Expression of Enzymatically Active Rabbit Hemorrhagic Disease Virus RNA-Dependent RNA Polymerase in
            <i>Escherichia coli</i>

Vázquez, Alicia; Alonso, J.M.; Casáis, Rosa; Boga, José Antonio; Parra, Francisco

doi:10.1128/jvi.72.4.2999-3004.1998

Cited by 42 publications

(8 citation statements)

References 24 publications

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“…Similar snap-back RNA synthesis was observed with the poliovirus RdRp, 3D pol , purified from infected HeLa cells (24,49), and recombinant RdRps of rabbit hemorrhagic disease virus and bovine viral diarrhea virus expressed in E. coli (43,50). RdRps from some animal viruses were able to synthesize template-size products, but only in the presence of an artificial primer (30,32,43). In contrast, RdRps from most of the plant RNA viruses can recognize the structured 3Ј end of plant viral RNA as a promoter and initiate RNA synthesis de novo.…”

supporting

confidence: 63%

“…A primer-dependent RNA synthesis using a snap-back RNA template is not expected to yield an authentic RNA product, because the 3Ј end of the RNA template will inevitably be excluded from the product, or extra nucleotides will be included in the product. Similar snap-back RNA synthesis was observed with the poliovirus RdRp, 3D pol , purified from infected HeLa cells (24,49), and recombinant RdRps of rabbit hemorrhagic disease virus and bovine viral diarrhea virus expressed in E. coli (43,50). RdRps from some animal viruses were able to synthesize template-size products, but only in the presence of an artificial primer (30,32,43).…”

supporting

confidence: 56%

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A Recombinant Hepatitis C Virus RNA-Dependent RNA Polymerase Capable of Copying the Full-Length Viral RNA

Ito

Lai

1999

J Virol

212

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All of the previously reported recombinant RNA-dependent RNA polymerases (RdRp), the NS5B enzymes, of hepatitis C virus (HCV) could function only in a primer-dependent and template-nonspecific manner, which is different from the expected properties of the functional viral enzymes in the cells. We have now expressed a recombinant NS5B that is able to synthesize a full-length HCV genome in a template-dependent and primer-independent manner. The kinetics of RNA synthesis showed that this RdRp can initiate RNA synthesis de novo and yield a full-length RNA product of genomic size (9.5 kb), indicating that it did not use the copy-back RNA as a primer. This RdRp was also able to accept heterologous viral RNA templates, including poly(A)- and non-poly(A)-tailed RNA, in a primer-independent manner, but the products in these cases were heterogeneous. The RdRp used some homopolymeric RNA templates only in the presence of a primer. By using the 3′-end 98 nucleotides (nt) of HCV RNA, which is conserved in all genotypes of HCV, as a template, a distinct RNA product was generated. Truncation of 21 nt from the 5′ end or 45 nt from the 3′ end of the 98-nt RNA abolished almost completely its ability to serve as a template. Inclusion of the 3′-end variable sequence region and the U-rich tract upstream of the X region in the template significantly enhanced RNA synthesis. The 3′ end of minus-strand RNA of HCV genome also served as a template, and it required a minimum of 239 nt from the 3′ end. These data defined the cis-acting sequences for HCV RNA synthesis at the 3′ end of HCV RNA in both the plus and minus senses. This is the first recombinant HCV RdRp capable of copying the full-length HCV RNA in the primer-independent manner expected of the functional HCV RNA polymerase.

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supporting

confidence: 63%

supporting

confidence: 56%

A Recombinant Hepatitis C Virus RNA-Dependent RNA Polymerase Capable of Copying the Full-Length Viral RNA

Ito

Lai

1999

J Virol

212

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“…The lack of an effective cell culture system for rabbit caliciviruses [ 2 , 39 ] prompted us to adopt cell-free in vitro assays for testing inhibitors of the RdRp, a protein that represents a prime target for antiviral drug design due to its essential role in the virus replication cycle and the fact that eukaryotic cells do not possess closely related enzymes. Sequence similarities between the 3D RdRp of picornaviruses and the RHDV polyprotein cleavage product p58 suggest that both polypeptides have a similar role in genome replication [ 40 , 41 ]. Expression of the respective coding region in Escherichia coli (E. coli) showed that p58 is indeed an enzymatically active RdRp [ 40 ], and did not demonstrate DNA-dependent RNA polymerase, reverse transcriptase or DNA-dependent DNA polymerase activities [ 41 ].…”

