The 1A protein of respiratory syncytial virus is an integral membrane protein present as multiple, structurally distinct species.

The respiratory syncytial virus (RSV) 1A protein was previously identified as a 7.5-kilodalton (kDa) nonglycosylated species that, on the basis of its predicted sequence determined from the sequence of its mRNA, contains a hydrophobic central domain that was suggestive of membrane interaction. Here, four major, structurally distinct intracellular species of the 1A protein were identified in cells infected by RSV or by a recombinant vaccinia virus expressing the 1A gene. The four species of 1A were: (i) the previously described, nonglycosylated 7.5-kDa species that appeared to be the full-length, unmodified 1A protein; (ii) a nonglycosylated 4.8-kDa species that was carboxy-coterminal with the 7.5-kDa species and might be generated by translational initiation at the second AUG in the sequence; (iii) a 13- to 15-kDa species that contained one or two N-linked carbohydrate side chains of the high-mannose type; and (iv) a 21- to 30-kDa glycosylated species that appeared to be generated from the 13- to 15-kDa species by further modification of the N-linked carbohydrate. All four forms of the 1A protein were synthesized and processed on intracellular membranes, and several lines of biochemical evidence showed that all four species were integral membrane proteins. Thus, the 1A protein is a third RSV integral membrane protein and is present as such in both glycosylated and nonglycosylated forms. With the use of antiserum raised against a synthetic peptide representing the C terminus of the 1A protein, indirect immunofluorescence showed that the 1A protein was expressed at the cell surface. Antibody-antigen complexes formed at the surface of intact infected cells were immunoprecipitated, showing that the 7.5-kDa, 13- to 15-kDa, and 21- to 30-kDa, but not the 4.8-kDa, species, were accessible to extracellular antibodies. Thus, the 1A protein is a candidate to be a viral surface antigen. The small size, gene map location integral membrane association, and cell surface expression of the 1A protein strongly suggested that it is a counterpart to the SH protein that has been described for simian virus type 5. We suggest that, in the future, the RSV 1A protein be given the same designation, namely, SH.

The envelope proteins from purified respiratory syncytial virus protect mice from intranasal virus challenge.

A lyophilized subunit vaccine prepared from purified respiratory syncytial virus, which contained the envelope glycoproteins F and G and the nonglycosylated matrix protein VPM, was tested in SJL mice for its ability to protect the lungs of mice from intranasal viral challenge. Initially, the mice were injected subcutaneously with one, two, or three doses of 5 or 25 micrograms of vaccine in 50% complete Freund's adjuvant or with complete Freund's adjuvant or phosphate-buffered saline only. Although none of the mice produced neutralizing serum antibody, three doses of 25 micrograms elicited antibodies to F, G, and VPM. Despite the absence of detectable neutralizing antibodies, the lungs of 93% of the vaccinated mice were protected from intranasal viral challenge. Because the initial protocol did not elicit neutralizing antibodies and a few single-dose animals were not protected, a second vaccine trial was carried out. For these studies the priming dose was increased to 50 micrograms, which was followed, in half the vaccine recipients, by a second dose of 25 micrograms. Mice given the priming dose of vaccine produced antibody to G and showed no neutralizing activity, whereas the mice given two doses of vaccine produced antibodies to G, F, and VPM and also displayed neutralizing activity for respiratory syncytial virus. The lungs of 100% of the vaccine recipients in this trial were protected from intranasal challenge. Although the vaccine elicited antibody to VPM, this response did not correlate with protection. In addition, examination of the sera from unimmunized mice recovering from respiratory syncytial virus infection revealed a serum antibody profile similar to that noted for humans, lacking antibody to VPM. Thus, the data show that a combined glycoprotein subunit vaccine affords complete protection to viral challenge and offers an approach to develop a multivalent subunit vaccine.

Characterization of bovine respiratory syncytial virus proteins and mRNAs and generation of cDNA clones to the viral mRNAs.

