Conformational Changes in the Solution Structure of the Dengue Virus 5′ End in the Presence and Absence of the 3′ Untranslated Region

Abstract

Dengue virus (DENV) is an ϳ10.7-kb positive-sense RNA virus that circularizes via RNA-RNA interactions between sequences in the 5 and 3 terminal regions. Complementarity between the cyclization sequence (CS) and the upstream AUG region (UAR) has been shown to be necessary for viral replication. Here, we present the solution structure of the 5 end of DENV type 2 in the presence and absence of the 3 end. We demonstrate that hybridization between the 5 and 3 CSs is independent of the UAR while the 5 UAR-3 UAR hybridization is dependent upon the 5 CS-3 CS interaction.Dengue virus (DENV) is a mosquito-borne, positivestranded RNA virus within the Flavivirus genus. The four serotypes (DENV1 to DENV4) cause major global public health problems, with tens of millions of dengue fever cases and hundreds of thousands of cases of life-threatening dengue hemorrhagic fever/dengue shock syndrome annually. The 10.7-kb viral RNA is translated into a single polyprotein, which is subsequently cleaved into three structural and seven nonstructural proteins by viral and cellular proteases. The open reading frame is flanked by an ϳ100-nucleotide (nt) 5Ј untranslated region (UTR) which contains a type I cap, and an ϳ450-nt 3Ј UTR lacking a poly(A) tail. The 5Ј and 3Ј ends interact via RNA-RNA hybridization of two regions, the cyclization sequence (CS) (6) and the upstream AUG region (UAR) (1), resulting in circularization of the viral genome. The CS has previously been shown to be crucial for DENV replication and RNA synthesis (18,19), and the UAR is proposed to play a similar role (1-3). RNA folding algorithms predict conserved secondary structures in the Flavivirus 5Ј UTR, and the CS is a highly conserved sequence among flaviviruses, suggesting an important function for these elements (4, 16). Both the CS and the UAR have recently been shown to play a role in 5Ј-to-3Ј end-to-end interaction and replication of the related West Nile virus (8,20,21).To define the solution structure of the 5Ј end and its interaction with the 3Ј UTR, we chemically and enzymatically probed the DENV2 5Ј end alone or hybridized to the 3Ј UTR. We resolved the structure of the 96-nt 5Ј UTR, as well as the 5Ј UTR including 55 nt of the capsid gene (5ЈUTRϩ55). The additional 55 nt in the coding region contain the CS region and the capsid hairpin (cHP), previously shown to be involved in viral translation start site selection (7) and RNA synthesis (6). Sequences were derived from the infectious clone of the prototype DENV2 strain 16681 (pD2/IC-30P-A; GenBank accession no. U87411) (11) and cloned into Litmus 28 (New England BioLabs, Beverly, MA) to generate the 5Ј UTR (104 nt, including AUG and an AflIII overhang) or 5ЈUTRϩ55 (151 nt, including an MluI overhang) downstream of the T7 RNA polymerase promoter. A predicted consensus structure for the 5Ј end of DENV1 to DENV4 (DENV1, GenBank accession no. U88536; DENV2, accession no. U87411; DENV3, accession no. M93130; DENV4, accession no. AF326825), corresponding to 5ЈUTRϩ55, was obtained by multiple alignment using CL...

Identification of Cis-Acting Elements in the 3′-Untranslated Region of the Dengue Virus Type 2 RNA That Modulate Translation and Replication

“…1 and supplemental Table 2). This is consistent with the observation that the 5Ј-3Ј-UAR binding occurs only after the 5Ј-3Ј-CS1 circularization has been established (73), suggesting that the latter is the primary driving force in genomic end-to-end communication. This conclusion is also supported by a stem trace plot of the minigenome RNA maturation in an MPGAfold run (data not shown).…”

Circularization of the HIV-1 genome facilitates strand transfer during reverse transcription

“…Circularization of flavivirus genomes was proposed to be mediated by direct RNA-RNA interactions. For the mosquito borne flaviviruses, the 39 UTR was found to interact with sequences in the capsid coding region near the 59 end of the genome (Hahn et al 1987;Khromykh et al 2001;Corver et al 2003;Alvarez et al 2005;Polacek et al 2009). The hepatitis C virus genome was also reported to circularize by a long-range RNA-RNA interaction between the 59 and 39 ends of the genome (Romero-Lopez and Berzal-Herranz 2009).…”

“…Alternatively, some viruses have developed RNA motifs located at both ends of the genome with complementary sequences that promote protein-independent circularization of the viral RNA. This is the case for flaviviruses (Hahn et al 1987;Khromykh et al 2001;Alvarez et al 2005;Polacek et al 2009), hepatitis C virus (Romero-Lopez and Berzal-Herranz 2009), and certain picornaviruses, such as FMDV (Serrano et al 2006), among others. Genome circularization is essential for replication of these positivestranded RNA viruses, and plays an important role in processes such as replication, transcription, and translation of the viral RNA genome.…”

A long-range RNA–RNA interaction between the 5′ and 3′ ends of the HCV genome

“…Alternatively, some viruses have developed RNA motifs located at both ends of the genome with complementary sequences that promote the protein-independent circularization of the mRNA. This is the case of flavivirus , certain picornaviruses, such as FMDV (Serrano et al 2006), retroviruses (Ooms et al 2007;Kenyon et al 2008), or Dengue virus (Polacek et al 2009), among others. With respect to HCV, conflicting results regarding the role of the 39 UTR in viral protein synthesis have been reported (Ito et al 1998;Ito and Lai 1999;Fang and Moyer 2000;Michel et al 2001;Murakami et al 2001;Kong and Sarnow 2002;McCaffrey et al 2002;Imbert et al 2003;Bradrick et al 2006;Song et al 2006;Lourenco et al 2008).…”