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Bioinformaticsproduct

Using BLAST and ExPASy for Genetic and Protein analysis of H1N1
variability, including mutations that confer resistance to antiviral
medications.
Objectives:
•Students will become familiar with the online databases available to researchers including GenBank, BLAST and ESPy utilities.
•Students will analyze normal and mutant strains of H1N1 viruses to look for nucleotide mutations that confer resistance to two antiviral therapies: zanamivir and oseltamivir.
•Students will observe the effect of nucleotide changes on viral protein structure•Students will hypothesize if a previously unknown, but sequenced resistant strain has a similar resistance mutation or a new type•Students will research the glycoprotein upon which the anti-viral therapies act, neuraminidase, to investigate the reason for antiviral resistance due to the known mutations Previous Knowledge:
1. In all cells and viruses, DNA is made of nucleotides and triplets of As, Ts, Cs and Gs code for specific amino acids2. Proteins are chains of amino acids3. The amino acid composition of the protein determines the shape, and therefore function of the protein, and changing the amino acids can change the shape and function of the protein4. Proteins are essential components of all cells and viruses5. Viruses are intracellular parasites and have molecules on their surface, that among other things, allow the viruses to infect cells, replicate themselves and leave cells thereby destroying them Introduction: Influenza is an infection of the upper respiratory tract which causes
sickness and death and widespread outbreaks also cause a significant economic impact
as well. The H1N1 influenza A pandemic of 2009 caused millions of people to become
sick, hundreds of thousands to be hospitalized and thousands of deaths in the United
States aloneThe new flu strain contained genes from influenza viruses from avian
(bird), swine (pig) and human. New strains often cause more infection because our
immune systems have been conditioned to respond to previously encountered strains.
When strains mutate it allows them to more easily slip by our bodyʼs defenses. In
addition, mutation can make previous vaccines and antiviral medications ineffective
against them.
1 Numbers are for United States only; worldwide projections are not given.
Background: Most of us are familiar now with the nomenclature “H1N1” but few of us
know the meaning of these letters and numbers. Scientists label flu viruses based on
the presence of two antigens on their surface. The first letter, “H” refers to the type of
Hemagglutinin present on the virusʼ surface. This molecule is primarily responsible for
the virusʼ ability to infect cells. The second letter, “N”, refers to the the type of
Neuraminidase present. This molecule helps the replicated virus leave from the cell in
which is has replicated, allowing the many copies to infect more cells. These two
proteins together determine much about the virus, including, in general, which species
the virus can infect and the virulence of the particular strain. Even though there are
many different strains of flu virus, they infect mammals and birds and there remains a lot
of similarity between them. These similarities can be exploited to develop vaccines and/
or anti-viral drug therapies that could be used against a wide array of similar viruses.
Between 1999 and 2002 two new antiviral drugs were introduced into the population:
zanamivir and oseltamivir. These same drugs were used to treat patients with H1N1. In
just about 10 years it is possible for flu viruses to evolve resistance to these drugs. In
order to monitor the potential development of resistant strains the Neuraminidase
Inhibitor Susceptibility Network was established.
Directions: You are a member of the Neuraminidase Inhibitor Susceptibility Network
and the World Health Organization has contacted you with information about a new viral
strain that might have resistance to the antiviral drugs zanamivir and oseltamivir
(Tamiflu). In this case study you will:
•Use BLAST to compare H1N1 sequences to observe conserved and variable regions•Align nucleotide sequences of normal strains of H1N1•Align multiple nucleotide and protein sequences to look for mutations.
•Compare the newly identified strain and hypothesize its resistance based on its mutations Part A: Introduction to BLASTn. Listed below are the identifying numbers for several
“normal” (non-resistant) strains of H1N1 that were sequenced during the 2009
pandemic. In part A of this activity you will use online database tools to look at virus
variation that does not confer antiviral resistance.
