Structures of flavivirus RNA promoters suggest two binding modes with NS5 polymerase

Abstract

Flaviviruses use a ~70 nucleotide stem-loop structure called stem-loop A (SLA) at the 5′ end of the RNA genome as a promoter for RNA synthesis. Flaviviral polymerase NS5 specifically recognizes SLA to initiate RNA synthesis and methylate the 5′ guanosine cap. We report the crystal structures of dengue (DENV) and Zika virus (ZIKV) SLAs. DENV and ZIKV SLAs differ in the relative orientations of their top stem-loop helices to bottom stems, but both form an intermolecular three-way junction with a neighboring SLA molecule. To understand how NS5 engages SLA, we determined the SLA-binding site on NS5 and modeled the NS5-SLA complex of DENV and ZIKV. Our results show that the gross conformational differences seen in DENV and ZIKV SLAs can be compensated by the differences in the domain arrangements in DENV and ZIKV NS5s. We describe two binding modes of SLA and NS5 and propose an SLA-mediated RNA synthesis mechanism.

The Role of the Stem-Loop A RNA Promoter in Flavivirus Replication

“…The NS5 RdRp and SLA interaction via the thumb subdomain of RdRp was also suggested by a refined yeast three-hybrid scan [39]. Thus, a model of the NS5 and SLA complex, wherein the 5 terminus of SLA binds the MTase active site and the top stem-loop binds in the thumb subdomain of RdRp was proposed [34]. This model allows the 3 end of viral genome to concomitantly bind to the template-binding channel of NS5 without the need of additional conformational change to accommodate the 3 end of the genome following genome cyclization (Figures 1C and 4B).…”

“…The structures show base pairing patterns near the 3-way junction (where the top stem-loop, side loop, and the bottom stem meet) that differ from their predicted secondary structures. In particular, self-complementary side loop sequences are involved in the intermolecular interactions with the side loop of another SLA molecule, rather than participating in the predicted intramolecular base-pair interaction [34]. Further, it was shown that SLA forms a dimer in solution, consistent with the structure, though the function of dimerization is currently unclear [34].…”

“…Recent crystal structures of DENV and ZIKV SLAs show that both SLAs consist of a top stem-loop, a side loop, and a bottom stem (Figure 3). Both SLAs form a letter 'L'-shaped structure with a different angle between the top and bottom stems of the L [34]. The DENV and ZIKV SLA structures differ in the lengths of the bottom stem and top stem-loop, and their relative orientations (Figure 3C,D).…”

“…In particular, self-complementary side loop sequences are involved in the intermolecular interactions with the side loop of another SLA molecule, rather than participating in the predicted intramolecular base-pair interaction [34]. Further, it was shown that SLA forms a dimer in solution, consistent with the structure, though the function of dimerization is currently unclear [34]. Mutational studies have identified nucleotides in SLA that are important for viral replication.…”

Dengue Virus Infection: A Tale of Viral Exploitations and Host Responses

“…The vesicle packets contain NS5 (RdRp), NS3 (possessing enzymatic properties: helicase), 5′ RNA-triphosphatase (RTP), a nucleoside triphosphatase (NTPase), and other viral NS proteins in addition to the host factors that catalyze RNA replication, collectively known as the replication complex [ 11 ]. The SLA at 5′UTR acts as a promoter for NS5, the viral RdRp [ 24 ]. The relocation of NS5 to the 3′end is facilitated by cyclization of the genome for the initiation of RNA synthesis.…”