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

Nomaguchi, Masako; Ackermann, Matt; Yon, Changsuek; You, Shihyun; Padmanabhan, Radhakrishnan

doi:10.1128/jvi.77.19.10730.2003

Cited by 13 publications

(23 citation statements)

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“…Structures in the 3'UTR, including the variable region (Alvarez et al, 2005; Chiu et al, 2005), dumbbell-shaped structures (DB1 and DB2) (Alvarez et al, 2005; Chiu et al, 2005) and 3' stem-loop (3'SL) (Holden and Harris, 2004; Holden et al, 2006; Tilgner et al, 2005; You et al, 2001; Yu and Markoff, 2005; Zeng et al, 1998) have been demonstrated to enhance both vRNA synthesis and translation. Both the 5'UTR and 3'UTR together have been shown to be required for translation when host cell cap-dependent translation has been shut off (Edgil and Harris, 2006; Edgil et al, 2006), in addition to playing a role in RNA synthesis (Filomatori et al, 2006b; Nomaguchi et al, 2003; Nomaguchi et al, 2004; You et al, 2001; You and Padmanabhan, 1999). The cHP, however, is the first element in the flavivirus coding region for which two distinct functions have been characterized: a role in translation start codon selection (Clyde and Harris, 2006) and, as shown here, a role in RNA synthesis.…”

Section: Discussionmentioning

confidence: 99%

“…The efficiency of first AUG selection is independent of the sequence of the cHP but is dependent on its position relative to the AUG and on its stability (Clyde and Harris, 2006). (B) Multiple structures at the 5' and 3' ends of flaviviruses have been shown to play an essential role in RNA synthesis (Alvarez et al, 2005; Filomatori et al, 2006a; Holden et al, 2006; Nomaguchi et al, 2003; Nomaguchi et al, 2004; Tilgner et al, 2005; You et al, 2001; You and Padmanabhan, 1999; Yu and Markoff, 2005; Zeng et al, 1998). The cHP element is also required for efficient RNA synthesis in a sequence-independent manner, possibly by forming part of the overall topology of the 5' end that initially binds the replicase complex or by stabilizing the cyclization interaction between the CS and UAR domains.…”

Section: Figures and Tablesmentioning

confidence: 99%

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The capsid-coding region hairpin element (cHP) is a critical determinant of dengue virus and West Nile virus RNA synthesis

2008

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Dengue virus (DENV) and West Nile virus (WNV) are members of the Flavivirus genus of positive-strand RNA viruses. RNA sequences and structures, primarily in the untranslated regions, have been shown to modulate flaviviral gene expression and genome replication. Previously, we demonstrated that a structure in the DENV coding region (cHP) enhances translation start codon selection and is required for viral replication. Here we further characterize the role of the cHP in the DENV life cycle. We demonstrate that the cHP is required for efficient viral RNA synthesis in a sequence-independent manner. Viruses with a disrupted cHP are rescued by a spontaneous compensatory mutation that restabilizes the structure. Furthermore, the cHP, which is predicted to be conserved among arthropod-borne flaviviruses, is required for WNV replication. We propose that the cHP is a multifunctional determinant of flavivirus replication, functioning in both translation and RNA synthesis.

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Section: Discussionmentioning

confidence: 99%

Section: Figures and Tablesmentioning

confidence: 99%

The capsid-coding region hairpin element (cHP) is a critical determinant of dengue virus and West Nile virus RNA synthesis

2008

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“…H798 forms stacking interactions with the initiating ATP nucleotide, and W795 binds a GTP molecule at the de novo GTP‐binding site (i‐1 position), which is located at a distance of 6–7 Å from the catalytic site . This GTP has been shown to be crucial for initiation, because the need of a high GTP concentration during de novo initiation has been observed, regardless of the nucleotide sequence of the template RNA . More precisely, this GTP nucleotide mimics a nascent RNA strand and interacts with the initiating ATP to stabilize it at the i position and thus to contribute to the proper positioning of its 3′ hydroxyl group which is involved in the formation of a phosphodiester bond with the second nucleotide at the i + 1 position .…”

Section: Nonstructural Proteinmentioning

confidence: 99%

Organization of theFlavivirusRNAreplicase complex

2017

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Flaviviruses, such as Dengue, Japanese encephalitis, West Nile, Yellow Fever, and Zika viruses, are serious human pathogens that cause significant morbidity and mortality globally each year. Flaviviruses are single-stranded, positive-sense RNA viruses, and encode two multidomain proteins, NS3 and NS5, that possess all enzymatic activities required for genome replication and capping. NS3 and NS5 interact within virus-induced replication compartments to form the RNA genome replicase complex. Although the individual enzymatic activities of both proteins have been extensively studied and are well characterized, there are still gaps in our understanding of how they interact to efficiently coordinate their respective activities during positive strand RNA synthesis and capping. Here, we discuss what is known about the structures and functions of the NS3 and NS5 proteins and propose a preliminary NS3:NS5:RNA interaction model based on a large body of literature about how the viral enzymes function, physical restraints between NS3 and NS5, as well as critical steps in the replication process.

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“…A second in vitro RdRP assay system relies on exogenously added subgenomic RNA templates containing the 5′- and 3′-terminal regions including the cyclization sequences (5′- and 3′-CS1 RNA elements) and infected cell lysates (You and Padmanabhan, 1999; You et. al., 2001) or purified NS5 protein (Tan et al,1996; Ackermann and Padmanabhan, 2001; Guyatt et al, 2001; Nomaguchi et. al., 2003; 2004; Yu, L. et.…”

Section: Targeting Critical Functions Of Individual Flaviviral Prmentioning

confidence: 99%

Molecular targets for flavivirus drug discovery

2009

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Flaviviruses are a major cause of infectious disease in humans. Dengue virus causes an estimated 50 million cases of febrile illness each year, including an increasing number of cases of hemorrhagic fever. West Nile virus, which recently spread from the Mediterranean basin to the Western Hemisphere, now causes thousands of sporadic cases of encephalitis annually. Despite the existence of licensed vaccines, yellow fever, Japanese encephalitis and tick-borne encephalitis also claim many thousands of victims each year across their vast endemic areas. Antiviral therapy could potentially reduce morbidity and mortality from flavivirus infections, but no effective drugs are currently available. This article introduces a collection of papers in Antiviral Research on molecular targets for flavivirus antiviral drug design and murine models of dengue virus disease that aims to encourage drug development efforts. After reviewing the flavivirus replication cycle, we discuss the envelope glycoprotein, NS3 protease, NS3 helicase, NS5 methyltransferase and NS5 RNA-dependent RNA polymerase as potential drug targets, with special attention being given to the viral protease. The other viral proteins are the subject of individual articles in the journal. Together, these papers highlight current status of drug discovery efforts for flavivirus diseases and suggest promising areas for further research.

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

Cited by 13 publications

References 0 publications

The capsid-coding region hairpin element (cHP) is a critical determinant of dengue virus and West Nile virus RNA synthesis

The capsid-coding region hairpin element (cHP) is a critical determinant of dengue virus and West Nile virus RNA synthesis

Organization of theFlavivirusRNAreplicase complex

Molecular targets for flavivirus drug discovery

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