We have characterized the proteins and mRNAs of bovine respiratory syncytial (BRS) virus strain 391-2 and constructed cDNA clones corresponding to 9 of the 10 BRS virus mRNAs. The proteins of BRS virus-infected cells were compared with the proteins from human respiratory syncytial (HRS) virus-infected cells. Nine proteins specific to BRS virus-infected cells, corresponding to nine HRS virus proteins, were identified. Only a BRS virus polymerase protein remains to be identified. The BRS virus G glycoprotein showed major antigenic differences from the HRS virus G glycoprotein by immunoprecipitation and Western (immuno-) blot analysis, whereas the BRS virus F, N, M, and P proteins showed antigenic cross-reactivity with their HRS virus counterparts. Analysis of RNAs from BRS virus-infected cells showed virus-specific RNAs which had electrophoretic mobilities similar to those of mRNAs of HRS virus but which hybridized poorly or not at all with HRS virus-specific probes in Northern (RNA) blot analysis. To analyze the BRS virus RNAs further, cDNA clones to the BRS virus mRNAs were generated. Nine separate groups of clones were identified and shown to correspond to nine BRS virus mRNAs by Northern blot analysis. A 10th BRS virus large mRNA was identified by analogy with the HRS virus polymerase mRNA. These data show that like HRS virus, BRS virus has 10 genes coding for 10 mRNAs.

Further characterization of the soluble form of the G glycoprotein of respiratory syncytial virus.

A soluble form of the G glycoprotein, the attachment protein, of respiratory syncytial virus is shed from infected HEp-2 cells. The Gs proteins of the Long and 18537 strains have apparent molecular sizes of 82 and 71 kilodaltons, respectively, 6 to 9 kilodaltons smaller than the virion-associated forms (Gv). The Gs protein of the Long strain was further characterized. Approximately one in six of all of the radiolabeled G molecules in these cultures at 24 h postinfection was present as the Gs protein. The Gs protein was clearly evident in culture fluids at 6 h postinfection, but the Gv protein could not be discerned until 12 h after infection, an observation that is consistent with the 12-h eclipse period for respiratory syncytial virus. Therefore, the Gs protein is shed, in part at least, from intact, infected cells and before the appearance of progeny virus. The appearance of a smaller Gs protein (74 kilodaltons) in fluids of infected calls which were incubated with tunicamycin shows that addition of N-linked oligosaccharides is not required for the genesis and shedding of the Gs protein. Sequencing of the amino terminus of purified Gs protein revealed two different termini, whose generations are consistent with cleavages of the full-length G protein between amino acids 65 and 66 and between residues 74 and 75. This result suggests that the Gs protein is present in two different forms which lack the proposed intracytoplasmic and transmembrane domains of the full-length G protein.

The purification of four respiratory syncytial virus proteins and their evaluation as protective agents against experimental infection in BALB/c mice.

The fusion (F) glycoprotein, large glyco- (G) protein, phospho- (P) protein and 22K protein of respiratory syncytial (RS) virus A2 strain were purified by a combination of immunoaffinity adsorption and preparative SDS-PAGE. All four proteins elicited serum antibody in mice after repeated inoculation in adjuvant, although the magnitude of the response as measured by ELISA varied from mouse to mouse. The F protein generated neutralizing antibodies in only 50% of the mice determined to be seropositive by ELISA. The G protein also induced neutralizing antibodies but in this instance neutralization tests and ELISA titres were more closely correlated. No neutralizing activity was detected in mice immunized with the P or 22K proteins although all produced antibody detectable by ELISA. Mice immunized with either the F or the G protein were found to be protected against subsequent RS virus challenge, whether they had developed neutralizing antibody or not. Mice inoculated with the P or 22K proteins were not protected.

Monoclonal antibodies demonstrate heterogeneity in the G glycoprotein of prototype strains and clinical isolates of respiratory syncytial virus.

In order to study variation among prototype strains and clinical isolates of respiratory syncytial (RS) virus, four prototype strains (Long, A2, CH18537, 9320) were used to produce monoclonal antibodies to this virus. The majority of monoclonals reacted with all four prototype strains by fluorescent antibody staining. Among the non-cross-reacting monoclonals, five additional patterns of reactivity with the prototype strains were recognized. Fourteen monoclonals, including ones representative of each of the patterns of reactivity with the prototype strains, were selected to use for typing prototype strains and community isolates. All 14 were found by immunoprecipitation to recognize the RS virus G glycoprotein. These monoclonals could uniquely identify each of the prototype strains. In addition to the antigenic differences among the prototype strains detected by the monoclonals, differences were also detected in the migration of the G glycoprotein of the prototype strains in polyacrylamide gel electrophoresis. Fluorescent antibody staining with panels of monoclonals distinguished two antigenic types among 114 isolates of RS virus recovered from children in St. Louis during the period 1981-86. The predominant type (80% of isolates) had a pattern of reactivity that resembled but differed from that of either the Long or A2 strains. The second type had a pattern of reactivity identical with that of 9320. The possible significance of this heterogeneity must be considered in developing diagnostic tests as well as active or passive immunotherapy for infections caused by RS virus.

