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

Abstract

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...

A comparison of AMPV subtypes A and B full genomes, gene transcripts and proteins led to reverse-genetics systems rescuing both subtypes

“…A previously comprehensive minigenome investigation of gene start signal efficiencies showed that the CCCUGUUCA was most efficient and that the variant sequence of CCCCGUUCA found on subtype B SH and G proteins would be expected to reduce transcription of those genes (Edworthy & Easton, 2005). The L gene transcription start sequences proved an exception and minigenome studies showed a reduced transcription efficiency (Edworthy & Easton, 2005), as might be expected for a gene coding a protein needed in smaller amounts.…”

“…The L gene transcription start sequences proved an exception and minigenome studies showed a reduced transcription efficiency (Edworthy & Easton, 2005), as might be expected for a gene coding a protein needed in smaller amounts. Surprisingly gene starts of the otherwise more distantly related subtype C viruses (Brown et al, 2014) like the subtype A viruses all used CCCUGUUCA, but again with the exception of the L gene.…”

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

“…In contrast, the PVM GS has one of five possible sequences (Table 1), the consensus sequence being AGGAyAArT, suggesting the possibility of differential expression of genes or a tolerance of variation in GS sequence (Chambers et al, 1990a;Chambers et al, 1990b). The GS sequences of both APV and RSV have been subjected to mutational analysis that identified residues important for the initiation of transcription (Edworthy and Easton, 2005;Kuo et al, 1997). Here, we describe a similar analysis of the role of each of the nine individual bases of the PVM GS sequence using a reverse genetics approach.…”

“…To assess the efficiency of initiation of transcription by different mutants of the PVM GS signal a dicistronic minigenome was constructed (Figure 1), containing two genes, similar to those described previously for APV and RSV (Edworthy and Easton, 2005;Kuo et al, 1996). The first gene in the PVM minigenome encoded bacterial chloramphenicol acetyl transferase (CAT) and was flanked by the N gene GS signal (AGGATAAAT) and the L gene GE signal (TAGTTAACAAAAAA).…”

“…Every position of the EGFP reporter gene GS signal was mutated to each of the three other possible bases using either quickchange mutagenesis (Stratagene) or standard two-step PCR mutagenesis, as described previously (Edworthy and Easton, 2005), resulting in a set of 28 mutants plus the N GS control construct. Mutagenic primers of 48 nucleotides in length were used for both techniques (all mutagenic primer sequences are available from authors on request).…”