Replacement Polymorphism Of N-linked        Glycosylation Sites         By Beau Grothendick
Dengue Fever Virus●Infectious RNA Flavivirus carried by thearthropods mosquitoes and ticks; an arbovirus●Contains about 11...
Virulence Factors: Envelope Proteins●   In many viruses, envelope proteins, or viral envelopes, assist    in infecting the...
N-linked Glycosylation●   Most common type of glycosidic bond●   Attachment of a sugar molecule (oligosaccharide) to a    ...
N-linked Glycosylation in Dengue Fever Virus●   Most prominent sites at residues ASN-67 and ASN-153/154    in DENV E prote...
Replacement Polymorphism●   Single nucleotide polymorphism (SNP) that produces a    different polypeptide sequence.●   SNP...
Can We Mutate Genes with Bioinformatic                  Software?●   I hypothesize that using the basic tools learned in a...
Protein Sequences Aligned with ClustalW
Multiple Alignment of Protein Sequences●   Each DENV E Protein showed conserved regions around    Asn-67 and throughout se...
GPP SERVER●   Utilized the GPP SERVER at    http://comp.chem.nottingham.ac.uk/cgi-bin/glyco/bin/getparams.cgi●   Server ru...
SIFT Sequence●   http://sift.bii.a-star.edu.sg/●   Sequence homology-based tool which separates tolerant    from intoleran...
How does SIFT sequence work?●   Takes a query sequence and searches for similar sequences●   Chooses most closely related ...
Interpreting SIFT sequence●   Scores of less than 0.05 predict poor or deleterious    mutations.●   Scores higher than 0.0...
DENV II E SIFT sequence score
Results of SIFT sequence●   DENV E proteins Asn-67 residue was substituted with    Lysine. Prediction scores were optimal ...
Confirmation of Successful Replacement          Polymorphism by GPP Server●   GPP Server was utilized again, this time to ...
GPP SERVER Results for DENV II"60 C     -""61 I     -""62 E     -""63 A     -""64 K     -""65 L     -""66 T     n""67 K   ...
GPP SERVER Results for DENV, W NILE, Y                 FEVER●   Negative control Yellow Fever virus E protein did not muta...
Further Questions●   The negative control did not yield a glycosylation site in    Yellow Fever virus E protein position 6...
Further applications of Replacement                  Polymorphism●   Agriculture●   Breading Livestock●   Assaying disease...
Cited Works●   Hanna Sl et al. N-linked glycosylation of west nile virus envelope proteins influences particle    assembly...
Cited Works (cont.)●   Burri DJ, Palma JR, Kunz S, Pasquato A. Envelope glycoprotein of arenaviruses. Viruses. 2012    Oct...
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Replacement Polymorphism of N-linked Glycosylation site Asn-67 in DENV E

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Replacement Polymorphism of N-linked Glycosylation site Asn-67 in DENV E

  1. 1. Replacement Polymorphism Of N-linked Glycosylation Sites By Beau Grothendick
  2. 2. Dengue Fever Virus●Infectious RNA Flavivirus carried by thearthropods mosquitoes and ticks; an arbovirus●Contains about 11,000 nucleotide bases ingenome●Related by genus to Yellow Fever virus andWest Nile Virus
  3. 3. Virulence Factors: Envelope Proteins● In many viruses, envelope proteins, or viral envelopes, assist in infecting the host cell by attaching and binding to receptor sites on the membrane● Enveloped viruses are highly adaptable and more protected from external influences● Viral envelopes encase the capsid and genome● Some contain viral glycoproteins
