RNA Aptamer Selection Against Dengue Virus Envelope Protein for Inhibition of Viral and Host Membrane Fusion

To view full proposal click here.

To view final manuscript for Fall 2010 DENG selection progress here.

The Dengue Virus(DENV) is a mosquito-borne pathogen that causes 100 million cases of dengue fever including up to 500,000 cases of life-threatening dengue hemorrhagic fever/dengue shock syndrome annually.(1,2) The virus’ positive -strand RNA genome is incased by a lipid bilayer and three structural proteins.(1,2) The major envelope glycoprotein E is the protein responsible for fusion of the viral membrane with the potential host cell membrane.(3,4,5) In the virus’ mature form E proteins occur in dimers on the viral membrane surface.(3,5) Upon reduction of pH the dimer pairs dissociate and form trimers with exposed fusion loops responsible for the attachment of the protein to the potential host membrane.(5) The conformational changes that take place are depicted in Figure 1(modified from Modis Y, et al). Inhibition of trimerization or binding of E protein trimers provides a potential for therapeutic treatment of the virus.(1)
Currently there are no antiviral treatment or vaccine commercially available.(6) Additionally there are no rapid diagnostic tests for dengue that provide a definitive diagnosis.(7) Successful therapeutics and diagnostics are needed for effective treatment of this disease.(8) Aptamers bind with high specificity and affinity to protein targets and offer potential as therapeutic and diagnostic agents.(9) An aptamer selected against DENV protein E is expected to provide either treatment of Dengue Fever(DENF) through inhibition of viral cell entry or diagnostic detection of the presence of DENV protein E.

Figure 1: Domain rearrangement in DENV protein E monomer in transition to trimer. a, Shows the tertiary structure of the DENV protein E
monomer in prefusion conformation. b, Shows the secondary structure and links between domains II and III of the protein. c, Shows the tertiary
structure of the DENV protein E monomer in postfusion trimer conformation. d, Shows the secondary structure and links between domains II and III.

Specific Aim 1: Selection of RNA aptamers against DENV protein E

DENV is a major public health concern that affects 112 countries.(1) DENV protein E is the virus' means of entrance in to host cells.(5) It has been shown that inhibition of viruses at the entry stage provides a route for treatment.(1) The selection of RNA aptamers that can bind to DENV protein E would provide molecules that can bind to and potentially inhibit DENV protein E. Recognition and/or inhibition of DENV protein E would provide a potential diagnostic or therapeutic tool.

1. Wang Q, Patel SJ, Vangrevelinghe E, Xu HY, Rao R, Jaber D, Schul W, Gu F, Heudi O, Ma NL, Poh MK, Phong WY, Keller TH, Jacoby E, Vasudevan SG. 2009. A small-molecule dengue virus entry inhibitor. Antimicrob Agents Chemother. 53(5):1823-1831.
2. Kyle JL, Harris E. 2008. Global spread and persistence of dengue. Annual Review of Microbiology. 62:71-92.
3. Kuhn RJ, Zhang W,Rossmann MG, Pletnev SV, Corver J, Lenches E, Jones, CT, Mukhopadhyay S, Chipman PR, Strauss EG, Baker TS, Strauss JH. 2002. Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell. 108:717-725.
4. Nayak V, Dessau M, Kucera K, Anthony K, Ledizet M, Modis Y. 2009. Crystal structure of dengue virus type 1 envelope protein in the postfusion conformation and its implications for membrane fusion. Journal of Virology. 83(9):4338-4344.
5. Modis Y, Ogata S, Clements D, Harrison SC. 2004. Structure of the dengue virus envelope protein after membrane fusion. Nature. 427:313-319.
6. Shrestha B, Brien JD, Sukupolvi-Petty S, Austin SK, Edeling MA, Kim T, O’Brien KM, Nelson CA, Johnson S, Fremont DH, Diamond MS. 2010. The development of therapeutic antibodies that neutralize homologous and heterologous genotypes of dengue virus type 1. PLoS Pthog. 6(4).
7. Tricou V, Vu HTT, Quynh NVN, Nguyen CVV, Tran HT, Farrar J, Wills B, Simmons CP. 2010. Comparison of two dengue NS1 rapid tests for sensitivity, specificity and relationship to viraemia and antibody responses. BMC Infectious Diseases. 10:142.
8. Whitehorn J, Farrar J. 2010. Dengue. British Medical Bulletin.
9. Stolenburg R, Reinemann C, Strehlitz B. 2007. SELEX-a revolutionary method to generate high-affinity nucleic acid ligands. Biomolecular Engineering. 24:381-403.


Brad Hall said...

Michael, this abstract is really well thought out and a very exciting project. You may consider shortening the title and I found a couple tense mismatches, but very well done. Can you also find some graphic off the web of either the virus or the specific protein to include? We will move forward on this project.

Brad Hall said...

In addition, it seems to me that either the envelope protein or serovar 3 would be useful from RayBiotech. It looks like they are all just versions of the same thing and serotype 1 may be more similar to Envelop than serotype 3 (per the alignment). Price is $231 for each, so lets go with the longer envelope protein, cat# 228-10309. I will order it today.

Travis Hughes said...

This is an awesome target Michael! Good luck.

Brad Hall said...

It was ordered today.

Brad Hall said...

Recombinant Dengue Envelope ST1 came in a few days ago (9-13-10?) from RayBiotech at a concentration of 100ug per 85ul. The solution was listed as 50mM Sodium chloride-phosphate buffer, pH 7.5 (PBS).

Since the size is 23000ug/umol, we had a concentration of 4347pmol per 85ul or 51pmol/ul. It was aliquoted at 2ul (100pmol) per PCR tube, labeled with DENG and placed in the -80C freezer in J3, first box. The LOT # was 710DENENV01