Section: Introductionmentioning

confidence: 99%

“…Sequence similarities between the 3D RdRp of picornaviruses and the RHDV polyprotein cleavage product p58 suggest that both polypeptides have a similar role in genome replication [ 40 , 41 ]. Expression of the respective coding region in Escherichia coli (E. coli) showed that p58 is indeed an enzymatically active RdRp [ 40 ], and did not demonstrate DNA-dependent RNA polymerase, reverse transcriptase or DNA-dependent DNA polymerase activities [ 41 ]. Crystal structure of RHDV RdRp revealed that this enzyme adopts a shape that resembles a right hand, with domains corresponding to the fingers, palm and thumb, as seen in the three-dimensional structures of many other polymerases [ 42 ].…”

Section: Introductionmentioning

confidence: 99%

Purification and Biochemical Characterisation of Rabbit Calicivirus RNA-Dependent RNA Polymerases and Identification of Non-Nucleoside Inhibitors

Urakova

Netzler

Kelly

et al. 2016

Viruses

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Rabbit haemorrhagic disease virus (RHDV) is a calicivirus that causes acute infections in both domestic and wild European rabbits (Oryctolagus cuniculus). The virus causes significant economic losses in rabbit farming and reduces wild rabbit populations. The recent emergence of RHDV variants capable of overcoming immunity to other strains emphasises the need to develop universally effective antivirals to enable quick responses during outbreaks until new vaccines become available. The RNA-dependent RNA polymerase (RdRp) is a primary target for the development of such antiviral drugs. In this study, we used cell-free in vitro assays to examine the biochemical characteristics of two rabbit calicivirus RdRps and the effects of several antivirals that were previously identified as human norovirus RdRp inhibitors. The non-nucleoside inhibitor NIC02 was identified as a potential scaffold for further drug development against rabbit caliciviruses. Our experiments revealed an unusually high temperature optimum (between 40 and 45 °C) for RdRps derived from both a pathogenic and a non-pathogenic rabbit calicivirus, possibly demonstrating an adaptation to a host with a physiological body temperature of more than 38 °C. Interestingly, the in vitro polymerase activity of the non-pathogenic calicivirus RdRp was at least two times higher than that of the RdRp of the highly virulent RHDV.

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“…Viral RdRp plays a central role in replication of the RNA genome, which is a critical step in the pathogenesis of many RNA viruses. To date, most of the knowledge regarding the biochemical properties of RdRp comes from studies of animal RNA viruses, such as poliovirus (2,4,14), hepatitis C virus (3,23), and rabbit hemorrhagic disease virus (38), and plant viruses, such as brome mosaic virus (17,31,33,35) and tobacco mosaic virus (25). Unlike that of the enzyme from animal viruses, the structural organization of plant viral RdRp is relatively less well characterized.…”

Section: Resultsmentioning

confidence: 99%

Identification and Characterization of the Escherichia coli -Expressed RNA-Dependent RNA Polymerase of Bamboo Mosaic Virus

Cheng²,

Huang

et al. 1998

J Virol

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Bamboo mosaic virus (BaMV), a member of the potexvirus group, infects primarily members of the Bambusoideae. The open reading frame 1 (ORF1) of BaMV encodes a 155-kDa polypeptide that was postulated to be involved in the replication and the formation of cap structure at the 5′ end of the viral genome. To characterize the activities associated with the 155-kDa viral protein, it was expressed in Escherichia coli BL21(DE3) cells with thioredoxin, hexahistidine, and S · Tag fused consecutively at its amino terminus, and the fusion protein was purified by metal affinity chromatography. Several RNA fragments, prepared by in vitro transcription, were tested as substrates for the RNA-dependent RNA polymerase (RdRp) activity. Among them, the expressed fusion enzyme was able to generate a 32P-labeled RNA product when 3′-end RNA fragments of the positive strand or negative strand of BaMV were included in the assay mixture. Dot hybridization assay revealed that the reaction products are complementary to their RNA substrates. Taken together, the evidence suggests that the 155-kDa protein encoded by ORF1 of BaMV has an RdRp activity and should be involved in the replication of BaMV. Mutational analyses demonstrate the importance of the GDD motif in the polymerase activity, and deletion studies suggest that the polymerase activity resides in the carboxyl terminus of the 155-kDa viral protein.

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Expression of Enzymatically Active Rabbit Hemorrhagic Disease Virus RNA-Dependent RNA Polymerase in Escherichia coli

Cited by 42 publications

References 24 publications

A Recombinant Hepatitis C Virus RNA-Dependent RNA Polymerase Capable of Copying the Full-Length Viral RNA

A Recombinant Hepatitis C Virus RNA-Dependent RNA Polymerase Capable of Copying the Full-Length Viral RNA

Purification and Biochemical Characterisation of Rabbit Calicivirus RNA-Dependent RNA Polymerases and Identification of Non-Nucleoside Inhibitors

Identification and Characterization of the Escherichia coli -Expressed RNA-Dependent RNA Polymerase of Bamboo Mosaic Virus

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