This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kuo, L.
Right arrow Articles by Collins, P. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kuo, L.
Right arrow Articles by Collins, P. L.

 Previous Article  |  Next Article 

J. Virol., Oct 1996, 6892-6901, Vol 70, No. 10
Copyright © 1996, American Society for Microbiology

Effects of mutations in the gene-start and gene-end sequence motifs on transcription of monocistronic and dicistronic minigenomes of respiratory syncytial virus

L Kuo, H Grosfeld, J Cristina, MG Hill and PL Collins
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892-0720, USA.

Preceding and following each gene of respiratory syncytial virus (RSV) are two conserved sequences, the gene-start (GS) and gene-end (GE) motifs, respectively, which are thought to be transcription signals. The functions and boundaries of these signals and the process of sequential transcription were analyzed with cDNA-encoded RNA analogs (minigenomes) of nonsegmented negative-sense RSV genomic RNA. Two minigenomes were used. The monocistronic RSV-CAT minigenome consists of the chloramphenicol acetyltransferase (CAT) translational open reading frame (ORF) bordered by the GS and GE motifs and flanked by the 3' leader and 5' trailer extragenic regions of genomic RNA. The dicistronic RSV-CAT-LUC minigenome is a derivative of RSV-CAT into which the ORF for luciferase (LUC), bordered by GS and GE motifs, was inserted downstream of the CAT gene with an intergenic region positioned between the two genes. Each minigenome was synthesized in vitro and transfected into RSV-infected cells, where it was replicated and transcribed to yield the predicted polyadenylated subgenomic mRNA(s). The only RSV sequences required for efficient transcription and RNA replication were the 44-nucleotide 3' leader region, the last 40 nucleotides of the 5' trailer region, and the 9- to 10-nucleotide GS and 12- to 13-nucleotide GE motifs. The GS and GE motifs functioned as self-contained, transportable transcription signals which could be attached to foreign sequences to direct their transcription into subgenomic mRNAs. Removal of the GS motif greatly reduced transcription of its gene, and the requirement for this element was particularly strict for the gene in the downstream position. Ablation of the promoter-proximal GS signal was not associated with increased antigenome synthesis. Consistent with its proposed role in termination and polyadenylation, removal of the CAT GE signal in RSV-CAT resulted in the synthesis of a nonpolyadenylated CAT mRNA, and in RSV-CAT-LUC the same mutation resulted in readthrough transcription to yield a dicistronic CAT-LUC mRNA. The latter result showed that a downstream GS signal is not recognized for reinitiation by the polymerase if it is already engaged in mRNA synthesis; instead, it is recognized only if the polymerase first terminates transcription at an upstream termination signal. This result also showed that ongoing transcription did not open the downstream LUC gene for internal polymerase entry. Removal of both the GS and GE signals of the upstream CAT gene in RSV- CAT-LUC silenced expression of both genes, confirming that independent polymerase entry at an internal gene is insignificant. Remarkably, whereas both genes were silent when the CAT GS and GE signals were both absent, restoration of the CAT GE signal alone restored a significant level (approximately 10 to 12% of the wild-type level) of synthesis of both subgenomic mRNAs. This analysis identified a component of sequential transcription that was independent of the promoter-proximal GS signal and appeared to involve readthrough from the leader region.


This article has been cited by other articles:

