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Journal of Virology, September 2004, p. 9064-9072, Vol. 78, No. 17
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.17.9064-9072.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

trans-Complementation Allows Recovery of Human Respiratory Syncytial Viruses That Are Infectious but Deficient in Cell-to-Cell Transmission

A. G. P. Oomens and Gail W. Wertz*

University of Alabama School of Medicine, Birmingham, Alabama

Received 9 January 2004/ Accepted 17 March 2004

Human respiratory syncytial virus (HRSV) expresses three transmembrane glycoproteins: small hydrophobic protein SH, attachment protein G, and fusion protein F. The genes encoding SH and G can be deleted from the HRSV genome and infectious virus recovered. In contrast, HRSVs lacking the F gene or a functional replacement thereof have not been reported. To generate a system with which to study the roles of the viral transmembrane glycoproteins, including F, in the HRSV life cycle, we generated a cell line expressing a heterologous viral glycoprotein for complementation of glycoprotein function in trans. We previously demonstrated that the baculovirus GP64 protein or a chimeric form of GP64 carrying the 12 C-terminal amino acids of the HRSV F protein (GP64/F) can efficiently mediate HRSV infectivity and improve its stability, when expressed from an engineered HRSV genome. Here, we report the development of a stably transfected Vero cell line (Vbac) constitutively expressing the GP64/F protein. From the Vbac cell line, viruses that lacked the SH and F open reading frames (ORFs) or the SH, G, and F ORFs could be recovered from cDNAs. These viruses, designated RS{Delta}SH,F and RS{Delta}SH,G,F, respectively, had place-keeper ORFs inserted in place of the deleted ORFs to maintain authentic transcription levels. In the Vbac cell line, RS{Delta}SH,F and RS{Delta}SH,G,F could be amplified to near wild-type-level titers, and the resulting viruses were infectious to Vero and HEp-2 cells. After entry into Vero or HEp-2 cells, however, neither virus RS{Delta}SH,F nor virus RS{Delta}SH,G,F was able to spread in the infected cultures. Growth analyses showed that infectious virions were not produced in Vero or HEp-2 cells infected with RS{Delta}SH,F and RS{Delta}SH,G,F. Combined, these data provide direct evidence that HRSV F is an essential viral protein required for cell-to-cell transmission and demonstrate that complementation with the GP64 protein in trans constitutes a powerful tool for the study of the role of individual HRSV transmembrane glycoproteins in virus assembly, morphogenesis, and pathogenesis. In addition, the ability to generate infectious but nonspreading viruses may provide an alternative approach for the development of safe and stable HRSV vaccine candidates.

* Corresponding author. Mailing address: Department of Microbiology, The Medical School, University of Alabama at Birmingham, BBRB Room 366, 845 19th St. South, Birmingham, AL 35294. Phone: (205) 934-0877. Fax: (205) 934-1636. E-mail: gailw{at}uab.edu.

Journal of Virology, September 2004, p. 9064-9072, Vol. 78, No. 17
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.17.9064-9072.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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