Dengue virus (DEN), a Category A priority pathogen, causes the most prevalent arthropod-borne viral illnesses in humans worldwide. Like other viruses, DEN lacks its own translational machinery and thus is dependent on the host cell for translation of viral RNA. DEN and other flaviviruses have a capped, nonpolyadenylated, positive-sense RNA genome. In contrast to many animal viruses, flaviviruses compete efficiently for the cellular translation apparatus without shutting off host protein synthesis. Our long-term goal is to define the mechanism of flavivirus translation, using DEN as a model, and to characterize the viral and cellular factors involved. Specifically, the proposed research investigates the sequence and structural determinants in the DEN 3' untranslated region (UTR) that stimulate viral translation and the factors that mediate this process. We have shown that the 3'UTR of the DEN genome stimulates translation in a manner similar to that reported for cellular and other viral systems but not yet for flaviviruses. In addition, we have identified sequence elements in the 3'UTR that modulate translation efficiency. The DEN 3'UTR can stimulate translation in the absence of viral proteins, leading to the hypothesis that the DEN 3'UTR stimulates viral translation by recruiting cellular factors. This will be addressed via the following two specific aims:
Specific Aim 1 : Characterize the role of the 3'UTR in stimulation of DEN translation and identify the sequences and structures in the DEN 3'UTR critical for viral translation. We have shown that the DEN 3'UTR acts as a translational enhancer in reporter constructs in both transfected cells and in vitro translation extracts and have begun to analyze sequences that contribute to this effect.
Specific Aim 1 will focus on further delineation of sequences and structures in the DEN type 2 (DEN2) 3'UTR that regulate translation in the context of reporter constructs, DEN2 replicons and a DEN2 infectious clone.
Specific Aim 2 : Identify and characterize proteins that mediate 3'UTR-stimulated DEN translation.
This aim focuses on isolation, identification, and characterization of the proteins that mediate viral translation via interaction with the DEN 3'UTR to begin to understand their mechanism of action. This work will provide insight into translation of DEN and other flaviviruses as well as into eukaryotic translational control. Moreover, these studies should identify targets for rational drug and vaccine development of this medically important pathogen.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI052324-02
Application #
6895407
Study Section
Experimental Virology Study Section (EVR)
Program Officer
Greenfield, Teri L
Project Start
2004-06-01
Project End
2009-05-31
Budget Start
2005-06-01
Budget End
2006-05-31
Support Year
2
Fiscal Year
2005
Total Cost
$257,791
Indirect Cost
Name
University of California Berkeley
Department
Type
Schools of Public Health
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
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Peña, José; Harris, Eva (2012) Early dengue virus protein synthesis induces extensive rearrangement of the endoplasmic reticulum independent of the UPR and SREBP-2 pathway. PLoS One 7:e38202
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Peña, José; Harris, Eva (2011) Dengue virus modulates the unfolded protein response in a time-dependent manner. J Biol Chem 286:14226-36
Friebe, Peter; Shi, Pei-Yong; Harris, Eva (2011) The 5' and 3' downstream AUG region elements are required for mosquito-borne flavivirus RNA replication. J Virol 85:1900-5
Parameswaran, Poornima; Sklan, Ella; Wilkins, Courtney et al. (2010) Six RNA viruses and forty-one hosts: viral small RNAs and modulation of small RNA repertoires in vertebrate and invertebrate systems. PLoS Pathog 6:e1000764
Paranjape, Suman M; Harris, Eva (2010) Control of dengue virus translation and replication. Curr Top Microbiol Immunol 338:15-34
Friebe, Peter; Harris, Eva (2010) Interplay of RNA elements in the dengue virus 5' and 3' ends required for viral RNA replication. J Virol 84:6103-18
Polacek, Charlotta; Friebe, Peter; Harris, Eva (2009) Poly(A)-binding protein binds to the non-polyadenylated 3' untranslated region of dengue virus and modulates translation efficiency. J Gen Virol 90:687-92
Clyde, Karen; Barrera, Julio; Harris, Eva (2008) The capsid-coding region hairpin element (cHP) is a critical determinant of dengue virus and West Nile virus RNA synthesis. Virology 379:314-23

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