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

Abstract

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 (GP ^64/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 GP ^64/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 RSΔ sh,f and RSΔ 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, RSΔ sh,f and RSΔ 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 RSΔ sh,f nor virus RSΔ 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 RSΔ sh,f and RSΔ 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.