Mutations in Conserved Domain II of the Large (L) Subunit of the Sendai Virus RNA Polymerase Abolish RNA Synthesis

ABSTRACT The negative-stranded RNA viral genome is an RNA-protein complex of helicoidal symmetry, resistant to nonionic detergent and high salt, in which the RNA is protected from RNase digestion. The 15,384 nucleotides of the Sendai virus genome are bound to 2,564 subunits of the N protein, each interacting with six nucleotides so tightly that the bases are poorly accessible to soluble reagents. With such a uniform structure, the question of template recognition by the viral RNA polymerase has been raised. In a previous study, the N-phase context has been proposed to be crucial for this recognition, a notion referring to the importance of the position in which the nucleotides interact with the N protein. The N-phase context ruled out the role of the template 3′-OH congruence, a feature resulting from the obedience to the rule of six that implies the precise interaction of the last six 3′-OH nucleotides with the last N protein. The N-phase context then allows prediction of the recognition by the RNA polymerase of a replication promoter sequence even if internally positioned, a promoter which normally lies at the template extremity. In this study, with template minireplicons bearing tandem replication promoters separated by intervening sequences, we present data that indeed show that initiation of RNA synthesis takes place at the internal promoter. This internal initiation can best be interpreted as the result of the polymerase entering the template at the internal promoter. In this way, the data are consistent with the importance of the N-phase context in template recognition. Moreover, by introducing between the two promoters a stretch of 10 A residues which represent a barrier for RNA synthesis, we found that the ability of the RNA polymerase to cross this barrier depends on the type of replication promoter, strong or weak, that the RNA polymerase starts on, a sign that the RNA polymerase may be somehow imprinted in its activity by the nature of the promoter on which it starts synthesis.

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Mutations in Conserved Domains IV and VI of the Large (L) Subunit of the Sendai Virus RNA Polymerase Give a Spectrum of Defective RNA Synthesis Phenotypes

Virology ◽

10.1006/viro.2000.0234 ◽

2000 ◽

Vol 269 (2) ◽

pp. 426-439 ◽

Cited By ~ 31

Author(s):

Joyce A. Feller ◽

Sherin Smallwood ◽

Sandra M. Horikami ◽

Sue A. Moyer

Keyword(s):

Rna Polymerase ◽

Rna Synthesis ◽

Sendai Virus ◽

Conserved Domains ◽

Virus Rna ◽

Defective Rna

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Viral RNA polymerase scanning and the gymnastics of Sendai virus RNA synthesis

Virology ◽

10.1016/j.virol.2003.10.031 ◽

2004 ◽

Vol 318 (2) ◽

pp. 463-473 ◽

Cited By ~ 81

Author(s):

Daniel Kolakofsky ◽

Philippe Le Mercier ◽

Frédéric Iseni ◽

Dominique Garcin

Keyword(s):

Rna Polymerase ◽

Rna Synthesis ◽

Sendai Virus ◽

Viral Rna ◽

Virus Rna

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Transcribing paramyxovirus RNA polymerase engages the template at its 3′ extremity

Journal of General Virology ◽

10.1099/vir.0.81353-0 ◽

2006 ◽

Vol 87 (3) ◽

pp. 665-672 ◽

Cited By ~ 5

Author(s):

Samuel Cordey ◽

Laurent Roux

Keyword(s):

Rna Polymerase ◽

Transcription Initiation ◽

Fluorescent Protein ◽

Sendai Virus ◽

Rna Viruses ◽

Mrna Transcription ◽

Gfp Expression ◽

Rnase Protection ◽

Virus Rna ◽

Green Fluorescent

For the non-segmented, negative-stranded RNA viruses, the mechanism controlling transcription or replication is still a matter of debate. To gain information about this mechanism and about the nature of the RNA polymerase involved, the length of an intervening sequence separating the 3′ end of Sendai virus minigenomes and a downstream transcription-initiation signal was increased progressively. It was found that transcription, as measured by green fluorescent protein (GFP) expression, decreased progressively in proportion to the increase in length of the intervening sequence. GFP expression correlated well with the levels of GFP mRNA in the cells, as measured by quantitative primer extension and by RNase protection. Thus, mRNA transcription was inversely proportional to the length of the inserted sequence. These data are evidence that the RNA polymerase initiating transcription at the downstream transcription signal somehow sees the distance separating this signal and the template 3′ extremity. Implication of this observation for the nature of the Sendai virus RNA polymerase and for the mechanism by which it synthesizes mRNAs or replication products is presented.

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RNA Synthesis by the Brome Mosaic Virus RNA-Dependent RNA Polymerase in Human Cells Reveals Requirements forDe NovoInitiation and Protein-Protein Interaction

Journal of Virology ◽

10.1128/jvi.00069-12 ◽

2012 ◽

Vol 86 (8) ◽

pp. 4317-4327 ◽

Cited By ~ 9

Author(s):

Chennareddy V. Subba-Reddy ◽

Brady Tragesser ◽

Zhili Xu ◽

Barry Stein ◽

C. T. Ranjith-Kumar ◽

...

