A Single Amino Acid Substitution in the Phosphoprotein of Respiratory Syncytial Virus Confers Thermosensitivity in a Reconstituted RNA Polymerase System

Marriott, Anthony C.; Wilson, Steven D.; Randhawa, J S; Easton, Andrew J.

doi:10.1128/jvi.73.6.5162-5165.1999

Cited by 14 publications

(9 citation statements)

References 24 publications

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“…Transfections were conducted as above but 48 hours post-transfection, mRNA was purified from the cells using a Sigma GenElute™ Direct mRNA Miniprep Kit. Purified mRNA was separated on a glyoxal-agarose gel and detected by northern blotting using non-radioactive DIG-labelled riboprobes specific for both CAT and EGFP (Marriott et al, 1999). CAT mRNA levels remained constant for the various mutants examined, as expected.…”

Section: Introductionsupporting

confidence: 64%

Mutational analysis of the gene start sequences of pneumonia virus of mice

Dibben

Easton

2007

Virus Research

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The transcriptional start sequence of pneumonia virus of mice is more variable than that of the other pneumoviruses, with five different nine-base gene start (GS) sequences found in the PVM genome. The sequence requirements of the PVM gene start signal, and the efficiency of transcriptional initiation of the different virus genes, was investigated using a reverse genetics approach with a minigenome construct containing two reporter genes. A series of GS mutants were created, where each of the nine bases of the gene start consensus sequence of a reporter gene was changed to every other possible base, and the resulting effect on initiation of transcription was assayed. Nucleotide positions 1, 2 and 7 were found to be most sensitive to mutation whilst positions 4, 5 and 9 were relatively insensitive. The L gene GS sequence was found to have only 20% of the activity of the consensus sequence whilst the published M2 gene start sequence was found to be non-functional. A minigenome construct in which the two reporter genes were separated by the F-M2 gene junction of PVM was used to confirm the presence of two alternative, functional, GS sequences that could both drive the transcription of the PVM M2 gene.

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Section: Introductionsupporting

confidence: 64%

Mutational analysis of the gene start sequences of pneumonia virus of mice

Dibben

Easton

2007

Virus Research

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“…As a significant human pathogen of the nonsegmented negative-sense (NNS) RNA viruses, RSV shares a common strategy for genome replication and gene expression with other NNS RNA viruses, such as measles, rabies, and Ebola (7)(8)(9)(10)(11). RSV RNA synthesis is catalyzed by a multifunctional RNA dependent RNA Polymerase (RdRP), which comprises of a large (L) protein that catalyzes three distinct enzymatic functions and an essential co-enzyme phosphoprotein (P) (12)(13)(14)(15)(16)(17). The polymerases catalyze both replications of viral genomes and transcriptions of viral genes, and the RNA synthesis is central to NNS RNA viral life cycles.…”

Section: Introductionmentioning

confidence: 99%

In Vitro Primer-Based RNA Elongation and Promoter Fine Mapping of the Respiratory Syncytial Virus

Cao

Gao

Roesler

et al. 2020

J Virol

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Respiratory syncytial virus (RSV) is a nonsegmented negative-sense (NNS) RNA virus and shares a similar RNA synthesis strategy with other members of NNS RNA viruses such as measles, rabies, and Ebola. RSV RNA synthesis is catalyzed by a multifunctional RNA dependent RNA Polymerase (RdRP), which comprises of a large (L) protein that catalyzes three distinct enzymatic functions and an essential co-enzyme phosphoprotein (P). Here, we successfully prepared highly pure, full-length wild-type and mutant RSV polymerase (L:P) complex. We demonstrated that the RSV polymerase could carry out both de novo and primer-based RNA synthesis. We defined the minimal length of the RNA template for in vitro de novo RNA synthesis using the purified RSV polymerase is 8 nucleotides (nts), shorter than previously reported. We showed the RSV polymerase catalyzes the primer-dependent RNA elongation with different lengths of primers on both short (10-nt) and long (25-nt) RNA templates. We compared the sequence specificity of different viral promoters and identified positions 3, 5, and 8 of the promoter sequence are essential to the in vitro RSV polymerase activity, consistent with the results previously mapped by the in vivo minigenome assay. Overall, these findings agree well with previous biochemical studies and extend our understanding of the promoter sequence and the mechanism of RSV RNA synthesis. IMPORTANCE As a major human pathogen, respiratory syncytial virus (RSV) affects 3.4 million children worldwide annually. However, no effective antivirals or vaccines are available. An in-depth mechanistic understanding of the RSV RNA synthesis machinery remains a high priority among the nonsegmented negative-sense (NNS) RNA viruses. There is a strong public health need for research on this virus due to major fundamental gaps in our understanding of NNS RNA virus replication. As the key enzyme executing transcription and replication of the virus, the RSV RNA dependent RNA polymerase (RdRP) is a logical target for novel antiviral drugs. Therefore, exploring the primer-dependent RNA elongation extends our mechanistic understanding of the RSV RNA synthesis. Further fine mapping of the promoter sequence paves the way to better understanding the function and structure of the RSV polymerase.

