Structural and functional analysis of dengue virus RNA

Diego E. Alvarez, Maria F. Lodeiro, Claudia V. Filomatori, Silvana Fucito, Juan A. Mondotte and Andrea V. Gamarnik1

Fundación Instituto Leloir, Patricias Argentinas 435, Buenos Aires, Argentina

Abstract. Sequences and structures present at the 5' and 3' UTRs of RNA viruses play crucial roles in the initiation and regulation of translation, RNA synthesis and viral assembly. In dengue virus, as well as in other mosquito-borne flaviviruses, the presence of complementary sequences at the ends of the genome mediate long-range RNA—RNA interactions. Dengue virus RNA displays two pairs of complementary sequences (CS and UAR) required for genome circularization and viral viability. In order to study the molecular mechanism by which these RNA—RNA interactions participate in the viral life cycle, we developed a dengue virus replicon system. RNA transfection of the replicon in mosquito and mammalian cells allows discrimination between RNA elements involved in translation and RNA synthesis. We found that mutations within CS or UAR at the 5' or 3' ends of the RNA that interfere with base pairing did not significantly affect translation of the input RNA but seriously compromised or abolished RNA synthesis. Furthermore, a systematic mutational analysis of UAR sequences indicated that, beside the role in RNA cyclization, specific nucleotides within UAR are also important for efficient RNA synthesis.

2006 New treatment strategies for dengue and other flaviviral diseases. Wiley, Chichester (Novartis Foundation Symposium 277) p 120—135

The genome-length RNA of dengue virus is infectious. Delivery of this RNA molecule into a susceptible cell triggers a complete round of viral replication. Once in the cytoplasm of the host cell, the viral genome participates in at least three different processes: it serves as mRNA to direct the synthesis of viral proteins, it acts as template for genome amplification, and it is packaged along with structural proteins during viral assembly. The molecular mechanisms controlling the utilization of the viral RNA in each step of the viral life cycle are still poorly understood. Because the 5' and 3' ends of the viral RNA are the places where translation initiation and synthesis of positive and negative strand RNA occur, we are interested

1 This paper was presented at the symposium by Andrea Gamarnik, to whom correspondence should be addressed.

in investigating the function of RNA structures and sequences located at the ends of the viral RNA.

The genome of dengue and other flaviviruses is about 11 kb long and encodes one open reading frame flanked by 5' and 3' untranslated regions (UTRs) (Rice 2001). The 5' UTR of dengue virus is around 100 nucleotides long and the sequence conservation is almost complete among different dengue virus serotypes (Markoff 2003). The predicted structure consists of a large stem loop (SLA) and a second short stem loop (SLB) containing at the 3'-terminal sequences the translation initiation codon (Fig. 1). It is likely that these sequences and structures influence translation initiation, which presumably takes place by a cap dependent scanning mechanism. Another major function of the 5' UTR probably resides in the negative strand, which serves as a site for positive strand RNA synthesis. Deletions engineered into the 5' UTR of dengue virus 4 were lethal (Cahour et al 1995), suggesting an important role of these RNA structures in viral replication.

The 3' UTR of dengue virus is around 450 nucleotides, lacks a poly(A) tail, but contains a number of conserved RNA structures (Fig. 1) (Shurtleff et al 2001). The viral genome ends in a very conserved 3' stem loop (3' SL). Detailed analysis of the structure-function of the 3' SL in West Nile virus, Kunjin virus, dengue virus and yellow fever virus revealed an absolute requirement of this RNA element for viral replication (Brinton et al 1986, Zeng et al 1998, Rauscher et al 1997, Proutski et al 1997, Men et al 1996, Yu & Markoff 2005, Tilgner et al 2005, Elgho-nemy et al 2005). Upstream of the 3' SL there is another essential RNA element for viral replication, the conserved sequence CS1 (Men et al 1996). This element contains the CS sequence, which is complementary to a sequence present within the coding region of protein C at the 5' end of the genome (Hahn et al 1987). 5'—3' long-range RNA—RNA interactions through these complementary sequences have been proposed to be necessary for replication of different mosquito-borne

FIG. 1. Schematic representation of dengue virus genome. The predicted secondary structures of defined domains at the 5' and 3' UTR are indicated: stem loop A (SLA), stem loop B (SLB), variable region (VR), domain A2, domain A3, and the 3' stem loop (3'SL). Also, the conserved sequences CS, CS2 and RCS2 are shown.

5'UTR 3'UTR

FIG. 1. Schematic representation of dengue virus genome. The predicted secondary structures of defined domains at the 5' and 3' UTR are indicated: stem loop A (SLA), stem loop B (SLB), variable region (VR), domain A2, domain A3, and the 3' stem loop (3'SL). Also, the conserved sequences CS, CS2 and RCS2 are shown.

flaviviruses (Alvarez et al 2005b, 2005a, Lo et al 2003, Khromykh et al 2001, Corver et al 2003). In addition, 5'—3' CS base pairing has also been reported to be important for in vitro activity of dengue and West Nile virus RNA polymerases (You et al 2001, Nomaguchi et al 2004). The mechanism by which the flavivirus replicase machinery initiates RNA synthesis specifically at the viral 3' UTR is still not clearly understood. The RNA replication complex assembles on cellular membranes and involves the viral RNA dependent RNA polymerase-methyltransferase NS5, the helicase-protease NS3, the glycoprotein NS1, the hydrophobic proteins NS2A and NS4A, and presumably host factors (Westaway et al 1999, 1997, Mackenzie et al 1998).

In addition to the 3' SL, other RNA structures and conserved motifs are present within dengue virus 3' UTR. Folding algorithms predicts two almost identical structures designed A2 and A3 preceding the 3' SL. Recent experiments using recombinant dengue virus 2 carrying deletions of domain A2 and/or A3 showed viral attenuation with defects in RNA synthesis, suggesting an important role of these RNA structures in viral replication (Alvarez et al 2005a). Within domains A3 and A2 there are highly conserved regions known as CS2 and repeated CS2 (RCS2), respectively (Shurtleff et al 2001). A recombinant virus with a deletion of 30 nucleotides between CS2 and RCS2 is currently under study as a dengue virus vaccine candidate (Durbin et al 2001). CS2 and RCS2 sequences can be found in Japanese encephalitis virus, West Nile virus, Murray Valley encephalitis virus, and dengue virus types 1 to 4 (for review see Markoff 2003), suggesting a conserved function of these elements in flavivirus replication. However, it is not clear the mechanisms by which these RNA structures participate in viral replication.

Here, we discuss the nature and requirements of long-range RNA—RNA interactions in the viral genome during dengue virus replication. Using genomic and subgenomic dengue virus RNAs together with biochemical tools, we analysed the role of secondary and tertiary structures of the viral RNA during translation and RNA synthesis.

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