Respiratory syncytial virus (RSV) is the most frequent cause of lower respiratory disease in infants, but no vaccine or effective therapy is available. The initiation of RSV infection of immortalized cells is largely dependent on cell surface heparan sulfate (HS), a receptor for the RSV attachment (G) glycoprotein in immortalized cells. However, RSV infects the ciliated cells in primary well differentiated human airway epithelial (HAE) cultures via the apical surface, but HS is not detectable on this surface. Here we show that soluble HS inhibits infection of immortalized cells, but not HAE cultures, confirming that HS is not the receptor on HAE cultures. Conversely, a “non-neutralizing” monoclonal antibody against the G protein that does not block RSV infection of immortalized cells, does inhibit infection of HAE cultures. This antibody was previously shown to block the interaction between the G protein and the chemokine receptor CX3CR1 and we have mapped the binding site for this antibody to the CX3C motif and its surrounding region in the G protein. We show that CX3CR1 is present on the apical surface of ciliated cells in HAE cultures and especially on the cilia. RSV infection of HAE cultures is reduced by an antibody against CX3CR1 and by mutations in the G protein CX3C motif. Additionally, mice lacking CX3CR1 are less susceptible to RSV infection. These findings demonstrate that RSV uses CX3CR1 as a cellular receptor on HAE cultures and highlight the importance of using a physiologically relevant model to study virus entry and antibody neutralization.

Budded virions (BV) of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) contain a major envelope glycoprotein (GP64) that is present on the plasma membrane of infected cells. GP64 is acquired by virions during budding through the plasma membrane, the final step in assembly of the budded virion at the cell surface. Previous studies (S. A. Monsma, A. G. P. Oomens, and G. W. Blissard (1996). J. Virol. 70, 4607-4616) showed that insertional inactivation of the AcMNPV gp64 gene resulted in a virus unable to move from cell to cell and nonlethal to orally infected Trichoplusia ni larvae. To determine whether GP64 is involved in virion budding, we measured BV production from Sf9 cells infected with a gp64null virus. Sf9 cells infected with gp64null virus vAc64- were pulse labeled, and progeny BV were isolated on equilibrium sucrose gradients and quantified. BV production from vAc64- was reduced to approximately 2% of that from wild-type AcMNPV. Thus the GP64 protein is important for efficient virion budding. To determine whether the highly charged 7-amino acid cytoplasmic tail domain (CTD) of GP64 was required for virion production, we generated a series of GP64 constructs containing C-terminal truncations or substitutions. Modified forms of GP64 were analyzed in transfected cells and in recombinant viruses in which the wild-type gp64 gene was replaced with a modified gp64. Deletion of 1-7 amino acids from the CTD did not affect GP64 trimerization, protein transport to the cell surface, or membrane fusion activity. However, deletions of 11 or 14 amino acids, which removed the CTD and portions of the predicted transmembrane (TM) domain, were trimerized but were present at lower levels on the cell surface due to shedding of these truncated proteins. Comparisons of growth curves and quantitative measurements of labeled progeny BV production from recombinant viruses expressing either wild-type or mutant GP64 proteins showed that deletion of the 7-residue CTD only moderately reduced the production of infectious virions ( approximately 50%). However, deletions of the C terminal 11 or 14 amino acids had more substantial effects. Removal of the C terminal 11 amino acids reduced titers of infectious virus by 78-96% and labeled progeny virions were reduced by 91-92%. Removal of 14 amino acids from the C terminus resulted in an approximately 98% reduction in progeny BV and a virus that was apparently incapable of efficient propagation in cell culture. Thus the GP64 CTD is not essential for production of infectious BV, but removal of the CTD results in a measurable reduction in budding efficiency. Deletion of the CTD plus small portions of the transmembrane domain resulted in shedding of GP64, reduced surface levels, and a dramatic reduction in the production of BV. Together, these data indicate that GP64 is an important and limiting factor in BV production.

GP64 is the major envelope glycoprotein from budded virions of the baculoviruses Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). To examine the potential role of GP64 as a viral attachment protein in host cell receptor binding, we generated, overexpressed, and characterized a soluble form of the OpMNPV GP64 protein, GP64solOp. Assays for trimerization, sensitivity to proteinase K, and reduction by dithiothreitol suggested that GP64solOp was indistinguishable from the ectodomain of the wild-type OpMNPV GP64 protein. Virion binding to host cells was analyzed by incubating virions with cells at 4 degrees C in the presence or absence of competitors, using a single-cell infectivity assay to measure virion binding. Purified soluble GP64 (GP64solOp) competed with a recombinant AcMNPV marker virus for binding to host cells, similar to control competition with psoralen-inactivated wild-type AcMNPV and OpMNPV virions. A nonspecific competitor protein did not similarly inhibit virion binding. Thus specific competition by GP64solOp for virion binding suggests that the GP64 protein is a host cell receptor-binding protein. We also examined the kinetics of virion internalization into endosomes and virion release from endosomes by acid-triggered membrane fusion. Using a protease sensitivity assay to measure internalization of bound virions, we found that virions entered Spodoptera frugiperda Sf9 cells between 10 and 20 min after binding, with a half-time of approximately 12.5 min. We used the lysosomotropic reagent ammonium chloride to examine the kinetics of membrane fusion and nucleocapsid release from endosomes after membrane fusion. Ammonium chloride inhibition assays indicated that AcMNPV nucleocapsids were released from endosomes between 15 and 30 min after binding, with a half-time of approximately 25 min.

