planned experiments SGE NS5 coIP -sent to Julien Pompon

Identification of Novel Protein:protein Interactions between Dengue Proteins  and Salivary proteome of Aedes aegypti using coimmunoprecipitation and mass spectrometry

Dengue virus (DENV) infects approximately 300 million each year throughout the tropics and the death rates are increasing every year.  Understanding the role of vector-virus interactions is becoming critical. The principal DENV vector Ae. aegypti saliva contains proteins with diverse functions to facilitate blood-feeding, like anti-hemostatics, inhibitors of platelet aggregation, vasodilators, allergens and immunomodulatory compounds. 10 days following blood-feeding from an infected human host, the female Aedes Aegypti mosquito, the virus penetrates several barriers: the midgut infection barrier (MIB), the midgut escape barrier (MEB) and finally infects the salivary glands and subsequently injected into a human host. The receptors for all DENV serotypes in mosquito salivary glands have never been formally identified. These receptors could potentially be key targets for transmission blocking. Mosquito salivary glands also have immune machineries to limit viral replication.  DENV-2 infected mosquitoes were found to have altered expression of salivary proteins especially anti-hemostatic and pain inhibitory functions were significantly reduced¹. Antibacterial peptides are strongly stimulated by DENV infection in A. aegypti salivary glands². Immune components belonging to the Imd and Toll signaling pathways are upregulated following DENV infection ^3,4. It is currently unknown how the immune system responds to or controls a high level of viral replication in the salivary glands.  

Several genome-wide studies of interactions between DENV and their hosts have been done using microarray, yeast 2 hybrid, RNAi screens and Virus overlay protein binding ^{5, 6, 7}. However the interactions of the DENV proteins in the Ae. aegypti  salivary glands has not been well documented. The 900 kDa non-structural protein 5 (NS5) is the most conserved amongst the viral proteins and plays vital roles in virus replication. Its N-terminal domain encodes a methyltransferase (MTase), and possibly guanylyl transferase (GTase) involved in RNA cap formation. The C-terminal region comprises a RNA-dependent RNA polymerase (RdRp) required for viral RNA synthesis. DENV NS5 interacts with importin beta and is exported from nucleus by exportin CRM1 and inhibits IFN signaling by binding to STAT2⁹. Based on Yeast two hybrid studies by Le Breton et al, NS5 was found to interact with several cellular proteins involved in the chromatin remodelling process in particular the kinesin family member KIF3B and the centrosomal components and proteins associated with the Golgi vesicle transport⁵. NS5 remains one of the most promising targets for anti-DENV intervention as RdRp inhibitor drugs are used for treatment of several viruses, for example, HIV-1, HBV, HCV and CMV⁸.

Proposed experiments:

·         We propose to identify novel interactions between DENV NS5 and Ae. aegypti  salivary proteins using direct physical interaction studies.

·        To achieve this, we will be carrying out co-immunoprecipitation of NS5 protein from infected mosquito Salivary gland extracts. NS5 antibody coupled to agarose resin will be added to DENV infected and uninfected Ae. aegypti salivary gland extracts and incubated with gentle end-over-end mixing for 2 hr at 4°C or overnight. Unbound proteins will be washed away and bound proteins will be eluted and identified by LC+MS/MS analysis. The experiment will be carried out based on Pierce Co-Immunoprecipitation Kit.

·        Once these interactions are identified, we will be carrying out coIP using recombinant NS5 to validate the in vivo interactions. The validated interactions will be further studied using genetic, biochemical, biophysical and structural techniques like gene knockout studies, ITC and protein x-ray crystallography.

·        Following the same methodology, we propose to carry out interaction studies for the other DENV structural and nonstructural proteins.

·         These studies will potentially lead to identification of novel candidates for vaccine or drug development. 

1.      Infection with dengue-2 virus alters proteins in naturally expectorated saliva of Aedes aegypti mosquitoes.Chisenhall DM, Christofferson RC,  McCracken MK, Johnson AM, Londono-Renteria B, Mores CN. Parasit Vectors. 2014 May 30;7:252.

2.      Luplertlop, N. et al. (2011) Induction of a peptide with activity against a broad spectrum of pathogens in the Aedes aegypti salivary gland, following infection with dengue virus. PLoS Pathog. 7, e1001252

3.      The Aedes aegypti toll pathway controls dengue virus infection. Xi Z, Ramirez JL, Dimopoulos G. PLoS Pathog. 2008 Jul 4;4(7)

4.      Mazzon M, Jones M, Davidson A, Chain B, Jacobs M (2009) Dengue virus NS5 inhibits interferon-alpha signaling by blocking signal transducer and activator of transcription 2 phosphorylation. J Infect Dis 200: 1261–1270.

5.      Le Breton M, Meyniel-Schicklin L, Deloire A, Coutard B, Canard B, et al. (2011) Flavivirus NS3 and NS5 proteins interaction network: a high-throughput yeast two-hybrid screen. BMC Microbiol 11: 234.

6.      Identification of new protein interactions between dengue fever virus and its hosts, human and mosquito. Mairiang D,  Zhang H,  Sodja A, Murali T, Suriyaphol P, Malasit P, Limjindaporn T, Finley RL Jr. PLoS One. 2013;8(1).

7.      Dengue-2-virus-interacting polypeptides involved in mosquito cell infection.

Paingankar MS1, Gokhale MD, Deobagkar DN. Arch Virol. 2010 Sep;155(9):1453-61.

8.      The dengue virus NS5 protein as a target for drug discovery, Antiviral Research, Volume 119, July 2015, Pages 57-67 Siew Pheng Lim, Christian G. Noble, Pei-Yong Shi

9.      Mazzon M, Jones M, Davidson A, Chain B, Jacobs M (2009) Dengue virus NS5 inhibits interferon-alpha signaling by blocking signal transducer and activator of transcription 2 phosphorylation. J Infect Dis 200: 1261–1270.