Expression of the ORF-2 protein of the human respiratory syncytial virus M2 gene is initiated by a ribosomal termination-dependent reinitiation mechanism

Ahmadian, Gholamreza; Randhawa, J S; Easton, Andrew J.

doi:10.1093/emboj/19.11.2681

Cited by 69 publications

(101 citation statements)

References 40 publications

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“…A similar phenomenon was described for the subgenomic mRNA found in RHDVinfected cells where translation of ORF3 of the genome is coupled to termination of translation of the upstream ORF (18). In both cases, it was shown that the nucleotide sequence of the second downstream ORF was not essential for the process to occur because the virus ORF could be replaced with other sequences without an effect on the coupled translation (17,18). For the RSV study, the gene encoding bacterial chloramphenicol acetyltransferase (CAT) was substituted for the M2-2 gene (Fig.…”

supporting

confidence: 63%

“…Transfection and Reporter Assays-HEp-2 cells were transfected as described previously (17). The total amount of DNA added to each well was reduced to 250 ng, and the transfection reagent Lipofectin (Invitrogen) was used as recommended.…”

Section: Methodsmentioning

confidence: 99%

“…Fig. 1B contains the entire coding sequence of the M2-1 ORF (17). Immediately downstream of the M2-1 stop codon, an XbaI site was used to fuse the CAT reporter gene in-frame with the three start codons for the M2-2 ORF that were present upstream of the M2-1 stop codon.…”

Section: Methodsmentioning

confidence: 99%

“…The first and second ORFs of the M2 mRNA overlap by 32 nucleotides (Fig. 1A), and Ahmadian et al (17) show that initiation of translation of the second, M2-2, ORF was coupled to termination of translation of the first ORF as moving the translational stop codon of the M2-1 ORF further downstream abrogated translation of the second ORF. A similar phenomenon was described for the subgenomic mRNA found in RHDVinfected cells where translation of ORF3 of the genome is coupled to termination of translation of the upstream ORF (18).…”

mentioning

confidence: 88%

“…For the RSV study, the gene encoding bacterial chloramphenicol acetyltransferase (CAT) was substituted for the M2-2 gene (Fig. 1B) (17). Meyers (18) has shown that the translation of the downstream ORF of RHDV subgenomic mRNA is dependent on a sequence located 84 nucleotides upstream of the region of overlap between the two ORFs.…”

mentioning

confidence: 99%

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Coupled Translation of the Respiratory Syncytial Virus M2 Open Reading Frames Requires Upstream Sequences

Gould

Easton

2005

Journal of Biological Chemistry

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We have investigated the mechanism of the translation of the second open reading frame (ORF) of the respiratory syncytial virus M2 transcript that uses a novel coupled translation process requiring prior translation of the upstream ORF. The second M2-2 ORF sequences play no role in the coupling process and can be replaced with other gene sequences. Surprisingly, the overlap region of the two ORFs alone was not sufficient for coupled translation to occur. An analysis of the sequences required for the coupling process showed that portions of the transcript located along the length of the first ORF M2-1, upstream of the ORF overlap region, were essential for coupled translation to occur. A critically important region for this process was centered ϳ150 nucleotides upstream of the ORF2 initiation codons. This region was shown to contain a significant degree of secondary structure, and mutation of this sequence to remove predicted areas of base pairing significantly reduced coupled translation, confirming that the secondary structure was important for the coupling process. Additional sequences further upstream increased the efficiency of the coupled translation process. These data indicate that upstream sequences act in conjunction with the M2-1/M2-2 overlap region to promote coupled translation.

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supporting

confidence: 63%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 88%

mentioning

confidence: 99%

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Coupled Translation of the Respiratory Syncytial Virus M2 Open Reading Frames Requires Upstream Sequences

Gould

Easton

2005

Journal of Biological Chemistry

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Translation initiation of viral mRNAs

López-Lastra

Ramdohr

Letelier

et al. 2010

Reviews in Medical Virology

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Viruses depend on cells for their replication but have evolved mechanisms to achieve this in an efficient and, in some instances, a cell-type-specific manner. The expression of viral proteins is frequently subject to translational control. The dominant target of such control is the initiation step of protein synthesis. Indeed, during the early stages of infection, viral mRNAs must compete with their host counterparts for the protein synthetic machinery, especially for the limited pool of eukaryotic translation initiation factors (eIFs) that mediate the recruitment of ribosomes to both viral and cellular mRNAs. To circumvent this competition viruses use diverse strategies so that ribosomes can be recruited selectively to viral mRNAs. In this review we focus on the initiation of protein synthesis and outline some of the strategies used by viruses to ensure efficient translation initiation of their mRNAs.

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Deletion and Substitution Analysis Defines Regions and Residues within the Phosphoprotein of Bovine Respiratory Syncytial Virus That Affect Transcription, RNA Replication, and Interaction with the Nucleoprotein

et al. 2001

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The phosphoprotein (P) of bovine respiratory syncytial virus (BRSV) is a multifunctional protein that plays a central role in transcription and replication of the viral genomic RNA. To investigate the domains and specific residues involved in different activities of the P protein, we generated a total of 22 deletion and 17 point mutants of the P protein. These mutants were characterized using an intracellular BRSV-CAT minigenome replication system for the ability to (1) direct minigenome transcription, (2) direct minigenome replication, and (3) form complexes with nucleocapsid protein (N) and large polymerase protein (L). These studies revealed that all the regions of P protein except amino acids 41-80 are essential for minigenome transcription and replication. Interestingly, amino acids 41-60 appeared to contain sequences that negatively regulate transcription and replication. Analysis of the N- or C-terminal ends indicated that deletion of up to 3 amino acids from the N- or C-terminus completely ablated the replication, while leaving substantial residual transcription. Single amino acid substitutions within the N-terminal 4 or C-terminal 13 amino acids showed that substitution at position 2, 4, 234, 236, 238, 240, or 241 was highly inhibitory to both transcription and replication, whereas substitution at position 3 was highly inhibitory to replication while leaving substantial residual transcription. Substitution of serine residues at the C-terminus indicated that loss of phosphorylation sites did not appear to have any effect on transcription and replication. Coimmunoprecipitation of P-N and P-L complexes with P-specific antiserum revealed that substitution mutations at the N- or C-terminus did not affect binding to N and L proteins, except that substitution mutation at C-terminus position 234, 236, 238, 240, or 241 affected binding to N protein by 10-fold.

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Expression of the ORF-2 protein of the human respiratory syncytial virus M2 gene is initiated by a ribosomal termination-dependent reinitiation mechanism

Cited by 69 publications

References 40 publications

Coupled Translation of the Respiratory Syncytial Virus M2 Open Reading Frames Requires Upstream Sequences

Coupled Translation of the Respiratory Syncytial Virus M2 Open Reading Frames Requires Upstream Sequences

Translation initiation of viral mRNAs

Deletion and Substitution Analysis Defines Regions and Residues within the Phosphoprotein of Bovine Respiratory Syncytial Virus That Affect Transcription, RNA Replication, and Interaction with the Nucleoprotein

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