In 1995 we described an intracellular transcription and replication system for respiratory syncytial virus (RSV) based on cDNA-encoded components. Tissue culture cells were transfected with (i) a plasmid encoding a helper-dependent RNA analog of either negative-sense genomic RNA or of positive-sense replicative intermediate (antigenome) RNA (called an RSV-CAT """"""""minigenome"""""""" or """"""""mini-antigenome"""""""", respectively), and (ii) various plasmids which each encode one of the RSV proteins. Expression of the plasmids was directed by T7 RNA polymerase supplied by a vaccinia virus recombinant. Three proteins, the nucleocapsid N, phosphoprotein P and large polymerase subunit L proteins, were necessary and sufficient for RNA replication (the synthesis of genome and antigenome). Unexpectedly, transcription (synthesis of subgenomic mRNA) by these three proteins alone yielded prematurely terminated mRNA. The coexpression of """"""""catalytic"""""""" amounts of the 5'-terminal ORF of the M2 mRNA restored authentic transcription. Thus, contrary to expectations, the RSV """"""""replicase"""""""" and """"""""transcriptase"""""""" are not identical: the latter requires a processivity factor. The naturally-occurring M2 mRNA contains a second, internal ORF which slightly overlaps the first and lacks a known protein product. Expression of ORF2 in the presence of L, N and P (with or without M2 ORF1) inhibited RNA replication and transcription. The NS1 protein also was a very potent inhibitor of RNA synthesis, whereas the M, SH, G and F proteins lacked detectable effect on RNA synthesis. The relative level of transcription versus RNA replication was insensitive to increase in the level of intracellular N and P protein. This showed that the shift from transcription to RNA replication is not mediated by increase in the intracellular accumulation of these proteins, in contrast to an accepted (but unproven) model for RNA replication of nonsegmented negative strand viruses. Preliminary experiments indicated that transmissible RSV-CAT particles were produced when the mix of complementing proteins included the four envelope-associated proteins, M, SH, F and G, in addition to N, P, L and M2 ORF1.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Intramural Research (Z01)
Project #
1Z01AI000498-10
Application #
2566787
Study Section
Special Emphasis Panel (LID)
Project Start
Project End
Budget Start
Budget End
Support Year
10
Fiscal Year
1996
Total Cost
Indirect Cost
City
State
Country
United States
Zip Code
McGivern, David R; Collins, Peter L; Fearns, Rachel (2005) Identification of internal sequences in the 3' leader region of human respiratory syncytial virus that enhance transcription and confer replication processivity. J Virol 79:2449-60
Zhang, Liqun; Bukreyev, Alexander; Thompson, Catherine I et al. (2005) Infection of ciliated cells by human parainfluenza virus type 3 in an in vitro model of human airway epithelium. J Virol 79:1113-24
Schomacker, Henrick; Collins, Peter L; Schmidt, Alexander C (2004) In silico identification of a putative new paramyxovirus related to the Henipavirus genus. Virology 330:178-85
Tran, Kim C; Collins, Peter L; Teng, Michael N (2004) Effects of altering the transcription termination signals of respiratory syncytial virus on viral gene expression and growth in vitro and in vivo. J Virol 78:692-9
Spann, Kirsten M; Collins, Peter L; Teng, Michael N (2003) Genetic recombination during coinfection of two mutants of human respiratory syncytial virus. J Virol 77:11201-11
Kotelkin, Alexander; Prikhod'ko, Elena A; Cohen, Jeffrey I et al. (2003) Respiratory syncytial virus infection sensitizes cells to apoptosis mediated by tumor necrosis factor-related apoptosis-inducing ligand. J Virol 77:9156-72
Zhang, Liqun; Peeples, Mark E; Boucher, Richard C et al. (2002) Respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology. J Virol 76:5654-66
Teng, Michael N; Collins, Peter L (2002) The central conserved cystine noose of the attachment G protein of human respiratory syncytial virus is not required for efficient viral infection in vitro or in vivo. J Virol 76:6164-71
Techaarpornkul, Sunee; Collins, Peter L; Peeples, Mark E (2002) Respiratory syncytial virus with the fusion protein as its only viral glycoprotein is less dependent on cellular glycosaminoglycans for attachment than complete virus. Virology 294:296-304
Gower, T L; Peeples, M E; Collins, P L et al. (2001) RhoA is activated during respiratory syncytial virus infection. Virology 283:188-96

Showing the most recent 10 out of 19 publications