Respiratory syncytial virus (RSV) is a major cause of severe pneumonia and bronchiolitis in infants and young children, and causes disease throughout life. Understanding the biology of infection, including virus binding to the cell surface, should help develop antiviral drugs or vaccines. The RSV F and G glycoproteins bind cell surface heparin sulfate proteoglycans (HSPGs) through heparin-binding domains. The G protein also has a CX3C chemokine motif which binds to the fractalkine receptor CX3CR1. G protein binding to CX3CR1 is not important for infection of immortalized cell lines, but reportedly is so for primary human airway epithelial cells (HAECs), the primary site for human infection. We studied the role of CX3CR1 in RSV infection with CX3CR1-transfected cell lines and HAECs with variable percentages of CX3CR1-expressing cells, and the effect of anti-CX3CR1 antibodies or a mutation in the RSV CX3C motif. Immortalized cells lacking HSPGs had low RSV binding and infection, which was increased markedly by CX3CR1 transfection. CX3CR1 was expressed primarily on ciliated cells, and ,50 % of RSV-infected cells in HAECs were CX3CR1 + . HAECs with more CX3CR1-expressing cells had a proportional increase in RSV infection. Blocking G binding to CX3CR1 with anti-CX3CR1 antibody or a mutation in the CX3C motif significantly decreased RSV infection in HAECs. The kinetics of cytokine production suggested that the RSV/CX3CR1 interaction induced RANTES (regulated on activation normal T-cell expressed and secreted protein), IL-8 and fractalkine production, whilst it downregulated IL-15, IL1-RA and monocyte chemotactic protein-1. Thus, the RSV G protein/CX3CR1 interaction is likely important in infection and infection-induced responses of the airway epithelium, the primary site of human infection.

An experimental system was developed to generate infectious human respiratory syncytial virus (HRSV) lacking matrix (M) protein expression (M-null virus) from cDNA. The role of the M protein in virus assembly was then examined by infecting HEp-2 and Vero cells with the M-null virus and assessing the impact on infectious virus production and viral protein trafficking. In the absence of M, the production of infectious progeny was strongly impaired. Immunofluorescence (IF) microscopy analysis using antibodies against the nucleoprotein (N), attachment protein (G), and fusion protein (F) failed to detect the characteristic virusinduced cell surface filaments, which are believed to represent infectious virions. In addition, a large proportion of the N protein was detected in viral replication factories termed inclusion bodies (IBs). High-resolution analysis of the surface of M-null virusinfected cells by field emission scanning electron microscopy (SEM) revealed the presence of large areas with densely packed, uniformly short filaments. Although unusually short, these filaments were otherwise similar to those induced by an M-containing control virus, including the presence of the viral G and F proteins. The abundance of the short, stunted filaments in the absence of M indicates that M is not required for the initial stages of filament formation but plays an important role in the maturation or elongation of these structures. In addition, the absence of mature viral filaments and the simultaneous increase in the level of the N protein within IBs suggest that the M protein is involved in the transport of viral ribonucleoprotein (RNP) complexes from cytoplasmic IBs to sites of budding.

To demonstrate the essential nature of the baculovirus GP64 envelope fusion protein (GP64 EFP) and to further examine the role of this protein in infection, we inactivated the gp64 efp gene of Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) and examined the biological properties of this virus in vivo. To provide GP64 EFP during construction of the recombinant GP64 EFP-null AcMNPV baculovirus, we first generated a stably transfected insect cell line (Sf9 OP64-6 ) that constitutively expressed the GP64 EFP of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV). The AcMNPV gp64 efp gene was inactivated by inserting the bacterial lacZ gene in frame after codon 131 of the gp64 efp gene. The inactivated gp64 gene was cloned into the AcMNPV viral genome by replacement of the wild-type gp64 efp locus. When propagated in the stably transfected insect cells (Sf9 OP64-6 cells), budded virions produced by the recombinant AcMNPV GP64 EFP-null virus (vAc 64Z ) contained OpMNPV GP64 EFP supplied by the Sf9 OP64-6 cells. Virions propagated in Sf9 OP64-6 cells were capable of infecting wild-type Sf9 cells, and cells infected by vAc 64Z exhibited a blue phenotype in the presence of X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside). Using cytochemical staining to detect vAc 64Z infected cells, we demonstrated that this GP64 EFP-null virus is defective in cell-to-cell propagation in cell culture. Although defective in cell-to-cell propagation, vAc 64Z produces occlusion bodies and infectious occlusion-derived virions within the nucleus. Occlusion bodies collected from cells infected by vAc 64Z were infectious to midgut epithelial cells of Trichoplusia ni larvae. However, in contrast to infection by a control virus, infection by vAc 64Z did not proceed into the hemocoel. Analysis of vAc 64Z occlusion bodies in a standard neonate droplet feeding assay showed no virus-induced mortality, indicating that occluded virions produced from vAc 64Z could not initiate a productive (lethal) infection in neonate larvae. Thus, GP64EFP is an essential virion structural protein that is required for propagation of the budded virus from cell to cell and for systemic infection of the host insect.

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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.