Flaviviruses and alphaviruses are spread by mosquito and tick vectors and cause severe human and animal illnesses such as encephalitis and hemorrhagic fever. These viruses include potential bioterrorist agents that are category A-C pathogens, such as the flavivirus dengue virus (DV) and the alphavirus Chikungunya virus (CV). DV is of particular concern as it has dramatically reemerged to become endemic in more than 100 countries including the US, and is now a global health problem. Alphaviruses can also be important emerging pathogens, as exemplified by the recent outbreak of CV infection with several million cases reported to date in India. Antiviral strategies for the flaviviruses and alphaviruses are urgently needed. The flavivirus and alphavirus membrane fusion proteins are members of the class II virus membrane fusion proteins. They are structurally very similar and refold to a homotrimer form to mediate virus fusion and infection. In collaboration with Dr. Felix Rey, we determined the structure of the homotrimer conformation of the fusion protein from the alphavirus Semliki Forest virus (SFV). The SFV homotrimer structure is strikingly similar to that of DV. Using the structures as a guide, we have recently developed recombinant forms of domain III (DUN) from the SFV and DV fusion proteins. These recombinant Dill proteins act as specific dominant-negative inhibitors of virus fusion and infection, and are the basis for our proposed screen for small molecule inhibitors of DV and CV.
In aim 1 we will use our recombinant protein systems to define the mechanism of DV and CV fusion protein trimerization and the steps in trimer formation.
In aim 2, we will develop fluorescence-based in vitro assays for the binding of DIM to the trimer core of DV and CV.
In aim 3 we will adapt this assay to a high throughput format. We will then use the assay in high throughput screens of small molecule libraries available through the NIH and the Northeast Biodefense Consortium, and validate hits by tests of virus fusion and infection. Ultimately, such inhibitors will be lead compounds for antiviral therapy, and important research tools to understand the class II virus fusion reaction.

Public Health Relevance

Our studies aim to discover novel antiviral therapies for the important viral pathogens dengue virus (category A) and Chikungunya virus (category C). Conservation of DIM contact sites leads to cross-genus inhibition by DIN proteins, suggesting that our proposed screens have the potential to identify broad-specificity inhibitors.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Specialized Center--Cooperative Agreements (U54)
Project #
5U54AI057158-10
Application #
8444621
Study Section
Special Emphasis Panel (ZAI1-DDS-M)
Project Start
Project End
2015-02-28
Budget Start
2013-03-01
Budget End
2015-02-28
Support Year
10
Fiscal Year
2013
Total Cost
$341,888
Indirect Cost
$25,641
Name
Columbia University (N.Y.)
Department
Type
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032
Charles, Jermilia; Firth, Andrew E; Loroño-Pino, Maria A et al. (2016) Merida virus, a putative novel rhabdovirus discovered in Culex and Ochlerotatus spp. mosquitoes in the Yucatan Peninsula of Mexico. J Gen Virol 97:977-87
Pham, Alissa M; Santa Maria, Felicia Gilfoy; Lahiri, Tanaya et al. (2016) PKR Transduces MDA5-Dependent Signals for Type I IFN Induction. PLoS Pathog 12:e1005489
Song, Jeongmin; Wilhelm, Cara L; Wangdi, Tamding et al. (2016) Absence of TLR11 in Mice Does Not Confer Susceptibility to Salmonella Typhi. Cell 164:827-8
Li, Melody M H; Bozzacco, Leonia; Hoffmann, Hans-Heinrich et al. (2016) Interferon regulatory factor 2 protects mice from lethal viral neuroinvasion. J Exp Med 213:2931-2947
Moser, Lindsey A; Lim, Pei-Yin; Styer, Linda M et al. (2016) Parameters of Mosquito-Enhanced West Nile Virus Infection. J Virol 90:292-9
Basu, Debaleena; Kahn, Jennifer N; Li, Xiao-Ping et al. (2016) Conserved Arginines at the P-Protein Stalk Binding Site and the Active Site Are Critical for Ribosome Interactions of Shiga Toxins but Do Not Contribute to Differences in the Affinity of the A1 Subunits for the Ribosome. Infect Immun 84:3290-3301
Steyer, Andrej; Jevšnik, Monika; Petrovec, Miroslav et al. (2016) Narrowing of the Diagnostic Gap of Acute Gastroenteritis in Children 0-6 Years of Age Using a Combination of Classical and Molecular Techniques, Delivers Challenges in Syndromic Approach Diagnostics. Pediatr Infect Dis J 35:e262-70
Uhde, Melanie; Ajamian, Mary; Li, Xueting et al. (2016) Expression of C-Reactive Protein and Serum Amyloid A in Early to Late Manifestations of Lyme Disease. Clin Infect Dis 63:1399-1404
Jacek, Elzbieta; Tang, Kevin S; Komorowski, Lars et al. (2016) Epitope-Specific Evolution of Human B Cell Responses to Borrelia burgdorferi VlsE Protein from Early to Late Stages of Lyme Disease. J Immunol 196:1036-43
Aguilar, Jorge L; Varshney, Avanish K; Pechuan, Ximo et al. (2016) Monoclonal antibodies protect from Staphylococcal Enterotoxin K (SEK) induced toxic shock and sepsis by USA300 Staphylococcus aureus. Virulence :1-10

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