scholarly journals Inhibition of coxsackievirus B3 and related enteroviruses by antiviral short interfering RNA pools produced using φ6 RNA-dependent RNA polymerase

2009 ◽  
Vol 90 (10) ◽  
pp. 2468-2473 ◽  
Author(s):  
Michaela Nygårdas ◽  
Tytti Vuorinen ◽  
Antti P. Aalto ◽  
Dennis H. Bamford ◽  
Veijo Hukkanen
Keyword(s):  
Rna Polymerase ◽  
Genomic Sequence ◽  
Coxsackievirus B3 ◽  
Human Enterovirus ◽  
Rna Dependent Rna Polymerase ◽  
Coxsackievirus B4 ◽  
Novel Approach ◽  
Experimental Therapies ◽  
Coxsackievirus A9 ◽  
Interfering Rna

Coxsackievirus B3 (CBV3) is a member of the human enterovirus B species and a common human pathogen. Even though much is known about the enteroviral life cycle, no specific drugs are available to treat enterovirus infections. RNA interference (RNAi) has evolved to be an important tool for antiviral experimental therapies and gene function studies. We describe here a novel approach for RNAi against CBVs by using a short interfering (siRNA) pool covering 3.5 kb of CBV3 genomic sequence. The RNA-dependent RNA polymerase (RdRP) of bacteriophage φ6 was used to synthesize long double-stranded RNA (dsRNA) from a cloned region (nt 3837–7399) of the CBV3 genome. The dsRNA was cleaved using Dicer, purified and introduced to cells by transfection. The siRNA pool synthesized using the φ6 RdRP (φ6–siRNAs) was considerably more effective than single-site siRNAs. The φ6–siRNA pool also inhibited replication of other enterovirus B species, such as coxsackievirus B4 and coxsackievirus A9.

scholarly journals Improved crystallization of the coxsackievirus B3 RNA-dependent RNA polymerase

2007 ◽  
Vol 63 (6) ◽  
pp. 495-498 ◽  
Author(s):  
Ilham Jabafi ◽  
Barbara Selisko ◽  
Bruno Coutard ◽  
Armando M. De Palma ◽  
Johan Neyts ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Coxsackievirus B3 ◽  
Rna Dependent Rna Polymerase

scholarly journals Space Constrained Homology Modelling: The Paradigm of the RNA-Dependent RNA Polymerase of Dengue (Type II) Virus

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Dimitrios Vlachakis ◽  
Dimitrios Georgios Kontopoulos ◽  
Sophia Kossida
Keyword(s):  
Rna Polymerase ◽  
Sequence Similarity ◽  
Homology Modelling ◽  
The Novel ◽  
Type Ii ◽  
3D Scaffold ◽  
Rna Dependent Rna Polymerase ◽  
X Ray ◽  
Novel Approach ◽  
Modelling Approach

Protein structure is more conserved than sequence in nature. In this direction we developed a novel methodology that significantly improves conventional homology modelling when sequence identity is low, by taking into consideration 3D structural features of the template, such as size and shape. Herein, our new homology modelling approach was applied to the homology modelling of the RNA-dependent RNA polymerase (RdRp) of dengue (type II) virus. The RdRp of dengue was chosen due to the low sequence similarity shared between the dengue virus polymerase and the available templates, while purposely avoiding to use the actual X-ray structure that is available for the dengue RdRp. The novel approach takes advantage of 3D space corresponding to protein shape and size by creating a 3D scaffold of the template structure. The dengue polymerase model built by the novel approach exhibited all features of RNA-dependent RNA polymerases and was almost identical to the X-ray structure of the dengue RdRp, as opposed to the model built by conventional homology modelling. Therefore, we propose that the space-aided homology modelling approach can be of a more general use to homology modelling of enzymes sharing low sequence similarity with the template structures.

scholarly journals The Crystal Structure of Coxsackievirus B3 RNA-Dependent RNA Polymerase in Complex with Its Protein Primer VPg Confirms the Existence of a Second VPg Binding Site on Picornaviridae Polymerases

