Infectivity of a Human Respiratory Syncytial Virus Lacking the SH, G, and F Proteins Is Efficiently Mediated by the Vesicular Stomatitis Virus G Protein

Abstract

To examine the requirements of the human respiratory syncytial virus (HRSV) SH (small hydrophobic), G (attachment), and F (fusion) proteins for virus infectivity and morphology, we used the prototype A2 strain of HRSV to generate a series of cDNAs from which (i) the SH open reading frame (ORF), (ii) the SH and G ORFs, or (iii) the SH, G, and F ORFs were deleted. Each deleted ORF was replaced as follows: the SH ORF was replaced with that of green fluorescent protein; the G ORF was replaced with that of G vsv , a chimeric glycoprotein consisting of the vesicular stomatitis Indiana virus (VSIV) G protein ecto-and transmembrane domains coupled to the HRSV F cytoplasmic tail; and the F ORF was replaced with that of marker protein ␤-glucuronidase. The number of genes and the intergenic junctions in the constructs were kept as found in A2 virus in order to maintain authentic levels of transcription. Infectious viruses were recovered from all three engineered cDNAs and designated RS⌬SH, RS⌬SH,G/G vsv , and RS⌬SH,G,F/G vsv , respectively. Low-pH-induced syncytium formation was observed in cells infected with viruses RS⌬SH,G/G vsv and RS⌬SH,G,F/G vsv , indicating that G vsv was expressed and functional. Neutralization of infectivity by anti-VSIV G antibodies and inhibition of entry by ammonium chloride showed that RS⌬SH,G,F/G vsv infectivity was mediated by G vsv and that an acidification step was required for entry into the host cell, similar to VSIV virions. All three engineered viruses displayed growth kinetics and virus yields similar to a wild-type A2 virus, both in Vero and HEp-2 cells. Abundant virus-induced filaments were observed at the surface of cells infected with each of the three engineered viruses or with virus A2, indicating that neither the SH and G proteins nor the F protein ecto-and transmembrane domains were required for the formation of these structures. This is the first report of the recovery of an infectious HRSV lacking a fusion protein of the Paramyxoviridae family and of manipulation of the HRSV entry pathway via incorporation of a nonparamyxoviral transmembrane glycoprotein.

The Human Respiratory Syncytial Virus Matrix Protein Is Required for Maturation of Viral Filaments

“…In this cDNA, the SH ORF was replaced with that of EGFP to allow the monitoring of infectious-virus recovery and cell-to-cell spread. SH was previously shown to be dispensable for virus replication in cell cultures, and EGFP expression from the SH location was shown to be an accurate and stable indicator of infectivity correlating with the number of PFU (10,43,44). While previous studies did not suggest a major role for the SH protein in viral assembly (2,3,(42)(43)(44)58), the reader should keep in mind that these studies were carried out in its absence.…”

“…2A). Note that the recWT cDNA is a second-generation cDNA with SH being replaced with EGFP, engineered such that artificial restriction sites (to facilitate glycoprotein exchange) contained within the 2003 version (43) are no longer present.…”

“…In previous work, we generated a cDNA (A2 strain) in which the SH ORF was replaced with that of EGFP (pRS⌬SH) (43). The virus recovered from cDNA pRS⌬SH (which contained artificial restriction sites to facilitate glycoprotein gene exchange) replicated to levels indistinguishable from those of an unaltered A2 virus, and EGFP expression from this location was shown previously to be an accurate indicator of infectivity correlating with the number of PFU (10,43,44). For this project, a second-generation cDNA was constructed which also had an exact replacement of the SH ORF with that of EGFP but which did not contain artificial restriction sites.…”

“…Total (cell-associated and released) progeny virus was harvested immediately after infection and at 1-day intervals thereafter by scraping cells into the medium and storing them at Ϫ80°C. Samples were assayed simultaneously by flow cytometry as previously described (43). Briefly, samples (20% of the total volume harvested) were thawed, mixed by gentle pipetting, cleared by low-speed centrifugation (5 min at 750 ϫ g), and used to infect freshly plated (receiver) HEp-2 cells.…”

“…These viral filaments are produced at the cell surface late in the infection cycle and remain largely attached to the infected cells, where they can be readily visualized by immunofluorescence (IF) or electron microscopy (EM). Viral filaments range in length from 2 to 8 m; contain high concentrations of N, G, and F proteins; and often appear as aggregates (1,16,31,43,51,53,55). In a previous study where anti-RhoA drugs were used to block the formation of viral filaments, the virion morphology was reported to shift from predominantly filamentous to predominantly round, without affecting the total yield of infectivity (22).…”