Replacement Polymorphism Of N-linked Glycosylation Sites

By Beau Grothendick

Dengue Fever Virus

Infectious RNA Flavivirus carried by the arthropods mosquitoes and ticks; an arbovirus

Contains about 11,000 nucleotide bases in genome

Related by genus to Yellow Fever virus and West Nile Virus

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

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 cell's membrane binds to viral envelopes asparagine (less commonly arginine) by oligosaccharyltransferase.

Also requires lipid dolichol phosphate

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

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

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

Protein Sequences Aligned with ClustalW

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

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

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.

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

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 E's Asn-67 site is not an optimal site, and mutates with ease.

DENV II E SIFT sequence score

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 protein's prediction score with asparagine was high (1.00) for replacement polymorphism. With lysine it was too low (~0.04)

W NILE E protein's 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. ( ~0.06)

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)

GPP SERVER Results for DENV II

"60C-""61I-""62E-""63A-""64K-""65L-""66Tn""67K-""68TG""69TG""70Tn"Figure 1.8 DENV II E Protein Post-K Replacement Polymorphism Glycoprediction by GPP Server

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

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?

Further applications of Replacement Polymorphism

Agriculture

Breading Livestock

Assaying diseases

Vaccination methods/Attenuation

Gene mapping

The future: Personalized medicine

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): 71367148

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.

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.