Dengue viruses are members of the Flaviviridae family (genus Flavivirus), which are small RNA viruses of 10–11 Kb in length with capped non-polyadenylated positive strand genomes. Dengue virus proteins are produced from a single open reading frame via translation of the genomic viral RNA as a single polyprotein that is co-translationally processed into 3 structural proteins (Capsid, prM, and Envelope) and 8 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, 2K, NS4B, and NS5). The non-structural proteins are responsible for directing viral genomic RNA replication, including synthesizing negative- and positive-strand RNAs and forming the viral RNA cap structure.
The flavivirus RNA cap is critical for viral polyprotein translation and RNA replication. The RNA cap allows the viral RNA to be efficiently translated by the cellular translational machinery and provides protection for the genome from cellular exonucleases. Flavivirus genomic RNA replication occurs on rough endoplasmic reticulum membranes in membranous compartments away from the cellular capping machinery, requiring the viruses to develop a mechanism for generating an RNA cap structure. Dengue and other flaviviruses have evolved a complete RNA capping machinery to form an RNA cap on the 5′ end of the positive-strand genomic RNA. Cellular RNA cap structures are formed via the action of an RNA triphosphatase (RTPase), guanylyltransferase (GTase), N7-methyltransferase (N7-MTase), and 2′-O methyltransferase (2′O-MTase) 
. Flavivirus genomic RNA is modified at the 5′ end of positive strand genomic RNA with a cap 1 structure (me7
) generated by the virus encoded RTPase (NS3), GTase (NS5), 2′-OMTase (NS5), and Guanine-N7-MTase (NS5) 
. X-ray crystal structures for each of these viral enzymes have been solved 
providing a wealth of information about how these enzymes may function. The RTPase resides within the helicase domain of NS3 and appears to utilize the helicase ATP hydrolysis site to remove the γ-phosphate from the 5′ end of the RNA 
. The NS5 N-terminal capping enzyme domain (dengue virus NS5 AA 1–265) possesses the 2′-O-MTase, Guanine-N7-MTase, and GTase activities and the NS5 C-terminal domain possesses the RNA dependent RNA polymerase 
During the GTase reaction, the NS5 N-terminal capping enzyme must bind to the 5′ end of the viral RNA. The GTase reaction uses two substrates, a covalently bound guanosine monophosphate (GMP) and the diphosphorylated 5′ end of the viral genomic RNA, to form the cap 0 structure (5′ GpppAGUAA…). We and others have studied how the capping enzyme binds GTP 
and the RNA requirements for cap methylation have been explored 
, but there is no empirical evidence for how the protein binds the uncapped diphosphorylated RNA substrate for the GTase reaction. The current location of the RNA binding region has been suggested based on the presence of basic residues and in silico
molecular dynamics docking of an RNA into the crystal structure of the dengue capping enzyme 
. A recent structure of the dengue virus type 3 capping enzyme in complex with an octomeric capped RNA demonstrated interactions between the guanosine cap structure and the capping enzyme showed no interactions between the RNA and the capping enzyme putative RNA binding region 
. This structure may represent the post-capping product, but does not shed light onto how the capping enzyme may bind diphosphorylated RNA during capping. The flavivirus NS5 capping enzyme does not encode a canonical Kx[D/N]G motif or any other known GTase motifs 
. Since the flavivirus capping enzyme is able to form a guanylated intermediate (a GMP linked to the protein via a phosphoamide bond) and transfer GMP to a diphosphorylated RNA 
, it stands to reason that the capping enzyme must have a non-canonical GTase motif. Understanding how the capping enzyme binds its diphosphorylated RNA substrate is critical for deciphering how this non-canonical GTase functions, but at this point how it binds diphosphorylated RNA is unclear.
In this manuscript we examine the binding of the viral 5′ diphosphorylated RNA substrate to the dengue virus capping enzyme. We developed a fluorescence polarization-based RNA binding assay to monitor the association of a short diphosphorylated RNA corresponding to the conserved 5′ end of the flavivirus genome and determined the RNA binding affinity to the capping enzyme. We assessed the effects of the various ligands used by the capping enzyme on RNA binding affinity, and determined that binding is negatively affected by GTP and ATP and positively affected by SAM. We also performed a structure-directed mutational analysis of the dengue 2 capping enzyme to determine which amino acids may be involved with RNA binding based on the structural similarity of the dengue virus capping enzyme with the Vaccinia virus VP39 methyltransferase protein bound to RNA. We identified several residues that are critical for binding to RNA and report their relative contribution to binding. We have also explored the contribution of the 5′ phosphates to RNA binding and found that the 5′ β- and α- phosphates are critical for diphosphorylated RNA binding to the capping enzyme.