Overlapping Local and Long-Range RNA-RNA Interactions Modulate Dengue Virus Genome Cyclization and Replication

Abstract

The dengue virus genome is a dynamic molecule that adopts different conformations in the infected cell. Here, using RNA folding predictions, chemical probing analysis, RNA binding assays, and functional studies, we identified new cis-acting elements present in the capsid coding sequence that facilitate cyclization of the viral RNA by hybridization with a sequence involved in a local dumbbell structure at the viral 3= untranslated region (UTR). The identified interaction differentially enhances viral replication in mosquito and mammalian cells. Dengue virus (DENV) is a member of the Flaviviridae family that includes other important pathogens such as yellow fever virus (YFV), West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), and Japanese encephalitis virus (JEV). The DENV genome is a plus-stranded RNA molecule that contains a single open reading frame flanked by highly structured 5= and 3= untranslated regions (UTRs) (1-3). RNA elements located within these regions are responsible for translation initiation and genome replication (4-7). The 5= UTR is about 100 nucleotides (nt) long and includes three different elements: (i) stem-loop A (SLA), which is the promoter for viral polymerase binding and activation (8-10); (ii) stem-loop B (SLB), which contains a sequence known as 5= upstream of the AUG region (5= UAR) that is complementary to a sequence present at the 3= UTR (3= UAR) and mediates longrange RNA-RNA interactions between the ends of the genome (11); and (iii) a spacer sequence between SLA and SLB rich in U's, which functions as an enhancer of viral replication (10). The viral 3= UTR is about 450 nucleotides long and comprises four defined domains: domain A1, which features a variable region (VR) (12); domains A2 and A3, which present two almost-identical dumbbell-like secondary structures (DB1 and DB2), which appear to work as enhancers for viral RNA replication (13-15); and domain A4, which contains a small hairpin (sHP) and the 3= stem-loop (3= SL), which are essential elements for viral replication (3,16). In addition to RNA structures defined in the UTRs that play different roles during infection, important RNA elements have been described in the protein coding region. In this regard, essential sequences that mediate long-range RNA-RNA interactions known as 5= cyclization sequence (5= CS) and 5= downstream of AUG region (5= DAR) are located within the capsid coding sequence (11,13,(17)(18)(19)(20)(21). Also, a hairpin known as cHP, located between 5= CS and 5= DAR, has been shown to be necessary for efficient RNA replication (22). The current model for viral RNA synthesis includes the interaction of the viral polymerase NS5 with the 5=-end SLA promoter and its transfer to the 3=-end initiation site by cyclization of the viral genome (9). Despite great advances in knowledge of cis-acting RNA elements in the flavivirus genomes, the molecular details and mechanisms by which many of them function during viral replication are still not well understood.Intrigued by dual roles of RNA sequences in v...

In-cell SHAPE uncovers dynamic interactions between the untranslated regions of the foot-and-mouth disease virus RNA

“…As shown for Dengue virus, RNA sequences involved in alternative long-range structures could constitute a mechanism to modulate the viral RNA 3D structure for efficient replication (75). In summary, our strategy comparing in-cell to in vitro SHAPE probing of functional monocistronic transcripts and in silico prediction of conserved helices revealed the conformational plasticity of the FMDV 3΄UTR region, and provided direct evidences for dynamic long-range interactions between this region and the IRES element.…”

RNA Structure Duplications and Flavivirus Host Adaptation

“…Regarding the DB elements, sequences involved in DB1 and DB2 formation were reported to play opposite roles in flavivirus genome cyclization. While DB1 bears nucleotides that are complementary to sequences present at the 5’ end of the genome that enhance long range RNA-RNA interactions and genome cyclization [34, 36], the sequence at the top-loop of DB2 competes with genome cyclization by forming a PK with nucleotides that overlap with 3’CS, one of the most conserved cyclization element among flaviviruses [32–34]. These observations support a diversification of function between the two SLs and between the two DBs.…”

“…Instead of a redundant function, as it was originally proposed, each of the duplicated RNA elements appear to play distinct roles during viral replication [31–34], and in some cases are directly linked to specific requirements in different host.…”

Functional Information Stored in the Conserved Structural RNA Domains of Flavivirus Genomes

“…Recently, a cis -acting element present in the capsid coding sequence of DENV was found to interact with 5′DB at the 3′UTR, forming a PK structural element. This interaction was proven to have a different effect on viral RNA replication in mosquito and mammalian cells (de Borba et al, 2015). …”