  • Wang, J. T., McElvain, L. E., Whelan, S. P. J. (2007). Vesicular Stomatitis Virus mRNA Capping Machinery Requires Specific cis-Acting Signals in the RNA. J. Virol. 81: 11499-11506 [Abstract] [Full Text]  
  • Rennick, L. J., Duprex, W. P., Rima, B. K. (2007). Measles virus minigenomes encoding two autofluorescent proteins reveal cell-to-cell variation in reporter expression dependent on viral sequences between the transcription units. J. Gen. Virol. 88: 2710-2718 [Abstract] [Full Text]  
  • Cowton, V. M., McGivern, D. R., Fearns, R. (2006). Unravelling the complexities of respiratory syncytial virus RNA synthesis. J. Gen. Virol. 87: 1805-1821 [Abstract] [Full Text]  
  • Edworthy, N. L., Easton, A. J. (2005). Mutational analysis of the avian pneumovirus conserved transcriptional gene start sequence identifying critical residues. J. Gen. Virol. 86: 3343-3347 [Abstract] [Full Text]  
  • Cheng, X., Park, H., Zhou, H., Jin, H. (2005). Overexpression of the M2-2 Protein of Respiratory Syncytial Virus Inhibits Viral Replication. J. Virol. 79: 13943-13952 [Abstract] [Full Text]  
  • Liuzzi, M., Mason, S. W., Cartier, M., Lawetz, C., McCollum, R. S., Dansereau, N., Bolger, G., Lapeyre, N., Gaudette, Y., Lagace, L., Massariol, M.-J., Do, F., Whitehead, P., Lamarre, L., Scouten, E., Bordeleau, J., Landry, S., Rancourt, J., Fazal, G., Simoneau, B. (2005). Inhibitors of Respiratory Syncytial Virus Replication Target Cotranscriptional mRNA Guanylylation by Viral RNA-Dependent RNA Polymerase. J. Virol. 79: 13105-13115 [Abstract] [Full Text]  
  • Cowton, V. M., Fearns, R. (2005). Evidence that the Respiratory Syncytial Virus Polymerase Is Recruited to Nucleotides 1 to 11 at the 3' End of the Nucleocapsid and Can Scan To Access Internal Signals. J. Virol. 79: 11311-11322 [Abstract] [Full Text]  
  • McGivern, D. R., Collins, P. L., Fearns, R. (2005). Identification of Internal Sequences in the 3' Leader Region of Human Respiratory Syncytial Virus That Enhance Transcription and Confer Replication Processivity. J. Virol. 79: 2449-2460 [Abstract] [Full Text]  
  • Easton, A. J., Domachowske, J. B., Rosenberg, H. F. (2004). Animal Pneumoviruses: Molecular Genetics and Pathogenesis. Clin. Microbiol. Rev. 17: 390-412 [Abstract] [Full Text]  
  • Mason, S. W., Aberg, E., Lawetz, C., DeLong, R., Whitehead, P., Liuzzi, M. (2003). Interaction between Human Respiratory Syncytial Virus (RSV) M2-1 and P Proteins Is Required for Reconstitution of M2-1-Dependent RSV Minigenome Activity. J. Virol. 77: 10670-10676 [Abstract] [Full Text]  
  • Cartee, T. L., Megaw, A. G., Oomens, A. G. P., Wertz, G. W. (2003). Identification of a Single Amino Acid Change in the Human Respiratory Syncytial Virus L Protein That Affects Transcriptional Termination. J. Virol. 77: 7352-7360 [Abstract] [Full Text]  
  • Zhou, H., Cheng, X., Jin, H. (2003). Identification of Amino Acids That Are Critical to the Processivity Function of Respiratory Syncytial Virus M2-1 Protein. J. Virol. 77: 5046-5053 [Abstract] [Full Text]  
  • Neumann, G., Whitt, M. A., Kawaoka, Y. (2002). A decade after the generation of a negative-sense RNA virus from cloned cDNA - what have we learned?. J. Gen. Virol. 83: 2635-2662 [Abstract] [Full Text]  
  • Fearns, R., Peeples, M. E., Collins, P. L. (2002). Mapping the Transcription and Replication Promoters of Respiratory Syncytial Virus. J. Virol. 76: 1663-1672 [Abstract] [Full Text]  
  • Cartee, T. L., Wertz, G. W. (2001). Respiratory Syncytial Virus M2-1 Protein Requires Phosphorylation for Efficient Function and Binds Viral RNA during Infection. J. Virol. 75: 12188-12197 [Abstract] [Full Text]  
  • Stope, M. B., Karger, A., Schmidt, U., Buchholz, U. J. (2001). Chimeric Bovine Respiratory Syncytial Virus with Attachment and Fusion Glycoproteins Replaced by Bovine Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase and Fusion Proteins. J. Virol. 75: 9367-9377 [Abstract] [Full Text]  
