Our main goal is to understand the mechanisms of dengue virus replication in molecular detail. Together with important human pathogens such as yellow fever, West Nile, Japanese encephalitis, and hepatitis C viruses, dengue virus is a member of the Flaviviridae family. It affects more than 50 million people annually and can produce clinical illness ranging from dengue fever, a nonspecific flu-like syndrome, to the severe and sometimes fatal dengue hemorrhagic fever. The World Health Organization continues to report outbreaks of severe forms of the disease in the Americas and Asia. Despite the wide morbidity and mortality associated with dengue infections, neither specific antiviral therapies nor a licensed vaccine exist. We believe that understanding the biology of the dengue virus at the molecular level will help us to design antiviral strategies to stop these viral diseases.
To address fundamental questions of the viral processes, we focused on structural and functional analyses of the genomic RNA, combining biochemistry, molecular biology, and classical virology. We made significant progress in understanding the function of intra-molecular RNA-RNA interactions in the viral RNA during dengue virus propagation. Our recent studies revealed the mechanisms by which dengue virus amplifies its genome in the infected cell. We also develop novel investigational tools to analyze specific steps of the viral life cycle and search for potential antiviral drugs.
After viral infection, the single-stranded RNA genome of dengue virus directs the synthesis of viral proteins. Once those proteins are synthesized, the viral RNA is copied to generate a complementary minus-strand RNA, which is transcribed into new molecules of plus strand. A virus-encoded RNA-dependent RNA polymerase (RdRp), in combination with other viral and cellular factors, catalyzes this process. Over the past few years, we have been studying how the viral polymerase discriminates between viral and cellular RNAs and where the signals are that modulate RdRp recognition and activity during dengue virus replication.
We have developed genomic and subgenomic viral cDNAs that carry reporter genes for rapid and sensitive analysis of viral replication. Using these tools in vivo, we identified an RNA element present at the 5′ end of the viral genome that is essential for viral RNA recognition and polymerase activity. The predicted stem-loop structure and specific nucleotides within this RNA element, named SLA, are crucial for dengue virus propagation in mosquito and mammalian cells and constitute the promoter for the viral RdRp.
We are also studying biochemical properties of the viral protein NS5, which contains two enzymatic activities: a methyltransferase at the amino terminus followed by the RdRp domain. Using RNA binding assays, we found that the viral protein forms highly stable ribonucleoprotein complexes with the promoter element, which can be visualized as single molecules by atomic force microscopy. Furthermore, we found that cyclization of RNA templates via long-range RNA-RNA contacts enhanced polymerase activity. We confirmed these in vitro observations in infected cells, in which demonstrated that circular conformations of the viral genome were directly involved in viral RNA amplification. On the basis of these findings, we propose the first model for dengue virus RNA synthesis: the SLA promoter present at the 5′ end of the viral RNA binds to the viral RdRp and facilitates template recognition at the 3′ end of the genome, which constitutes the initiation site, via long-range RNA-RNA interactions. Given that the SLA structure and the complementary sequences that mediate genome cyclization are conserved in all mosquito-borne flaviviruses, the mechanism proposed for dengue virus may be applicable to other members of the family.
The 5′ and 3′ untranslated regions (UTRs) of the dengue virus genome contain several RNA domains that act as cis-acting elements regulating viral replication. Using deletions and mutations in the context of infectious dengue virus RNAs, we have been dissecting specific roles of RNA sequences and structures present at the viral UTRs. These analyses allow identification of RNA structures within the viral 3′ UTR that are essential for viral RNA replication and elements that function as enhancers of the viral processes.
Recently we became interested in the molecular interactions that take place during dengue virus entry into the host cell. The interaction of viral envelope protein E with host cell receptor(s) directs dengue virus particles to the endocytic pathway. The acidic environment in the endosome is believed to produce major conformational changes in the E protein, changes that induce fusion of the viral and host cell membranes, which in turn releases the viral genome into the cytoplasm. The E protein bears several glycosylation sites, and interactions of the carbohydrates with cellular lectins enhance viral entry. We have been studying the relevance of each N-glycosylation site of the E protein during different stages of viral replication by removing individual sites in the context of infectious RNAs. Our results show that some glycosylation sites are important for viral infectivity and that certain glycans are also required during early steps of viral morphogenesis. In addition, we found the role of sugar moieties in the E protein to be different during dengue virus infection of mammalian and mosquito cells, highlighting the involvement of distinct host functions during the propagation of dengue virus.
Last updated September 2008
