Replication of positive strand flaviviruses is mediated by the viral RNA-dependent RNA polymerases (RdRP). To study replication of dengue virus (DEN), a flavivirus family member, an in vitro RdRP assay was established using cytoplasmic extracts of DEN-infected mosquito cells and viral subgenomic RNA templates containing 5-and 3-terminal regions (TRs). Evidence supported that an interaction between the TRs containing conserved stem-loop, cyclization motifs, and pseudoknot structural elements is required for RNA synthesis. Two RNA products, a template size and a hairpin, twice that of the template, were formed. To isolate the function of the viral RdRP (NS5) from that of other host or viral factors present in the cytoplasmic extracts, the NS5 protein was expressed and purified from Escherichia coli. In this study, we show that the purified NS5 alone is sufficient for the synthesis of the two products and that the template-length RNA is the product of de novo initiation. Furthermore, the incubation temperature during initiation, but not elongation phase of RNA synthesis modulates the relative amounts of the hairpin and de novo RNA products. A model is proposed that a specific conformation of the viral polymerase and/or structure at the 3 end of the template RNA is required for de novo initiation.The dengue virus, which is the causative agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome, is estimated to infect 100 million people per year worldwide (1-3). The virus is spread by the mosquito, Aedes agypti, which puts ϳ40% of the world at risk for dengue infection (1). Approximately 5% of infected individuals worldwide develop hemorrhagic or shock manifestations, which can commonly result in death (1). The dengue virus type 2 (DEN2) 1 is the most prevalent of the four dengue serotypes.The virus contains a positive strand, 5Ј-capped RNA, 10,723 nucleotides in length (for New Guinea-C strain; Ref. 4), which encodes a single polyprotein precursor, arranged in the order, C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5 (for a review, see Ref. 5). This precursor is processed in the endoplasmic reticulum by a combination of the signal peptidase and the viral serine protease to generate three structural proteins of the virion, C, prM, and E (6 -8) and at least seven nonstructural (NS) proteins.NS3, the second largest protein encoded by the virus, contains a serine catalytic triad within the N-terminal 180 amino acids, and it requires NS2B for protease activity (9 -18). The crystal structures of the protease domain alone and in complex with an inhibitor have been reported (19,20). However, the function of other nonstructural proteins in viral replication is poorly understood.NS3 also contains conserved motifs found in several NTPase/ RNA helicases (21-23). According to the current model, replication is initiated by the viral RNA-dependent RNA polymerase (RdRP) by synthesis of minus (Ϫ) strand to form a doublestranded RNA intermediate, which then serves as a template for genomic positive strand (24 -27). The vira...

By using a purified dengue virus RNA-dependent RNA polymerase and a subgenomic 770-nucleotide RNA template, it was shown previously that the ratio of the de novo synthesis product to hairpin product formed was inversely proportional to increments of assay temperatures (20 to 40°C). In this study, the components of the de novo preinitiation complex are defined as ATP, a high concentration of GTP (500 M), the polymerase, and the template RNA. Even when the 3-terminal sequence of template RNA was mutated from -GGUUCU-3 to -GGUUUU-3, a high GTP concentration was required for de novo initiation, suggesting that high GTP concentration plays a conformational role. Furthermore, utilization of synthetic primers by the polymerase indicated that AGAA is the optimal primer whereas AG, AGA, and AGAACC were inefficient primers. Moreover, mutational analysis of the highly conserved 3-terminal dinucleotide CU of the template RNA indicated that change of the 3-terminal nucleotide from U to C reduced the efficiency about fivefold. The order of preference for the 3-terminal nucleotide, from highest to lowest, is U, AϳG, and C. However, change of the penultimate nucleotide from C to U did not affect the template activity. A model consistent with these results is that the active site of the polymerase switches from a "closed" form, catalyzing de novo initiation through synthesis of short primers, to an "open" form for elongation of a double-stranded template-primer.Dengue viruses are members of the Flavivirus family of positive-strand RNA viruses. These viruses infect as many as 100 million individuals per year and are the causative agents of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome (24,35,53). Approximately 500,000 cases of dengue hemorrhagic fever are reported worldwide, with as many as 25,000 deaths annually (23). The dengue virus, the most prevalent tropical infectious agent after malaria, is spread by the mosquito Aedes Aegypti, which is a common day-biting mosquito in tropical climates (23,24). Epidemiological studies have demonstrated that dengue virus type 2 (DEN2) is the most prevalent of the four serotypes (DEN1 to -4).The genome of the New Guinea-C strain of DEN2 is 10,723 nucleotides long and contains a type 1 cap structure at the 5Ј terminus but lacks a poly(A) tail at the 3Ј end (30; for a review see reference 12). The genome encodes a single polyprotein, NH 2 -C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-COOH, which is processed within the endoplasmic reticulum (ER), producing the capsid (C), precursor membrane (prM) protein, and envelope protein (E) through cotranslational processing by the ER-resident signal peptidase (49,55,66). The C-terminal portion of the polyprotein, NS1 to NS5, is processed into at least seven nonstructural proteins in the ER by both the host protease(s) and the virally encoded serine protease, NS2B/NS3, of the trypsin family.NS3 is the second largest protein encoded by the DEN2 virus and contains at least three enzymatic properties. Contained within the N-terminal 180 amin...

No abstract

No abstract