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“…This will affect the observed activity of the Luc enzyme produced from these transcripts, and hence the level of expression for this mutant cannot be considered to be truly representative of transcriptional capacity. Results of CAT ELISA and Luc enzyme assays have previously been shown to closely match levels of mRNA transcription from minigenomes measured by Northern blot analysis (Marriott et al, 1999). To clarify the effect of the introduction of this start codon, and to confirm that the mutations were affecting mRNA transcription rather than having an indirect effect on another aspect of gene expression, Northern blot analysis was carried out using mRNA isolated from HEp2 cells previously infected with recombinant vaccinia virus expressing T7 RNA polymerase in a plasmid-based rescue system as described by Marriott et al (1999), with plasmid amounts optimized for APV (0?4 mg of the N plasmid and dicistronic minigenome, 0?2 mg of the P and L plasmids and 0?02 mg of the M2-1 plasmid) transfected into approximately 2610 6 cells using Lipofectin (Invitrogen).…”

mentioning

confidence: 60%

“…3). A riboprobe containing sequences from both CAT and Luc genes was generated by transcription from an APV dicistronic minigenome and the blots were processed as described by Marriott et al (1999). It can be seen that the levels of Luc mRNA relative to those of CAT mRNA are consistent with the protein levels determined for the mutants.…”

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confidence: 84%

Mutational analysis of the avian pneumovirus conserved transcriptional gene start sequence identifying critical residues

Edworthy

Easton

2005

Journal of General Virology

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Seven of the eight genes in the avian pneumovirus (APV) genome contain a conserved 9 nt transcriptional start sequence with the virus large (L) polymerase gene differing from the consensus at three positions. The sequence requirements of the APV transcriptional gene start sequence were investigated by generating a series of mutations in which each of the nine conserved bases was mutated to each of the other three possible nucleotides in a minigenome containing two reporter genes. The effect of each mutation was assessed by measuring the relative levels of expression from the altered and unaltered gene start sequences. Mutations at positions 2, 7 and 9 significantly reduced transcription levels while alterations to position 5 had little effect. The L gene start sequence directed transcription at levels approximately 50 % below that of the consensus gene start sequence. These data suggest that there are common features in pneumovirus transcriptional control sequences.Avian pneumovirus (APV) causes acute respiratory infection in domestic poultry throughout most of the world (Jones, 1996) and it has been detected in several wild bird species, although the disease impact in these species is not yet clear (Bennett et al., 2002(Bennett et al., , 2004Lwamba et al., 2002;Shin et al., 2000Shin et al., , 2002. APV is classified as a member of the family Paramyxoviridae, the subfamily Pneumovirinae and the genus Metapneumovirus (Cavanagh & Barrett, 1988;Collins & Gough, 1988;Ling & Pringle, 1988). It is distinguished from members of the genus Pneumovirus by the order of its genes -39-N-P-M-F-M2-SH-G-L-59 (Ling et al., 1992;Randhawa et al., 1996Randhawa et al., , 1997 Yu et al., 1992a, b) and the absence of non-structural protein genes (Randhawa et al., 1997).RNA synthesis in pneumoviruses requires a ribonucleoprotein complex comprising the nucleocapsid (N) protein bound to the genomic RNA together with the phosphoprotein (P) and large (L) polymerase protein. Efficient, progressive, transcription is enhanced by the M2-1 protein, a feature that is unique to the members of the subfamily Pneumovirinae (Collins et al., 1995;Naylor et al., 2004). Transcription of virus mRNA is carried out by the virionassociated polymerase complex in a progressive stop-start manner from the 39 to the 59 end of the genome (Dickens et al., 1984;, and is directed by gene start and gene end sequences that flank the transcription units. The gene order in non-segmented negativestrand RNA viruses is significant, as their transcription strategy results in more mRNA transcripts from genes at the 39 end of the genome, with levels of mRNA progressively decreasing in a step-wise manner to the 59 end. In Human respiratory syncytial virus (HRSV) and APV, the gene start sequence is conserved in all of the genes except for the L gene (Li et al., 1996;Ling et al., 1992Ling et al., , 1995Randhawa et al., 1996;Yu et al., 1991Yu et al., , 1992a, although the HRSV gene start sequence is 10 nt in length (Collins et al., 1986;Kuo et al., 1996). The APV consensus is GG...

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A Single Amino Acid Substitution in the Phosphoprotein of Respiratory Syncytial Virus Confers Thermosensitivity in a Reconstituted RNA Polymerase System

Cited by 14 publications

References 24 publications

Mutational analysis of the gene start sequences of pneumonia virus of mice

Mutational analysis of the gene start sequences of pneumonia virus of mice

In Vitro Primer-Based RNA Elongation and Promoter Fine Mapping of the Respiratory Syncytial Virus

Mutational analysis of the avian pneumovirus conserved transcriptional gene start sequence identifying critical residues

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