Effects of Altering the Transcription Termination Signals of Respiratory Syncytial Virus on Viral Gene Expression and Growth In Vitro and In Vivo

The 241 aa human respiratory synctyial virus (HRSV) Long strain P protein is phosphorylated at serines 116, 117 and/or 119, and 232. Phosphates added to these residues have slow turnover and can be detected in the absence of protein phosphatase inhibition. Inhibition of phosphatases PP1 and PP2A increases the level of phosphorylation at serines 116, 117 and/or 119, suggesting a more rapid turnover for phosphates added to these residues compared to that of S232. High-turnover phosphorylation is detected in the P-protein NH 2 -terminal region, mainly at S54 and, to a lesser extent, at S39, in the Long strain. When the P protein bears the T46I substitution (in the remaining HRSV strains), phosphates are added to S30, S39, S45 and S54. Phosphatase PP1 removes phosphate at residues in the central part of the P-protein molecule, whereas those in the NH 2 -terminal region are removed by phosphatase PP2A. The significance of the phosphorylation of the NH 2 -terminal region residues for some P-protein functions was studied. The results indicated that this modification is not essential for P-protein oligomerization or for its role in viral RNA synthesis. Nonetheless, dephosphorylation at S54 could facilitate P-M protein interactions that probably occur during the egress of viral particles.

Section: Discussionmentioning

confidence: 77%

Determination of phosphorylated residues from human respiratory syncytial virus P protein that are dynamically dephosphorylated by cellular phosphatases: a possible role for serine 54

Asenjo

Rodríguez

Villanueva

2005

“…Alterations of transcription termination signals have been shown to have an effect on transcriptional readthrough, but did not result in any significant difference in the ability of the altered viruses to replicate in vitro or in vivo (Tran et al, 2004). An AAG substitution (genomic sense) at position 5937 (strain #8544 numbering) in the SH gene end inadvertently introduced into a recombinant AMPV has been shown to produce increased readthrough at the SH-G gene junction and reduced expression of G mRNA (R. Ling and A. J. Easton, unpublished data).…”

Section: Discussionmentioning

confidence: 95%

Avian metapneumovirus SH gene end and G protein mutations influence the level of protection of live-vaccine candidates

Naylor

Ling

Edworthy

et al. 2007

A prototype avian metapneumovirus (AMPV) vaccine (P20) was previously shown to give variable outcomes in experimental trials. Following plaque purification, three of 12 viruses obtained from P20 failed to induce protection against virulent challenge, whilst the remainder retained their protective capacity. The genome sequences of two protective viruses were identical to the P20 consensus, whereas two non-protective viruses differed only in the SH gene transcription termination signal. Northern blotting showed that the alterations in the SH gene-end region of the non-protective viruses led to enhanced levels of dicistronic mRNA produced by transcriptional readthrough. A synthetic minigenome was used to demonstrate that the altered SH gene-end region reduced the level of protein expression from a downstream gene. The genomes of the remaining eight plaque-purified viruses were sequenced in the region where the P20 consensus sequence differed from the virulent progenitor. The seven protective clones were identical, whereas the non-protective virus retained the virulent progenitor sequence at two positions and contained extensive alterations in its attachment (G) protein sequence associated with a reduced or altered expression pattern of G protein on Western blots. The data indicate that the efficacy of a putative protective vaccine strain is affected by mutations altering the balance of G protein expression.

“…In contrast to the VSV GE signals, those of RSV are somewhat divergent and vary in their termination efficiencies (Kuo et al, 1997;Hardy et al, 1999;Tran et al, 2004). Most are not maximally efficient and, so, readthrough mRNAs representing two or more genes are found in infected cells (Collins & Wertz, 1983).…”

Section: Transcription Termination At the Ge Signalsmentioning

confidence: 99%

Unravelling the complexities of respiratory syncytial virus RNA synthesis

Cowton

McGivern

Fearns

2006

106

Human respiratory syncytial virus (RSV) is the leading cause of paediatric respiratory disease and is the focus of antiviral-and vaccine-development programmes. These goals have been aided by an understanding of the virus genome architecture and the mechanisms by which it is expressed and replicated. RSV is a member of the order Mononegavirales and, as such, has a genome consisting of a single strand of negative-sense RNA. At first glance, transcription and genome replication appear straightforward, requiring self-contained promoter regions at the 39 ends of the genome and antigenome RNAs, short cis-acting elements flanking each of the genes and one polymerase. However, from these minimal elements, the virus is able to generate an array of capped, methylated and polyadenylated mRNAs and encapsidated antigenome and genome RNAs, all in the appropriate ratios to facilitate virus replication. The apparent simplicity of genome expression and replication is a consequence of considerable complexity in the polymerase structure and its cognate cis-acting sequences; here, our understanding of mechanisms by which the RSV polymerase proteins interact with signals in the RNA template to produce different RNA products is reviewed. Background and scope of the reviewThe World Health Organization estimates that Human respiratory syncytial virus (RSV) is responsible for 64 million infections and 160 000 deaths per annum. Its victims are mostly young infants, but it is increasingly recognized as a significant cause of disease in the elderly population and can often be fatal for patients with compromised immune systems . RSV is a member of the subfamily Pneumovirinae in the family Paramyxoviridae, order Mononegavirales, i.e. the non-segmented, negativestrand RNA viruses. This order includes several exotic pathogens, such as the Ebola and Nipah viruses, and other, more familiar ones, such as the parainfluenza, measles and mumps viruses. Most of our initial understanding of mononegavirus transcription and genome replication stemmed from studies with the paramyxovirus Sendai virus (SeV) and the rhabdovirus Vesicular stomatitis virus (VSV). These viruses can be grown to very high titres, which has facilitated analysis of their RNA-synthesis mechanisms using biochemical techniques. These studies still provide a blueprint for mononegavirus RNA synthesis; however, sequence analysis of different virus genomes and advances in reverse-genetics techniques have opened up this field. Recent investigations have revealed that mononegaviruses differ in the layout of their promoter and gene-junction regions, template structure and polymerase composition. Therefore, although the overall strategy of transcription and replication is similar for all non-segmented, negative-strand RNA viruses, it is possible that the molecular mechanisms that individual viruses use to achieve this differ. Given that RSV is an important human pathogen, it is worth consideration in its own right and, in this review, we aim to examine the data regarding RSV specifically, h...