Alanine scanning mutagenesis of poliovirus protein 2C around the 252 protein position

    Poliovirus belongs to the picornaviridae family of plus strand RNA viruses, which include a large number of important human and animal pathogens. Picornaviridae has thirteen genera that include the enterovirus. Human enteroviruses cause over three billion infections a year, and have some well-known viruses such as poliovirus, foot-and-mouth viruses and Hepatitis A viruses.  Although Poliovirus genome consists of only 7500 nucleotide bases, the same genome contains all the necessary proteins necessary for a virus to hijack host cells biome and replicate itself. 

     Due to poliovirus’s small genome size, it depends highly on the host in order to survive because poliovirus need and uses other cells resources in order to replicate. The virus binds to a host cells cellular receptor CD155, then the virion is internalized into the cell and uncoated. The encoding plus strand RNA is translated into a polyprotein, which is additionally processed into precursors and mature proteins. Some of these viral proteins shut down host cellular translations. Some of the mature viral proteins, particularly 3A and 2C, reorganizes the cytoplasm to create an environment that is suitable for viral replication. Newly made viral RNAs are used for genome replication, and some of the plus strand RNAs are encapsidated into empty capsid protein, which later becomes a mature poliovirus.

     At the five prime end (which marks the beginning of the sequence) of the polyprotein, there is a non-translational region.  This region is used because there is an internal ribosome entry site (IRES) that binds to the ribosome so that translation could begin. The start codon AUG  (which codes for methionine) is where translation begins. The viral RNA is translated into one long polyprotein, then followed by a stop codon and another non-translation region that includes a poly A tail. With the help of cellular proteins like heat shock protein 90 (hsp90) and viral proteases, the long polyprotein cleaves into three main regions, and the regions are additional cleave to ultimately form mature proteins, which is an individual viral protein. The three main segments are noted P1, P2, and P3 regions. P1 the region encodes the segment of the viral genome that encodes for the capsid proteins. P1 further cleaves in order to form mature proteins VP1, VP3, and VP0. The viruse matures to an infectious particle when VP0 is further cleaved to form VP2 and VP4. The capsid proteins form the protective shell that protects the viral RNA, and also has the receptor protein that binds to CD155 during virion entry into cell.

     The P2 and P3 regions encodes for mature proteins that are involved in the genomic replication and viral packaging.  The P3 region contains mature proteins 3D, which is a RNA polymerase protein, and 3A that is involved in the reorganization of the cytoplasm.  The P2 region also has a particularly important protein 2C, which is an ATPase protein that is involved the replication and encapsidation of the viral RNA into the capsid proteins. Work from the Wimmer lab have shown that mature protein 2C is very critical to the packaging of RNA, particularly at an asparagine at the 252 position. Additional experiments were performed where three amino acids were changed to alanine, which is a neutral amino acid that was conventionally substituted for other amino acids to find their biological function. Results from these experiments showed that the three amino acid changes upstream and downstream of the 252 asparagine made the virus temperature sensitive where the virus could not grow at higher temperature, but grew perfectly fine at lower temperatures. However, there were two amino acids further upstream and downstream of the 252 positions that resulted in non-viable viruses or “dead” virus. 

     In my research project I will be working with the two mutants that resulted in non-viable viruses.  In these viruses’ three amino acids were changed at a time and it possible that not all the amino acid contributes to the lethality of the virus. For this summer, I would like the opportunity to investigate the specific amino acid that caused the mutants to be non-viable. I am proposing to change each of the amino acid individual to investigate the biological function of the mutants separately. I hypothesize the single amino acid would yield viable mutants with notable biological functions.

     Through this process I will learn essential molecular genetics procedures such as Polymerase Chain Reaction (PCR), transcription, and transfecting.  I will use PCR to replicate the mutants, which will introduce specific site mutations in the DNA plasmid that encodes the poliovirus genome. Through T7 promoter directed transcription, I will convert the DNA into RNA, which would more resembles poliovirus RNA. I will then use transfection to introduce the poliovirus RNA into eukaryotic cells, specifically the human cell line Hela  R19.  I will transfect wild type poliovirus and the various mutant viruses RNA in order to see the underlying affects of the modification. Last I will conduct a plaque assay, which will enable me to quantify the concentration and phenotype of each. Through all these processes I will figure out which amino acid(s) caused the virus to be non-viable. With this project I will acquire all the fundamental skills that are utilized in molecular biology laboratories that studies virology.