2008 ◽  
Vol 82 (19) ◽  
pp. 9577-9590 ◽  
Author(s):  
Arnaud Gruez ◽  
Barbara Selisko ◽  
Michael Roberts ◽  
Gérard Bricogne ◽  
Cécile Bussetta ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Rna Polymerase ◽  
Binding Site ◽  
Complex Structure ◽  
Coxsackievirus B3 ◽  
Primer Binding Site ◽  
Genome Replication ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Mouth Disease Virus

ABSTRACT The RNA-dependent RNA polymerase (RdRp) is a central piece in the replication machinery of RNA viruses. In picornaviruses this essential RdRp activity also uridylates the VPg peptide, which then serves as a primer for RNA synthesis. Previous genetic, binding, and biochemical data have identified a VPg binding site on poliovirus RdRp and have shown that is was implicated in VPg uridylation. More recent structural studies have identified a topologically distinct site on the closely related foot-and-mouth disease virus RdRp supposed to be the actual VPg-primer-binding site. Here, we report the crystal structure at 2.5-Å resolution of active coxsackievirus B3 RdRp (also named 3Dpol) in a complex with VPg and a pyrophosphate. The pyrophosphate is situated in the active-site cavity, occupying a putative binding site either for the coproduct of the reaction or an incoming NTP. VPg is bound at the base of the thumb subdomain, providing first structural evidence for the VPg binding site previously identified by genetic and biochemical methods. The binding mode of VPg to CVB3 3Dpol at this site excludes its uridylation by the carrier 3Dpol. We suggest that VPg at this position is either uridylated by another 3Dpol molecule or that it plays a stabilizing role within the uridylation complex. The CVB3 3Dpol/VPg complex structure is expected to contribute to the understanding of the multicomponent VPg-uridylation complex essential for the initiation of genome replication of picornaviruses.

scholarly journals Sequence analysis of human rhinoviruses in the RNA-dependent RNA polymerase coding region reveals large within-species variation

2004 ◽  
Vol 85 (8) ◽  
pp. 2271-2277 ◽  
Author(s):  
Carita Savolainen ◽  
Pia Laine ◽  
Mick N. Mulders ◽  
Tapani Hovi
Keyword(s):  
Rna Polymerase ◽  
Respiratory Infections ◽  
Amino Acid Level ◽  
Human Rhinovirus ◽  
Human Enterovirus ◽  
Species Variation ◽  
Coding Region ◽  
Rna Dependent Rna Polymerase ◽  
Field Isolates ◽  
The Difference

Human rhinoviruses (HRVs; family Picornaviridae), the most frequent causative agents of respiratory infections, comprise more than 100 distinct serotypes. According to previous phylogenetic analysis of the VP4/VP2-coding sequences, all but one of the HRV prototype strains distribute between the two established species, Human rhinovirus A (HRV-A) and Human rhinovirus B (HRV-B). Here, partial sequences of the RNA-dependent RNA polymerase (3D polymerase)-coding gene of 48 HRV prototype strains and 12 field isolates were analysed. The designated division of the HRV strains into the species HRV-A and HRV-B was also seen in the 3D-coding region. Phylogenetically, HRV-B clustered closer to human enterovirus (HEV) species HEV-B, HEV-C and poliovirus than to HRV-A. Intraspecies variation within both HRV-A and HRV-B was greater in the 3D-coding region than in the VP4/VP2-coding region, with the difference maxima reaching 48 % at the nucleotide level and 36 % at the amino acid level in HRV-A and 53 and 35 %, respectively, in HRV-B. Within both species, a few strains formed a separate cluster differing from the majority of strains as much as HEV-B from HEV-C. Furthermore, the tree topology within HRV-A differed from that for VP4/VP2, suggesting possible recombination events in the evolutionary history of the strains. However, all 12 field isolates clustered similarly, as in the capsid region. These results showed that the within-species variation in the 3D region is greater in HRV than in HEV. Furthermore, HRV variation in the 3D region exceeds that in the capsid-coding region.

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