The Baculovirus GP64 Protein Mediates Highly Stable Infectivity of a Human Respiratory Syncytial Virus Lacking Its Homologous Transmembrane Glycoproteins

Abstract

Baculovirus GP64 is a low-pH-dependent membrane fusion protein required for virus entry and cell-to-cell transmission. Recently, GP64 has generated interest for practical applications in mammalian systems. Here we examined the membrane fusion function of GP64 from Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expressed in mammalian cells, as well as its capacity to functionally complement a mammalian virus, human respiratory syncytial virus (HRSV). Both authentic GP64 and GP 64/F , a chimeric protein in which the GP64 cytoplasmic tail domain was replaced with the 12 C-terminal amino acids of the HRSV fusion (F) protein, induced low-pH-dependent cell-cell fusion when expressed transiently in HEp-2 (human) cells. Levels of surface expression and syncytium formation were substantially higher at 33°C than at 37°C. The open reading frames (ORFs) encoding GP64 or GP 64/F , along with two marker ORFs encoding green fluorescent protein (GFP) and ␤-glucuronidase (GUS), were used to replace all three homologous transmembrane glycoprotein ORFs (small hydrophobic SH, attachment G, and F) in a cDNA of HRSV. Infectious viruses were recovered that lacked the HRSV SH, G, and F proteins and expressed instead the GP64 or GP 64/F protein and the two marker proteins GFP and GUS. The properties of these viruses, designated RS⌬SH,G,F/GP64 or RS⌬SH,G,F/GP 64/F , respectively, were compared to a previously described HRSV expressing GFP in place of SH but still containing the wild-type HRSV G and F proteins (RS⌬SH [A. G. Oomens, A. G. Megaw, and G. W. Wertz, J. Virol., 77:3785-3798, 2003]). By immunoelectron microscopy, the GP64 and GP 64/F proteins were shown to incorporate into HRSV-induced filaments at the cell surface. Antibody neutralization, ammonium chloride inhibition, and replication levels in cell culture showed that both GP64 proteins efficiently mediated infectivity of the respective viruses in a temperature-sensitive, low-pH-dependent manner. Furthermore, RS⌬SH,G,F/GP64 and RS⌬SH,G,F/GP 64/F replicated to higher levels and had significantly higher stability of infectivity than HRSVs containing the homologous HRSV G and F proteins. Thus, GP64 and a GP64/HRSV F chimeric protein were functional and efficiently complemented an unrelated human virus in mammalian cells, producing stable, infectious virus stocks. These results demonstrate the potential of GP64 for both practical applications requiring stable pseudotypes in mammalian systems and for studies of viral glycoprotein requirements in assembly and pathogenesis.

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.…”

“…While M-containing virus (designated recWT) was amplified to a relatively high titer in two passages in HEp-2 cells, the M-lacking virus (designated M null) required four passages in H2-M cells to obtain a titer of 1 ϫ 10 5 PFU/ml. Virus stocks used for the experiments described above were verified by reverse transcription-PCR on RNA harvested from infected cells at passage 5 (M null) or passage 3 (recWT), followed by sequence analysis as previously described (44). No unintended changes were found (data not shown).…”

“…In this experiment, we examined the impact of the absence of M on the subcellular targeting of In the "M-null" virus, the M ORF was replaced with a modified M ORF in which AUGs were mutated to ablate expression, and the SH ORF was replaced with the EGFP ORF for tracking purposes. (A virus with the SH ORF being replaced with that of EGFP was previously shown to replicate in a manner indistinguishable from that of the wt virus [10,43,44].) The properties of the M-null virus were compared to those of the "recWT" control virus.…”

“…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.…”

“…Relative protein expression levels were determined by a cell enzyme-linked immunosorbent assay (ELISA) as previously described (42,44), with minor modifications. Briefly, infected cells were fixed at 26 h postinfection (hpi) and permeabilized with 0.2% Triton (for N, G, and F) or 0.1% SDS (for M).…”