  4. 4. N-linked Glycosylation● Most common type of glycosidic bond● Attachment of a sugar molecule (oligosaccharide) to a nitrogen atom in an amino acid residue● Lack of N-linked glycosylation sites in viral proteins diminish replication rate of virus● Oligosaccharide on host cells membrane binds to viral envelopes asparagine (less commonly arginine) by oligosaccharyltransferase.● Also requires lipid dolichol phosphate
  5. 5. N-linked Glycosylation in Dengue Fever Virus● Most prominent sites at residues ASN-67 and ASN-153/154 in DENV E protein sequence.● Ablation of residues ASN-67 and ASN-153/154 reduces virulence but does not prevent infection of virus into host cells.● Absence of ASN-67 reduces replication in mammalian host cells● Stability and function of E protein can be maintained with removal of glycosylation sites. Previous research maintains the replacement of asparagine with glutamine yields stability
  6. 6. Replacement Polymorphism● Single nucleotide polymorphism (SNP) that produces a different polypeptide sequence.● SNP – Change in DNA by variation in a single nucleotide● May result in missense or nonsense mutations
  7. 7. Can We Mutate Genes with Bioinformatic Software?● I hypothesize that using the basic tools learned in an introductory bioinformatics course, it is possible to remove the ASN-67 glycosylation site from the DENV E protein while keeping the E protein functioning and stable● Protein Sequences collected from databases NCBI, UniProt, and SwissProt● DENV I, II, III, and IV E were saved in Fasta Format● Yellow Fever Virus E Protein and West Nile Virus E Protein were saved in Fasta Format as negative controls
  8. 8. Protein Sequences Aligned with ClustalW
  9. 9. Multiple Alignment of Protein Sequences● Each DENV E Protein showed conserved regions around Asn-67 and throughout sequence● W Nile and Yellow Fever E Proteins did not show minimal conserved residues compared to DENV E Proteins● W Nile and Yellow Fever E Proteins did not share a common Asn-67 residue with DENV E Proteins
  10. 10. GPP SERVER● Utilized the GPP SERVER at http://comp.chem.nottingham.ac.uk/cgi-bin/glyco/bin/getparams.cgi● Server run by University of Nottingham● Predicts Glycosylsation sites for a given sequence● Quick and easy processing, results forwarded by e-mail● All protein sequences were ran to verify ASN-67 was a valid glycosylation site for DENV I,II,III, and IV E Proteins● No glycosylation sites on W NILE E or Y FEVER E
  11. 11. SIFT Sequence● http://sift.bii.a-star.edu.sg/● Sequence homology-based tool which separates tolerant from intolerant amino acid substitutions● Submit a sequence to sift, deleterious mutations will be highlighted in red● For lab assays, phenotypic changes may also be predicted● SIFT is based on the premise that protein evolution is correlated with protein function.
  12. 12. How does SIFT sequence work?● Takes a query sequence and searches for similar sequences● Chooses most closely related sequences in function to the query● Obtains alignment of sequences● Calculates possibilities for ALL single amino acid substitutions for each residue number in a sequence
  13. 13. Interpreting SIFT sequence● Scores of less than 0.05 predict poor or deleterious mutations.● Scores higher than 0.05 predict functional mutations● Regions that do not support many substitutions are highlighted in red; optimal areas for target mutation.● DENV Es Asn-67 site is not an optimal site, and mutates with ease.