Step 1: Go to the National Center for Biotechnology Informationʼ. BLAST stands
for “Basic Local Alignment Search Tool” and it is one of the many web-based
applications available to investigate, analyze, research and identify genes and proteins
of interest. The applications of BLAST and other tools are almost limitless! All of these
things are free and open to the public. This particular site is maintained by the National
Institutes of Health (NIH).
Step 2: Choose nucleotide blast from the menu of choices under the heading “Basic
BLAST”. To become familiar with BLAST output we will compare the nucleotide
sequences of two strains of H1N1. Enter this accession number in the box, CY056295.
Give your job the title, “align two non-resistant H1N1 strains”.
Step 3: Click on the box in front of “Align two or more sequences”. This will open a
new box. Enter accession number CY062530 in this box.
Step 4: At the bottom section called “Program Selection” select the choice “Highly
similar sequences (megablast). Then click “show results in a new window” so we can
refer back to our search criteria if needed.
Step 5: Click the “BLAST” button. Your search will show up in a new window or tab
and if you scroll down you can see the aligned nucleotides for the two neuraminidase
genes in our two strains.
Answer the questions for Part A once your report is finished.
Part A Questions:
1. What are the lengths of the two sequences used in this comparison? Query: _________ nucleotides Subject: _________ nucleotides 2. How many differences can be found between the two strands? (count them!) 3. Since every 3 bases codes for 1 amino acid, what is the maximum number of amino acids in the protein that would be made from the shorter of the two sequences? 4. Will every nucleotide difference result in an amino acid difference? Explain your answer.
5. Since neither of these strains is resistant to anti-viral therapies, what can be concluded about the variation in the gene for neuraminidase between these two strains? Part B: Collecting Info on your sequences. There are a lot of details recorded about
the sequences that are uploaded to the GenBank database. In Part B of this activity we
will collect information about our strains, including a reference number to their translated
amino acid (protein) sequence. We will use these sequences in Pact C of this activity.
Step 1: From the results of your BLAST page click on the link after “Query ID”. This
will bring up all of the information about this sequenced gene. Look over the information
and answer the questions:
Part B Questions:
2. Who was the host of this virus? Be as specific as possible? 4. If you scroll down to the bottom you can find the entire sequence and translated amino acid sequence. See appendix A for a list of amino acid abbreviations. Write down the protein_id number:___________________ Step 2: Use your back button to return to your BLAST results. Scroll down and click on
the accession number of our subject sequence, cy062530.1. Answer the same 4
questions about this viral strain.
2. Who was the host of this virus? Be as specific as possible? 4. If you scroll down to the bottom you can find the entire sequence and translated amino acid sequence. See appendix A for a list of amino acid abbreviations. Write down the protein_id number:___________________ Part C: Using BLASTp. In Part C of this activity we will use BLASTp to align two
protein sequences.
Step 1: Navigate back to the BLAST input page. From the tabs at the top click on
.
From here choose “blast” and then “protein blast”.
Step 2: Input the first of the two protein sequences from Part B into the box. Click on
“align two or more sequences” and enter the second protein reference number.
Step 3: Choose “show results in a new window” and click the BLAST button.
Step 4: Scroll down to the bottom to see the two sequences aligned. It is difficult to
look for amino acid differences in this view. To change the view to make it easier, scroll
to the very top. Click “Formatting options”. In the “alignment view” pull down menu,
choose “query-anchored with dots for identitites”. Click the blue “reformat” button to see
the changed view.
Step 5: Scroll back all the way to the bottom. Now you can see the identical amino
acids marked with a dot, and the differences are marked with their respective amino
acid letters.
Answer the questions for Part C once your report is finished.
Part C Questions:
1. How many amino acid differences are there between the two sequences?________ 2. List the differences here. Scientists use the numbers of the amino acids to label them. Record the differences here: 3. Use Appendix A to record what change in the type of amino acids has occurred at each site. Note your conclusions below: 4. Even though we know these strains do not cause anti-viral resistance, which variation is more likely to confer resistance? why? 5. What then can be concluded about this variation? Part D: Aligning multiple protein sequences. In Part D of this activity we will align
multiple neuraminidase protein sequences to find the nature of the mutation that confers
resistance to the two antiviral drugs, zanamivir and oseltamivir.
