Molecular Biology of Dengue Virus

IGLESIAS NG.; BYK L.; GAMARNIK A.

Dengue Fever and Dengue Hemorrhagic Fever

CABI, Nosworthy Way, Wallingford OX10 8DE

Año: 2014; p. 342 - 366

Dengue virus (DENV) isa major arthropod-borne pathogen causing a significant burden of disease intropical and subtropical areas of the world (Beatty et al., 2009). About 3.6billion people are currently living in areas at risk. It is estimated more than400 million infections and 100 million symptomatic cases of DENV annually(Bhatt et al., 2013). DENV is a positive-sense RNAvirus, belonging to the Flavivirus genus of the Flaviviridae family (Lindenbachet al., 2007) The Flavivirus genus comprises 53 species, from which 40 areimportant human pathogens including West Nile virus (WNV), Japaneseencephalitis virus (JEV), yellow fever virus (YFV), and tick-borne encephalitisvirus (TBEV). In recent years a great deal of research on DENV biology hasprovided an enormous amount of information about molecular aspects of itsreplication. Despite this great effort there are no therapeutics approvedagainst DENV. The DENV genome is infectious; transfectionof the full-length viral RNA into a susceptible cell results in a completecycle of viral replication. A great advance in our knowledge of DENV molecularbiology has been possible by the construction of full-length cDNA clones of thefour serotypes (Blaney et al., 2004, Gualano et al., 1998, Kapoor et al., 1995a,Kinney et al., 1997, Lai et al., 1991, Polo et al., 1997, Puri et al., 2000,Sriburi et al., 2001, Suzuki et al., 2007). These infectious clones have beenused to genetically manipulate different regions of the viral RNA to definefunctions of viral proteins and RNA structures, to study virus-cellinteractions, and to generate live attenuated vaccine candidates. An importantadvance has been the development of genomic and subgenomic DENV RNAs containingreporter genes, such as luciferase or GFP (Mondotte et al., 2007, Samsa et al.,2009, Schoggins et al., 2012, Zou et al., 2011). These tools have been used todissect functions of viral proteins and RNAs in each step of the viral lifecycle (Samsa et al., 2012). Subgenomic RNAs, also known as replicons, contain areporter gene replacing one or all of the structural proteins (capsid,pre-membrane prM, and envelope E) (Alvarez et al., 2005a, Jones et al., 2005,Alcaraz-Estrada et al., 2010, Ng et al., 2007, Qing et al., 2010). Thereplicons are useful to evaluate translation and viral RNA amplification, whichcan be followed by measuring reporter activity as a function of time after RNAtransfection. Cell lines stably expressing DENV replicons have also beendeveloped. Full-length reporter viruses have the additional ability ofgenerating new viral particles still encoding the reporter gene that can beused for subsequent infections (Mondotte et al., 2007, Samsa et al., 2009).Reporter containing viruses have been employed to study viral functions andhost-virus interactions during viral entry, viral translation, RNA synthesis,and formation of infectious viral particles. Replicons may also be packagedinto virus like particles (VLPs) by providing the structural proteins in trans.This trans packaging system was first used for Kunjin virus (KUNV) and thenextended to other flaviviruses including DENV (Suzuki et al., 2009, Scholle etal., 2004, Gehrke et al., 2003, Khromykh et al., 1998, Lai et al., 2008). Dueto the high error rate of the viral polymerase, a valuable tool to definefunctions in the viral genome is the selection, in cell culture, of spontaneousnucleotide changes restoring a function impaired by an artificially introducedmutation. Sequencing of these selected viruses provides a great deal of informationabout genetic interactions between different parts of the genome and activitiesof conserved RNA structures (Lodeiro et al., 2009, Teramoto et al., 2008,Villordo et al., 2010). Furthermore, utilization of modern microscopictechniques, including electron microscopy (EM), electron tomography (ET) andtransmission EM (TEM), together with classical virology, allowed great advancesin understanding how cellular membranous structures are modified by DENVinfection (Mackenzie et al., 1996, Welsch et al., 2009).This chapter willexamine the multiple functions of the DENV genome, not only as a codingmolecule for viral protein synthesis but also as a main regulator of viralprocesses. Recent advances on functions of viral RNAstructures, studies on the biogenesis and architecture of replication complexesand mechanistic studies about viral RNA replication are the topics that will behighlighted.