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J Virol, July 1998, p. 5707-5716, Vol. 72, No. 7
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Identification of the Respiratory Syncytial Virus Proteins Required for Formation and Passage of Helper-Dependent Infectious Particles

Michael N. Teng and Peter L. Collins*

Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892-0720

Received 22 January 1998/Accepted 30 March 1998

We developed a system to identify the viral proteins required for the packaging and passage of human respiratory syncytial virus (RSV) by reconstructing these events with cDNA-encoded components. Plasmids encoding individual RSV proteins, each under the control of a T7 promoter, were cotransfected in various combinations together with a plasmid containing a minigenome into cells infected with a vaccinia virus recombinant expressing T7 RNA polymerase. Supernatants from these cells were passaged onto fresh cells which were then superinfected with RSV. Functional reconstitution of RSV-specific packaging and passage was detected by expression of the reporter gene carried on the minigenome. As expected, the four nucleocapsid proteins N, P, L, and M2-1 failed to direct packaging and passage of the minigenome. Passage was achieved by further addition of plasmids expressing three membrane-associated proteins, M, G, and F; inclusion of the fourth envelope- associated protein, SH, did not alter passage efficiency. Passage was reduced 10- to 20-fold by omission of G and was abrogated by omission of either M or F. Coexpression of the nonstructural NS1 or NS2 protein had little effect on packaging and passage except through indirect effects on RNA synthesis in the initial transfection. The M2-1 transcription elongation factor was not required for the generation of passage-competent particles. However, addition of increasing quantities of M2-1 to the transfection mediated a dose-dependent inhibition of passage which was alleviated by coexpression of the putative negative regulatory factor M2-2. Omission of the L plasmid reduced passage 10- to 20-fold, most likely due to reduced availability of encapsidated minigenomes for packaging. However, the residual level of passage indicated that neither L protein nor the process of RSV-specific RNA synthesis is required for the production and passage of particles. Omission of N or P from the transfection abrogated passage. Thus, the minimum RSV protein requirements for packaging and passaging a minigenome are N, P, M, and F, although the efficiency is greatly increased by addition of L and G.

* Corresponding author. Mailing address: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, 7 Center Dr. MSC 0720, Bethesda, MD 20892-0720. Phone: (301) 496-3481. Fax: (301) 496-8312. E-mail: pcollins{at}atlas.niaid.nih.gov.

J Virol, July 1998, p. 5707-5716, Vol. 72, No. 7
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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Copyright © 1998 by the American Society for Microbiology. All rights reserved.