to several human pathogens including human parainfluenza viruses (HPIV), respiratory syncytial virus (RSV), measles virus, and mumps virus. My research has focused on a specific protein that is produced by SeV, and all viruses related to SeV, known as the large (L) polymerase protein. SeV and its related viruses hijack the host cell’s machinery (e.g. proteins) for the majority of their functions, however these viruses produce 5-10 proteins that allow them to infect and survive within host cells.The few proteins that are produced by these viruses are distinct from human proteins making them excellent targets for antiviral therapy and vaccine development. I focus on studying the molecular mechanisms of the SeV L protein to gain a better understanding of how this protein functions in context of a host cell. I have created specific mutations in a certain domain of the L protein to observe the effects these mutations have on the protein alone and within an infectious virus particle. These mutations effect a specific viral function, 5’cap methylation, which is required for efficient translation of viral proteins. Eukaryotic mRNA requires a 5’cap structure for mRNA stability and this cap must be methylated for efficient translation to occur. Methylation occurs at the guanine-N7 position (Cap0) and the 2’O-ribose of the 5’penultimate nucleotide residue (Cap1). It has been well established that methylation of the 5’ cap at the guanine-N7 position is absolutely required for efficient translation, but significance of methylation at the 2’O-ribose has remained unclear. Recent studies have demonstrated that the presence or absence of 2’O-ribose methylation has an evolutionary basis and plays a role in distinguishing self from non-self mRNA. Therefore several viruses have evolved strategies for ensuring that their viral mRNAs are capped and methylated, mimicking host mRNAs and evading antiviral responses. It has recently been shown that lack of 2’O-ribose methylation in certain RNA viruses led to increased immune responses in host cells thus indicating the importance of methylation at this position of the mRNA cap structure. To date, the relationship between 2’O-ribose cap methylation and host immune responses in paramyxoviruses has not been explored. In the lab of Dr. Sakaguchi I had the opportunity to examine the functional role of cap methylation defective SeV mutants in primary cell cultures (derived directly from mice, the natural host of SeV). To evaluate the significance of cap methylation in SeV infection, primary mouse embryonic fibroblasts (MEFs), wild-type (wt) as well as several cell types that are deficient in immune components important for antiviral responses against RNA viruses, were used. I hypothesized that antiviral immune responses will be greater in wt MEFs infected with SeV mutants lacking cap methylation. Following infection of MEFs, I analyzed viral protein expression levels, the number of infectious virus particles budding from the cells, and induction of immune response stimulated genes as a downstream indicator of recognition of SeV. Interestingly, I did not observe any increased immune responses to SeV mutants lacking cap methylation in wt MEFs. However, we did find that certain cellular sensors of viral RNA seem to play a role in detecting viral RNA lacking cap methylation. Further collaboration and future experiments are needed to further investigate the importance of 2’O-ribose methylation in antiviral responses against paramyxoviruses. The EAPSI fellowship provided me with a unique and exciting opportunity to pursue international research collaborations while experiencing an orientation to the science community, culture and language of Japan.

National Science Foundation (NSF)
Office of International and Integrative Activities (IIA)
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Carter Kimsey
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Murphy Andrea M
United States
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