  • Marriott, A. C., Smith, J. M., Easton, A. J. (2001). Fidelity of Leader and Trailer Sequence Usage by the Respiratory Syncytial Virus and Avian Pneumovirus Replication Complexes. J. Virol. 75: 6265-6272 [Abstract] [Full Text]  
  • Karger, A., Schmidt, U., Buchholz, U. J. (2001). Recombinant bovine respiratory syncytial virus with deletions of the G or SH genes: G and F proteins bind heparin. J. Gen. Virol. 82: 631-640 [Abstract] [Full Text]  
  • Parks, G. D., Ward, K. R., Rassa, J. C. (2001). Increased Readthrough Transcription across the Simian Virus 5 M-F Gene Junction Leads to Growth Defects and a Global Inhibition of Viral mRNA Synthesis. J. Virol. 75: 2213-2223 [Abstract] [Full Text]  
  • Harmon, S. B., Megaw, A. G., Wertz, G. W. (2001). RNA Sequences Involved in Transcriptional Termination of Respiratory Syncytial Virus. J. Virol. 75: 36-44 [Abstract] [Full Text]  
  • Finke, S., Cox, J. H., Conzelmann, K.-K. (2000). Differential Transcription Attenuation of Rabies Virus Genes by Intergenic Regions: Generation of Recombinant Viruses Overexpressing the Polymerase Gene. J. Virol. 74: 7261-7269 [Abstract] [Full Text]  
  • Fearns, R., Collins, P. L., Peeples, M. E. (2000). Functional Analysis of the Genomic and Antigenomic Promoters of Human Respiratory Syncytial Virus. J. Virol. 74: 6006-6014 [Abstract] [Full Text]  
  • Peeples, M. E., Collins, P. L. (2000). Mutations in the 5' Trailer Region of a Respiratory Syncytial Virus Minigenome Which Limit RNA Replication to One Step. J. Virol. 74: 146-155 [Abstract] [Full Text]  
  • Kato, A., Kiyotani, K., Hasan, M. K., Shioda, T., Sakai, Y., Yoshida, T., Nagai, Y. (1999). Sendai Virus Gene Start Signals Are Not Equivalent in Reinitiation Capacity: Moderation at the Fusion Protein Gene. J. Virol. 73: 9237-9246 [Abstract] [Full Text]  
  • Fearns, R., Collins, P. L. (1999). Role of the M2-1 Transcription Antitermination Protein of Respiratory Syncytial Virus in Sequential Transcription. J. Virol. 73: 5852-5864 [Abstract] [Full Text]  
  • Finke, S., Conzelmann, K.-K. (1999). Virus Promoters Determine Interference by Defective RNAs: Selective Amplification of Mini-RNA Vectors and Rescue from cDNA by a 3' Copy-Back Ambisense Rabies Virus. J. Virol. 73: 3818-3825 [Abstract] [Full Text]  
  • Rassa, J. C., Parks, G. D. (1999). Highly Diverse Intergenic Regions of the Paramyxovirus Simian Virus 5 Cooperate with the Gene End U Tract in Viral Transcription Termination and Can Influence Reinitiation at a Downstream Gene. J. Virol. 73: 3904-3912 [Abstract] [Full Text]  
  • Hardy, R. W., Harmon, S. B., Wertz, G. W. (1999). Diverse Gene Junctions of Respiratory Syncytial Virus Modulate the Efficiency of Transcription Termination and Respond Differently to M2-Mediated Antitermination. J. Virol. 73: 170-176 [Abstract] [Full Text]  
  • Fearns, R., Collins, P. L. (1999). Model for Polymerase Access to the Overlapped L Gene of Respiratory Syncytial Virus. J. Virol. 73: 388-397 [Abstract] [Full Text]  
  • Muhlberger, E., Lotfering, B., Klenk, H.-D., Becker, S. (1998). Three of the Four Nucleocapsid Proteins of Marburg Virus, NP, VP35, and L, Are Sufficient To Mediate Replication and Transcription of Marburg Virus-Specific Monocistronic Minigenomes. J. Virol. 72: 8756-8764 [Abstract] [Full Text]  
  • Atreya, P. L., Peeples, M. E., Collins, P. L. (1998). The NS1 Protein of Human Respiratory Syncytial Virus Is a Potent Inhibitor of Minigenome Transcription and RNA Replication. J. Virol. 72: 1452-1461 [Abstract] [Full Text]  
  • Hardy, R. W., Wertz, G. W. (1998). The Product of the Respiratory Syncytial Virus M2 Gene ORF1 Enhances Readthrough of Intergenic Junctions during Viral Transcription. J. Virol. 72: 520-526 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»