Ex vivo promoter analysis of antiviral heat shock cognate 70B gene in Anopheles gambiae

Seokyoung Kang,Cheolho Sim,Young S Hong,Brian D Byrd,Frank H Collins

doi:10.1186/1743-422x-5-136

Abstract

BackgroundThe Anopheles gambiae heat shock cognate gene (hsc70B) encodes a constitutively expressed protein in the hsp70 family and it functions as a molecular chaperone for protein folding. However, the expression of hsc70B can be further induced by certain stimuli such as heat shock and infection. We previously demonstrated that the An. gambiae hsc70B is induced during o'nyong-nyong virus (ONNV) infection and subsequently suppresses ONNV replication in the mosquito. To further characterize the inducibility of hsc70B by ONNV infection in An. gambiae, we cloned a 2.6-kb region immediately 5' upstream of the starting codon of hsc70B into a luciferase reporter vector (pGL3-Basic), and studied its promoter activity in transfected Vero cells during infection with o'nyong-nyong, West Nile and La Crosse viruses.ResultsSerial deletion analysis of the hsc70B upstream sequence revealed that the putative promoter is likely located in a region 1615–2150 bp upstream of the hsc70B starting codon. Sequence analysis of this region revealed transcriptional regulatory elements for heat shock element-binding protein (HSE-bind), nuclear factor κB (NF-κB), dorsal (Dl) and fushi-tarazu (Ftz). Arbovirus infection, regardless of virus type, significantly increased the hsc70B promoter activity in transfected Vero cells.ConclusionOur results further validate the transcriptional activation of hsc70B during arbovirus infection and support the role of specific putative regulatory elements. Induction by three taxonomically distinct arboviruses suggests that the HSC70B protein may be expressed to cope with cellular stress imposed during infection.

Highlights

The Anopheles gambiae mosquito is the principle vector of the malaria parasite Plasmodium falciparum in sub-Saharan Africa
Since membrane fusion is one of the protein maturation processes mediated by molecular chaperones, such as the HSP70 family, it is possible that HSP70 may enhance or suppress maturation of viral proteins [12,13,14]
Deletion analysis of the hsc70B promoter To identify the critical elements required for transcription, various deletions of the 5' upstream region of the hsc70B locus were generated and ligated into the pGL3-Basic vector

Summary

Introduction

Alphaviruses, including ONNV, Sindbis virus, and Chikungunya virus have class II fusion proteins such as E glycoproteins that mediate membrane fusion between virus and host cells during virus entry [8,9]. The Anopheles gambiae heat shock cognate gene (hsc70B) encodes a constitutively expressed protein in the hsp family and it functions as a molecular chaperone for protein folding. We previously demonstrated that the An. gambiae hsc70B is induced during o'nyongnyong virus (ONNV) infection and subsequently suppresses ONNV replication in the mosquito. To further characterize the inducibility of hsc70B by ONNV infection in An. gambiae, we cloned a 2.6-kb region immediately 5' upstream of the starting codon of hsc70B into a luciferase reporter vector (pGL3-Basic), and studied its promoter activity in transfected Vero cells during infection with o'nyong-nyong, West Nile and La Crosse viruses

Methods

Results

Discussion

Conclusion