De Novo Synthesis of Negative-Strand RNA by Dengue Virus RNA-Dependent RNA Polymerase In Vitro: Nucleotide, Primer, and Template Parameters

Abstract

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...

Requirements for West Nile Virus (–)- and (+)-Strand Subgenomic RNA Synthesis in Vitro by the Viral RNA-dependent RNA Polymerase Expressed in Escherichia coli

“…The de novo initiation events required incubation of the components, the (ϩ)-strand template RNA, NS5, ATP, and GTP at lower temperatures. Once the heparin-resistant, de novo initiation complex is formed, then 3Ј-elongation was insensitive to temperature variations and the de novo product was preferentially formed (40,53). In the context of viral infection, only the de novo synthesized RNA by the viral replicase is physiologically important to faithfully replicate viral RNA through multiple rounds.…”

“…In the context of viral infection, only the de novo synthesized RNA by the viral replicase is physiologically important to faithfully replicate viral RNA through multiple rounds. Using the in vitro system described here for WNV and for DEN2 described in previous studies (25,40,41,53), it may be possible to delineate other parameters that modulate the de novo synthesis of viral RNA in vitro.…”

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

“…This sequence-specific requirement of PK1 in the 3Ј-CS1 and its complementary sequence in the 5Ј-CS in replication is not yet understood. Moreover, the in vitro RdRP assays using (ϩ)-strand templates containing WT and 5Ј-and 3Ј-DB mutant sequences and purified full-length DENV2 NS5 polymerase (54,59) revealed that the minus strand synthesis was not affected by these mutations (Fig. 7).…”

“…Previous studies have shown that this RNA molecule contains the essential cis-acting elements required for efficient translation (15,29,30,58) as well as negative strand RNA synthesis in vitro (10,41,54,59). MPGAfold utilizes the genetic operators of mutation, recombination, and selection in parallel on a high performance computer.…”

“…On the right arm of the DBs are duplicated conserved sequences RCS2 and CS2. C, a minigenome of DENV2 RNA sequence containing both 5Ј-and 3Ј-UTR sequences and other essential elements for RNA synthesis in vitro (10,54) was used for analysis of secondary structures by MPGAfold as described under "Experimental Procedures." Based on this analysis, a stable best fit structure (E ϭ Ϫ226.2 kcal/mol) has only the 3Ј-DB, whereas the appearance of the 5Ј-DB is visible in metastable structures such as in D (Ϫ225.6 kcal/mol).…”