Keyword(s):

Rna Polymerase ◽

Mosaic Virus ◽

Protein Interaction ◽

Rna Synthesis ◽

Brome Mosaic Virus ◽

Human Cells ◽

Rna Dependent Rna Polymerase ◽

Protein Protein Interaction ◽

Virus Rna

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Initiation of RNA Synthesis by the Hepatitis C Virus RNA-Dependent RNA Polymerase Is Affected by the Structure of the RNA Template

Biochemistry ◽

10.1021/bi5006656 ◽

2014 ◽

Vol 53 (44) ◽

pp. 7002-7012 ◽

Cited By ~ 4

Author(s):

Stefan Reich ◽

Michael Kovermann ◽

Hauke Lilie ◽

Paul Knick ◽

René Geissler ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Rna Synthesis ◽

Hepatitis C Virus Rna ◽

Rna Dependent Rna Polymerase ◽

Virus Rna

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Sendai virus RNA polymerase scanning for mRNA start sites at gene junctions

Virology ◽

10.1016/j.virol.2006.12.033 ◽

2007 ◽

Vol 362 (2) ◽

pp. 411-420 ◽

Cited By ~ 11

Author(s):

Philippe Plattet ◽

Laura Strahle ◽

Philippe le Mercier ◽

Stéphane Hausmann ◽

Dominique Garcin ◽

...

Keyword(s):

Rna Polymerase ◽

Sendai Virus ◽

Virus Rna

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Enisamium Reduces Influenza Virus Shedding and Improves Patient Recovery by Inhibiting Viral RNA Polymerase Activity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02605-20 ◽

2021 ◽

Vol 65 (4) ◽

Author(s):

Aartjan J. W. te Velthuis ◽

Tatiana G. Zubkova ◽

Megan Shaw ◽

Andrew Mehle ◽

David Boltz ◽

...

Keyword(s):

Influenza Virus ◽

Placebo Group ◽

Rna Polymerase ◽

Rna Synthesis ◽

Respiratory Infections ◽

Respiratory Viruses ◽

Virus Shedding ◽

Virus Rna ◽

Viral Respiratory Infections ◽

Viral Respiratory

ABSTRACT Infections with respiratory viruses constitute a huge burden on our health and economy. Antivirals against some respiratory viruses are available, but further options are urgently needed. Enisamium iodide (laboratory code FAV00A, trade name Amizon) is an antiviral, marketed in countries of the Commonwealth of Independent States for the treatment of viral respiratory infections, but its clinical efficacy and mode of action are not well understood. In this study, we investigated the efficacy of enisamium in patients aged between 18 and 60 years with confirmed influenza virus and other viral respiratory infections. Enisamium treatment resulted in reduced influenza virus shedding (at day 3, 71.2% in the enisamium group tested negative versus 25.0% in placebo group [P < 0.0001]), faster patient recovery (at day 14, 93.9% in the enisamium group had recovered versus 32.5% in placebo group [P < 0.0001]), and reduced disease symptoms (from 9.6 ± 0.7 to 4.6 ± 0.9 score points in enisamium group versus 9.7 ± 1.1 to 5.6 ± 1.1 score points in placebo group [P < 0.0001]) compared to those in the placebo group. Using mass spectrometry, and cell-based and cell-free viral RNA synthesis assays, we identified a hydroxylated metabolite of enisamium, VR17-04. VR17-04 is capable of inhibiting influenza virus RNA synthesis and is present in plasma of patients treated with enisamium. VR17-04 inhibits the activity of the influenza virus RNA polymerase more potently than its parent compound. Overall, these results suggest that enisamium is metabolized in humans to an inhibitor of the influenza virus RNA polymerase that reduces viral shedding and improves patient recovery in influenza patients. (This study has been registered at ClinicalTrials.gov under identifier NCT04682444.)

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Template-Dependent Initiation of Sindbis Virus RNA Replication In Vitro

Journal of Virology ◽

10.1128/jvi.72.8.6546-6553.1998 ◽

1998 ◽

Vol 72 (8) ◽

pp. 6546-6553 ◽

Cited By ~ 64

Author(s):

Julie A. Lemm ◽

Anders Bergqvist ◽

Carol M. Read ◽

Charles M. Rice

Keyword(s):

Rna Polymerase ◽

Sindbis Virus ◽

Rna Synthesis ◽

Rna Replication ◽

Functional Assay ◽

Minus Strand ◽

Virus Rna ◽

A Genome ◽

Strand Initiation

ABSTRACT Recent insights into the early events in Sindbis virus RNA replication suggest a requirement for either the P123 or P23 polyprotein, as well as mature nsP4, the RNA-dependent RNA polymerase, for initiation of minus-strand RNA synthesis. Based on this observation, we have succeeded in reconstituting an in vitro system for template-dependent initiation of SIN RNA replication. Extracts were isolated from cells infected with vaccinia virus recombinants expressing various SIN proteins and assayed by the addition of exogenous template RNAs. Extracts from cells expressing P123C>S, a protease-defective P123 polyprotein, and nsP4 synthesized a genome-length minus-sense RNA product. Replicase activity was dependent upon addition of exogenous RNA and was specific for alphavirus plus-strand RNA templates. RNA synthesis was also obtained by coexpression of nsP1, P23C>S, and nsP4. However, extracts from cells expressing nsP4 and P123, a cleavage-competent P123 polyprotein, had much less replicase activity. In addition, a P123 polyprotein containing a mutation in the nsP2 protease which increased the efficiency of processing exhibited very little, if any, replicase activity. These results provide further evidence that processing of the polyprotein inactivates the minus-strand initiation complex. Finally, RNA synthesis was detected when soluble nsP4 was added to a membrane fraction containing P123C>S, thus providing a functional assay for purification of the nsP4 RNA polymerase.

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

Journal of Virology ◽

10.1128/jvi.73.9.7694-7702.1999 ◽

1999 ◽

Vol 73 (9) ◽

pp. 7694-7702 ◽

Cited By ~ 156

Author(s):

Jong-Won Oh ◽

Takayoshi Ito ◽

Michael M. C. Lai

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Rna Synthesis ◽

De Novo ◽

Full Length ◽

Viral Rna ◽

Hcv Rna ◽

Independent Manner ◽

Virus Rna

ABSTRACT 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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