Mentor: Adam Lauring
Reviewers: D. Kirschner & J. Platt
Background: RNA-dependent RNA polymerase (RdRp) is an essential protein for RNA viruses. It is in charge of both genome replication and mRNA transcription, directly influencing replication rate, replication fidelity, and viral fitness. The structure of RdRp is highly reserved in most RNA viruses. It features the right-hand shape with the fingers, palm, and thumb subdomains. In influenza viruses, RdRp (PB1) binds to the other two proteins - PB2 and PA - to form the polymerase complex. The exquisite interactions between the subunits during replication and transcription impose extra constraints on the local structure of RdRp. Previous research has identified some mutations in the influenza RdRp that have a significant impact on viral fitness. However, it is largely unknown how mutations on other sites of RdRp contribute to viral fitness.
Methods: To identify the key mutations on influenza A virus RdRp for viral fitness, I will build up a variant library of influenza A viruses in which each virus has a distinct single amino acid substitution on RdRp. To create this variant virus library, I used deep mutational scanning, a novel large scale mutagenesis technology to introduce target mutations into PB1 segments. I then integrated the variant segments into plasmids and transfected the plasmids with the aid of the “helper virus”. After getting the variant library of viruses, I let the variants compete against each other through four passages. I will collect the samples before and after each passage and use barcoded-subamplicon sequencing to measure the frequency change of every variant genome over time. The variants with higher fitness are expected to increase in frequency during the competition, while the genome frequencies of the variants with lower fitness are expected to decrease.
Results: By the time of the presentation, I created a mini-library for viruses containing 96 codon variants. I have completed two passages of competition and used MinION, a less precise but much faster sequencing method to obtain a general idea of transfection efficiency and codon diversity in the library. According to the sequencing results, all targeted mutations were recovered in the plasmid library, and the virus library before passage adopted a similar codon frequency pattern as the plasmid library. However, different codon variants were recovered at different levels. Some variants were much more abundant than the others in the plasmid library.
Conclusions: Taken together, these results indicate that the methods I used are promising in generating an influenza A virus PB1 variant library with a high codon diversity. But more benchmarking is required for optimal coverage.