Step 1: Navigate back to the blastp tool (see part C). Keep the query sequence the
same as in Part C (ADD52538.1). Now click align two or more sequences and enter all
of the following numbers, each followed by the enter/return key. Be sure to keep them
in order so we will know which ones are resistant.
4 normal strains:ADG27998ADB98137ACT67242BAJ05804 and 4 mutant strains:ADB98138ADF97837ACT10319ADG28013 Step 2: Choose “Show results in a new window” and click the BLAST button.
Step 3: The default view for the results will show EACH sequence compared to the
query sequence. We want to see them all together. At the top, change the formatting
options to “query anchored with dots for identities”. Donʼt forget to click the “Reformat”
button for your changes to take effect. Then scroll down to view all of your amino acid
sequences aligned. Imagine doing this analysis without a computer!
Part D Questions:
1. What conclusions can you draw from the information. Be as specific and complete in your explanation as possible.
Part E: Analyzing a new mutant strain. You have been asked to analyze a new
resistant strain of H1N1. You have been provided with the nucleotide sequence. In Part
D of this activity you will translate the nucleotide sequence into an amino acid sequence
to draw conclusions about the nature of its mutation.
Step 1: Navigate to Scroll to the bottom and choose translate. It can be found in
section labeled DNA-->protein.
Step 2: Copy and paste the sequence from appendix B into the box.
Step 3: Click on translate sequence. This will translate the nucleotide sequence in all
of the possible “frames”, 3 from the top strand and 3 from the bottom strand.
Step 4: Examine the translated sequences. Your protein will not have many stop
codons in the middle of the sequence and it should start with a “Met” for Methionine.
Once you have located the most likely reading frame, click on this frame number.
Step 5: You will see a new window with your sequence. Copy this protein sequence
starting with the earliest M and ending with the last letter before the STOP codon.
Step 6: Return to the BLASTp search page from Part D. Paste this new protein
sequence into the second box. Be sure it is separate from the last entry by a enter/
return.
Step 7: Choose results in a new window and click BLAST.
Step 8: Reformat the results like you did in Part D to view all of the sequences aligned
together. Donʼt forget to click the reformat button.
Part E Questions:
1. What conclusions can you draw from the aligned sequences on the original non-mutant, mutant and new mutant strains? Be as specific as possible in your analysis. Be sure to include where researchers should focus their study in the future to further analyze the nature of this new mutant strain.
EXTENSION--Part F: The anti-viral drugs zanamivir and oseltamivir target the active
site of the catalytic component of neuraminidase. This active site has several key
amino acids that function in the chemical reaction. In Part F of this activity you will
research the new mutations amino acid substitutions to draw conclusions about which
mutation is the likely cause of the antiviral resistance.
Step 1: Use google to search for “PMCID: PMC1563878”. Click on the first result. This
will bring up the Journal of Virology article, “Importance of Neuraminidase Active-Site
Residues to the Neuraminidase Inhibitor Resistance of Influenza Viruses”.
Step 2: Scroll down about half-way looking for figure 1. Click to enlarge this figure.
This image shows the active site of neuraminidase and its interactions with its substrate,
sialic acid (yellow). Antiviral drugs, such as Tamiflu act by mimicking the structure of
sialic acid and blocking this active site.
Part F Questions:
1. List below the neuraminidase amino acids that seem to play important roles in binding of sialic acid: 2. Which, if any, of these amino acids are mutated in our new strain? 3. How should researchers use this new information? 1. Campbell , A. Malcolm . "Amino Acids: Their Properties and Structures". Davidson College. 5/25/2010 < 2. Decker, PhD, Janet M. "Case 3: The Case of the Variable Virus." Department of Veterinary Science and Microbiology. University Of Arizona, n.d. Web. 5/20/2010. <>. 3. "ExPASy Proteomics Server". Swiss Institute of Bioinformatics (SIB). 5/24/2010 <>.