  14. 14. DENV II E SIFT sequence score
  15. 15. Results of SIFT sequence● DENV E proteins Asn-67 residue was substituted with Lysine. Prediction scores were optimal (~0.06); protein continued to function● Y FEVER E proteins prediction score with asparagine was high (1.00) for replacement polymorphism. With lysine it was too low (~0.04)● W NILE E proteins prediction score with asparagine high (1.00) and with lysine it was non-existent ( 0.00)● DENV II E was replaced with glutamine as a positive control, and scored sufficiently. ( ~0.04)
  16. 16. Confirmation of Successful Replacement Polymorphism by GPP Server● GPP Server was utilized again, this time to see if the glycosylation sites had been removed by the mutation or were still intact.● Previous mutations of ASN-67 showed ability to still glycogen with amino acids in triplet peptide other than asparagine and thymine● For current mutations made, results showed the ability to glycogen had been removed from Asn-67 (now Lys-67)
  17. 17. GPP SERVER Results for DENV II"60 C -""61 I -""62 E -""63 A -""64 K -""65 L -""66 T n""67 K -""68 T G""69 T G""70 T n"Figure 1.8 DENV II E Protein Post-K Replacement Polymorphism Glycoprediction by GPPServer
  18. 18. GPP SERVER Results for DENV, W NILE, Y FEVER● Negative control Yellow Fever virus E protein did not mutate with the ability to glycolate on position 67 and West Nile virus E protein DID mutate the ability to glycolate on site 67● Positive control DENV II with glutamine substitution did not mutate with ability to glycolate on site 67● Experiment group successful mutated with no ability to glycolate on site 67
  19. 19. Further Questions● The negative control did not yield a glycosylation site in Yellow Fever virus E protein position 67. Looking at the larger surrounding peptide structure needed for N-linked glycosylation, why did this happen?● Do lysine substitutions on position 67 reduce virulence any more in mammalian cells than glutamine substitutions?● If we assayed these mutations of Dengue Fever Virus, how would their replication rate adjust in arthropods?
  20. 20. Further applications of Replacement Polymorphism● Agriculture● Breading Livestock● Assaying diseases● Vaccination methods/Attenuation● Gene mapping● The future: Personalized medicine
  21. 21. Cited Works● Hanna Sl et al. N-linked glycosylation of west nile virus envelope proteins influences particle assembly and infectivity. J Virol.” 2005 Nov;79(21):13262-74● Moudy Rm et al. West Nile virus envelope protein glycosylation is required for efficient viral transmission by Culex vectors. Virology. 2009 Apr 25;387(1):222-8. Epub 2009 Feb 27.● Mondotte, A. Juan et al. Essential Role of Dengue Virus Envelope Protein N Glycosylation at Asparagine-67 during Viral Propagation. J Virol. 2007 July; 81(13): 7136–7148● Lee E. et al. Both E protein glycans adversely affect dengue virus infectivity but are beneficial for virion release. J Virol. 2010 May;84(10):5171-80. Epub 2010 Mar 10.● Rodenhuis-Zybert IA, Wilschut J, Smit JM. Dengue virus life cycle: viral and host factors modulating infectivity. Cell Mol Life Sci. 2010 Aug;67(16):2773-86. Epub 2010 Apr 6.● Fan YH et al. A missense polymorphism in porcine interferon-gamma cDNA affects antiviral activity of the protein variant. Mol Immunol. 2007 Jul;44(13):3297-304. Epub 2007 Apr 9.● Lee E, Weir RC, Dalgarno L. Changes in the dengue virus major envelope protein on passaging and their localization on the three-dimensional structure of the protein. Virology. 1997 Jun 9;232(2):281-90.● Tajima S, Takasaki T, Kurane I. Characterization of Asn130-to-Ala mutant of dengue type 1 virus NS1 protein. Virus Genes. 2008 Apr;36(2):323-9. Epub 2008 Feb 21.
  22. 22. Cited Works (cont.)● Burri DJ, Palma JR, Kunz S, Pasquato A. Envelope glycoprotein of arenaviruses. Viruses. 2012 Oct 17;4(10):2162-81. doi: 10.3390/v4102162.● Alen MM Et al. Crucial role of the N-glycans on the viral E-envelope glycoprotein in DC-SIGN- mediated dengue virus infection. Antiviral Res. 2012 Dec;96(3):280-7. doi: 10.1016/j.antiviral.2012.10.007. Epub 2012 Oct 31.● Li J et al Naturally mutated envelope protein domain I of Chinese B dengue virus attenuated human dendritic cell maturation. Int Immunopharmacol. 2012 Dec;14(4):683-9. doi: 10.1016/j.intimp.2012.09.003. Epub 2012 Sep 28.

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