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Functional RNA Elements in the Dengue Virus Genom Featured

Leopoldo G. Gebhard, Claudia V. Filomatori and Andrea V. Gamarnik

Abstract: Dengue virus (DENV) genome amplification is a process that involves the viral RNA, cellular and viral proteins, and a complex architecture of cellular membranes. The viral RNA is not a passive template during this process; it plays an active role providing RNA signals that act as promoters, enhancers and/or silencers of the replication process. RNA elements that modulate RNA replication were found at the 5' and 3' UTRs and within the viral coding sequence. The promoter for DENV RNA synthesis is a large stem loop structure located at the 5' end of the genome. This structure specifically interacts with the viral polymerase NS5 and promotes RNA synthesis at the 3' end of a circularized genome. The circular conformation of the viral genome is mediated by long range RNA-RNA interactions that span thousands of nucleotides. Recent studies have provided new information about the requirement of alternative, mutually exclusive, structures in the viral RNA, highlighting the idea that the viral genome is flexible and exists in different conformations. In this article, we describe elements in the promoter SLA and other RNA signals involved in NS5 polymerase binding and activity, and provide new ideas of how dynamic secondary and tertiary structures of the viral RNA participate in the viral life cycle. (Viruses 2011, 3, 1739-175)

Dengue virus (DENV) is a member of the Flavivirus genus of the Flaviviridae family [1]. The Flavivirus genus includes other important human pathogens such as yellow fever (YFV), West Nile (WNV), Japanese encephalitis (JEV), and tick borne encephalitis (TBEV) [1]. Flaviviruses are enveloped viruses with a single stranded, ~11 kb, positive-sense RNA genome. The genome encodes a single long open reading frame (ORF), flanked by highly structured 5' and 3' untranslated regions (UTRs). The virus enters the host cell by receptor mediated endocytosis. Upon internalization and acidification of the endosome, fusion of viral and vesicular membranes allows release of the genomic RNA into the cytoplasm, which serves as mRNA. Translation of the single ORF at the rough ER produces a large polyprotein that is cleaved co- and posttranslationally into the mature proteins. The N-terminal of the polyprotein encodes the three structural proteins (C-prM-E), followed by at least seven non-structural (NS) proteins (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5) (Figure 1A) [2]. After translation of the RNA, virus-induced hypertrophy of intracellular membranes occurs, originating structures known as convoluted membranes and vesicle packets [3–5]. Flavivirus RNA synthesis occurs in close association with cellular membranes inside the vesicle packets in so-called viral replication complexes. The process begins with the synthesis of a negative strand RNA, which serves as template for the amplification of additional positive strand genomic RNA. The enzymatic reaction is catalyzed by the RNA-dependent RNA polymerase (RdRp) activity of the viral NS5 protein, in association with the viral protease/helicase NS3, other viral NS proteins, and presumably host factors. The newly synthesized RNA associates to the capsid (C) protein by a mechanism still unknown. The RNA-C complex buds into the ER lumen acquiring the lipid bilayer, and the viral E and prM proteins. Furin-mediated proteolysis of prM in the trans-Golgi network [6] triggers rearrangement, homodimerization of E, and formation of new viral particles [7].

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