Right after the first world war, the world was swept with the deadly Spanish Flu which killed an estimated 2.5 - 5% of the human population and infected over 20% of the world population. The disease killed more people the first world war and more people than the infamous plague of 1300s. Spanish Flu was a mutant of bird-specific H1N1 virus. The famous avian flu, H5N1, is also a mutant of a bird-specific flu virus. Here you will see how to study this virus in-silico.

• Go to http:// ncbi.nlm.nih.gov/



• Type avian influenza flu virus polymerase in the query box on the NCBI home page.



• The data base would return the Entrez page which happens to be the life sciences search engine



• Clicking the protein sequence database option on the Entrez page would provide with list of reports which contain information about the proteins that the Entrez Database has generated in response to our query.



• Clicking the third protein in the list would open the information about the polymerase that we are looking for. The information page contains accession number of the protein and nucleotide sequence.



• Open http://www.uniprot.org/uniprot, the Uniprot ID of the protein is P26108. All the information available about the polymerase is available on the Uniprot web page.



• The page reveals the name which is polymerase basic protein 2 in the Names and origin section of the uniprot page. The same page also reveals the organism to which the protein belongs which happens to be Influenza A virus. The function of the protein is mentioned in the general annotation section on the same page along with its subunits. The polymerase basic 2 subunit is involved in transcription initiation and cap-stealing mechanism, in which cellular capped pre-mRNA are used to generate primers for viral transcription. It binds the cap of the target pre-RNA which is subsequently cleaved by PB1. May play a role in genome replication.



• On the uniprot page in section of sequences the amino acid sequence of the whole protein is present.



• In the sequence annotation section of the uniprot page the sequence responsible for nuclear localization is also highlighted which contains specified amino acids from position 736 to 739 in the protein sequence they are highlighted in blue. The sequence is KRKRD.



• On the same Uniprot page in the cross reference section in 3D structure database subsection position 685 to 759 are mentioned along with the ID through which the 3D structure of the protein could be viewed.



• Clicking 685-759 reveals the protein sequence again along with a highlighted region in Yellow that sequence from 685-759 is a motif which is conserved in almost all isolated strains of influenza A virus.



• This could be verified by running a BLAST of the highlighted sequence. The option of BLAST is all ready present on the top of the same page. Click on BLAST.



• Running a BLAST would list all the similar proteins in the Uniprot data base and results reveal that the sequence is conserved among all the resultant proteins.



• The 3D structure (courtesy gizmag) reveals atomic model of the conserved motif (736-739) has the nuclear localizing signal (lysine arginine lysine arginine) which is recognized by importin alpha which also has a leader peptide recognized by importin B which ultimately transports the protein to the nuclease.



• Inorder to computationally cleave the leader peptide or NLS sequence, go to http://expasy.org.



• The expasy server has proteomics and sequence analysis tools, by clicking on that open a page containing different tools.



• The expasy server has a peptide cutter tool which is included in the proteomics tools.


Clicking peptide cutter would direct us to the tool.



• Pasting the Uniprot protein ID or the amino acid sequence of the protein in the query box would result in listing the enzymes that could cut amino acids from the polypeptide chain at particular positions.



• Our target is to cut the NLS (nuclear localizing signal) so that the protein is not transported into the nuclease. Hence trypsin perfectly cleaves the region.

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Refrences
[1] http://gizmag.com/go/6904/
[2] http://embl.org/aboutus/news/press/2009/04feb09/index.html

Contributed by:Maria Bashir