Identification of the Influenza A nucleoprotein sequence that interacts with the viral polymerase
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Influenza A is a negative stranded RNA virus with a segmented genome. Once the virus infects a cell it must replicate its full length viral genomic RNA (vRNA) through a positive sense complementary intermediate RNA (cRNA) as well as transcribe viral messenger RNA (mRNA) using the vRNA as a template. The regulation of whether the viral polymerase replicates the genome by synthesizing cRNA, or produces mRNA in order to make viral protein involves, the viral nucleoprotein (NP). We tried to find the sequence residues of NP that directly interact with the viral polymerase. We mutated to alanine several residues on NP that are surface exposed on recently solved crystal structures as well as those thought to be oriented toward the viral polymerase complex in cryo-EM studies. As a first screen, we tested these mutants in a mini-genome assay where the NP stimulation of the viral polymerase can be studied in transfected cells. Through this screen we found that the NP mutants that hindered its ability to stimulate polymerase activity the most were located in a loop between two alpha helixes in the head domain of NP located at residues 203 to 209. Specifically, the NP single mutants of R204, W207, and R208 were inactive in the mini-genome assay. Using RT-PCR we found that the cRNA to vRNA step of replication is severely inhibited by these mutations. Immunoprecipitation using transfected cells showed that the NP mutants lost the ability to bind all three polymerase subunits. This indicates that this loss of polymerase binding may be the reason the NP mutant fails to stimulate polymerase activity. To make sure that this loss of polymerase stimulation was not due to altering other functions of NP we made sure that the protein had proper cellular localization, oligomerization, and RNA binding abilities. Using immuniflourescence we found that mutant NP localized to the nucleus just like wild type. In order to test RNA binding and oligomerization we tested NP purified from a baculovirus expressing system. Using fluorescence polarization we found that NP binds single stranded RNA with similar affinity to wild type. Using gel filtration we found that mutant NP forms oligomers just like wild type. Using covariation analysis of how different positions in an amino acid alignment change relative to each other we predicted possible binding sites between NP and the three polymerase subunits PA, PB1 and PB2. Due to more complete crystal structure data we focused on the PA-NP interaction and found that covariation aided in finding binding sequence residues on PA but not NP. Another outcome of developing the covariation method was developing a program to view broad primary structure changes in large sequence alignments. This method has been informative in evaluating how amino acid positions in influenza have changed over time, as well as what defines specific residues as belonging to human or avian viruses.