4. "GenBank sequences from pandemic (H1N1) 2009 viruses". NCBI. 5/20/10 <>.
6. McKimm-Breschkin, J., T. Trivedi, A. Hampson, A. Hay, A. Kilmov, M. Tashiro, F. Hayden, and M. Zambon. "Neuraminidase Sequence Analysis and Susceptibilities of Influenza Virus Clinical Isolates to Zanamivir and Oseltamivir." Antimicrobial Agents and Chemotherapy 47(7).July (2003): 2264–2272. Web. 5/20/2010. < 7. "National Center For Biotechnology Information". NCBI. 5/20/10 <>.
9. "Updated CDC Estimates of 2009 H1N1 Influenza Cases, Hospitalizations and Deaths in the United States, April 2009 – April 10, 2010". Centers For Disease Control and Prevention. 5/24/2010 < 11. Yen, Hui-Ling, Erich Hoffman, Garry Taylor, Christoph Scholtissek, Arnold S. Monto, Robert G. Webster, and Elena A. Govorkova. "Importance of Neuraminidase Active-Site Residues to the Neuraminidase Inhibitor Resistance of Influenza Viruses." American Society for Microbiology 80(17).September (2006): 8787–8795. Web. 5/20/2010. <>. Appendix A
Amino Acids: Their Properties and Structures Electrically Charged (negative and hydrophilic) Electrically Charged (positive and hydrophilic) Appendix B
>gi|NewResistantH1N1|2010|agtttaaaat gaatccaaac caaaagataa taaccattgg ttcggtctgt atgacaattggaatggctaa cttaatatta caaattggaa acataatctc aatatggatt agccactcaattcaacttgg gaatcaaaat cagattgaaa catgcaatca aagcgtcatt acttatgaaaacaacacttg ggtaaatcag acatatgtta acatcagcaa caccaacttt gctgctggacagtcagtggt ttccgtgaaa ttagcgggca attcctctct ctgccctgtt agtggatgggctatatacag taaagacaac agtataagaa tcggttccaa gggggatgtg tttgtcataaggggaccatt catatcatgt tcccccttgg aatgcagaac cttcttctcg actcaaggggccttgctaaa tgacaaacat tccaatggaa ccattaaagt taggagccca tatcgaaccctaatgagctg tcctattggt gaagttccct ctccatacaa ctcaagattt gagtcagtcgcttggtcagc aagtgcttgt catgatggca tcaattggct aacaattgga atttctggcccagacaatgg ggcagtggct gtgttaaagt acaacggcat aataacagac actatcaagagttggagaaa caatatattg agaacacaag agtctgaatg tgcatgtgta aatggttcttgctttactgt aatgaccgat ggaccaagtg atggacaggc ctcatacaag atcttcagaatagaaaaggg aaagatagtc aaatcagtcg aaatgaatgc ccctaattat cactatgaggaatgctcctg ttatcctgat tctagtgaaa tcacatgtgt gtgcagggat aactggcatggctcgaatcg accgtgggtg tctttcaatc agaatctgga atatcagata ggatacatatgcagtgggat tttcggagac aatccacgcc ctaatgataa gacaggcagt tgtggtccagtatcgtctaa tggagcaaat ggagtaaaag gattttcatt caaatacggc aatggtgtttggatagggag aactaaaagc attagttcaa gaaacggttt tgagatgatt tgggatccgaacggatggac tgggacagac aataacttct caataaagca agatatcgta ggaataaatgagtggtcagg atatagcggg agttttgtyc agcatccaga actaacaggg ctggattgtataagaccttg cttctgggtt gaactaatca gagggcgacc caaagagaac acaatctggactagcgggag cagcatatcc ttttgcggtg taaacagtga cactgtgggt tggtcttggccagacggtgc tgagttgcca tttaccattg acaagtaatt tgttcaaaaa act

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