RNA Protein Interactions in RNA Viruses
We are interested in studying the replication mechanism of dengue virus and the molecular mechanism of RNA structure modulation of by viral proteins.
Dengue virus (DV) is a human pathogen that causes a serious public health threat in tropic and subtropic regions of the world. DV is a member of positive strand RNA virus of the Flaviviridae family that causes dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Neither an effective vaccine nor an effective therapy for DV infection in humans is available. DV virions are spherical in shape with a diameter of 50 nm. The nucleocapsid consists of capsid and genomic RNA and is surrounded by a lipid bilayer in which the viral envelope and membrane proteins are embedded. DV has a single stranded RNA genome of ~11Kb that contains a 5' untranslated region (5' UTR), a single open reading frame (ORF), and a 3' UTR. The ORF of flavivirus encodes a polyprotein precursor of ~3,400 amino acids that is processed in the endoplasmic reticulum by host and viral protease into three structural (core [C], pre-membrane [prM] or membrane [M], and envelope [E]) proteins and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. The nonstructural proteins are involved in viral replication.
Virus subgenomic replicons (SGR) that are capable of replicating autonomously and efficiently in culture cells can be used as an alternative system to study virus replication and to screen antiviral drugs. We successfully established stable cell lines harboring selectable subgenomic replicon of a DV strain. The adaptive mutation in DV SGR generated during the establishment of the DV SGR cell lines will be identified. The effect of the viral gene mutation on viral replication and host cell defense will be studied. Currently, we are using the DV SGR system to investigate the role of viral NS proteins on viral replication via a reverse genetics approach. In addition to NS3 that has protease and helicase activities and NS5 that methyl transferase and RNA dependent RNA polymerase activities, we put more effort on studying the function of other DV NS proteins that have no obvious structural or functional motifs in viral replication. Both UTR regions of DV RNA are important for viral polyprotein synthesis and viral RNA replication, and we will identify essential RNA elements resided in these nontranslated regions that ensure efficient translation and replication.
RNA chaperones are proteins that aid in the process of RNA folding by preventing mis-folding or by resolving mis-folded species in an ATP-independent manner, while how a protein exerts its RNA chaperone activity is not well characterized. Several viral proteins have been shown to have RNA chaperone activity in vitro, although the biological function of this activity is not clear. We found that DV core protein (DV C protein) and the helicase domain of dengue virus NS3 protein (DV NS3H) possessed RNA chaperone activity. Both DV proteins interact with a variety of RNA species and can modulate RNA conformation arrangement in an ATP-independent manner, although they have distinct size, sequence and structure. DV C protein and DV NS3H will be used as the model molecules to explore the molecular mechanism of RNA chaperone action.