Expression of the respiratory syncytial virus 22K protein on the surface of infected HeLa cells.

Immunofluorescent staining of unfixed respiratory syncytial virus-infected HeLa cells with monoclonal antibodies (MAbs) demonstrated that the 22K protein is expressed on the cell membrane along with the fusion (F) protein and large glycoprotein (G). All three proteins were detected in the cytoplasm at 17 h post-infection and in the case of the F and G proteins this coincided with their appearance on the cell surface. However, the 22K protein could not be detected on the surface until approximately 16 h after its detection in the cytoplasm, when cytopathic effect was extensive. No evidence for the surface expression of the phosphoprotein (P), matrix (M) or nucleocapsid (N) proteins was found. Trypsin treatment of infected cells prior to unfixed immunofluorescent staining and Western blot analysis indicated that, unlike the G protein, the quantity of 22K protein detected on the cell surface constituted only a small proportion of the total present in the cell. A comparison of the patterns of immunofluorescent staining produced by MAbs on acetone-fixed infected cells suggested that the N, P and 22K proteins, but not the M protein, may be associated with the same intracellular structures.

Heterogeneity of the respiratory syncytial virus 22K protein revealed by Western blotting with monoclonal antibodies.

Respiratory syncytial (RS) virus-infected HeLa, HEp-2, Vero and BS-C-1 cell lysates were electrophoresed on SDS-polyacrylamide gels under reducing conditions and analysed by Western blotting and immunoperoxidase using monoclonal antibodies specific for the 22K protein (relative mol. wt. of 23,000 in our gel system). Three novel polypeptides with mol. wt. of 24,000, 21,000 and 17,000 were stained in addition to the 23,000 polypeptide which was present in the greatest amount in all three virus strains tested regardless of host cell line. When samples were electrophoresed under non-reducing conditions each of the three higher mol. wt. polypeptides seen in reducing gels migrated as two bands (total of six bands) with altered electrophoretic mobilities. In experiments using the alkylating agent iodoacetamide under conditions where the novel 24,000, 21,000 and 17,000 polypeptides were not visible, the number of mobility variants of the 23,000 polypeptide which could be detected in non-reducing conditions was increased from two to four. At least one, and possibly three, of these variants was the result of conformational variation in the 23,000 polypeptide caused by the generation or rearrangement of intrachain disulphide bonds after the infected cells were lysed in SDS-PAGE sample buffer. Post-lysis conformational changes were minimized by treatment of the infected cells with iodoacetamide before solubilization or by decreasing the SDS concentration or using milder detergents in the lysis buffer.

Structural differences between subtype A and B strains of respiratory syncytial virus.

Differences in the properties of homologous intracellular structural components of eight strains of subtype A and eight strains of subtype B of human respiratory syncytial (RS) virus were examined. The size of the fusion (F) protein cleavage products and the phosphoprotein (P) showed systematic differences between virus strains representing the two subtypes. The apparent mol. wt. in SDS-polyacrylamide gels under reducing conditions was 48,000 (48K) and 46K to 47K for the cleavage product F1 in subtype A and B strains, respectively. The size of the F2 protein was 18K to 20K. The subtype B strains showed a slightly higher mol. wt. of this protein compared to the subtype A strains. The size of the P protein was 36K in subtype A strains, but only 34K in subtype B strains. Variations also occurred in the size of the glycoprotein (G) and the 22K to 24K structural protein. These variations did not correlate with the virus subtypes, but were strain-specific. The size of non-glycosylated forms of the F protein cleavage products was determined by use of material from tunicamycin-treated cells. A 44K to 45K non-glycosylated form of the F1 protein was detected with subtype A virus strains, but the corresponding protein of subtype B strains was not reproducibly identified, presumably due to instability in the absence of glycosylation or altered antigenicity. Monoclonal antibody immunosorbent-bound viral glycoproteins were partially digested with proteases. The pattern of breakdown products of the F1 protein was distinctly different between subtype A and B strains, but it was similar among strains of the same subtype. No subtype-specific pattern was seen in proteolytic digests of monoclonal antibody-bound G protein.

Nucleotide sequence of the gene encoding the fusion glycoprotein of Newcastle disease virus.

The nucleotide sequence of the gene encoding the fusion (F) glycoprotein of the Beaudette C strain of Newcastle disease virus (NDV) has been determined from cDNA clones obtained from virion RNA. The gene is 1792 nucleotides long, including mRNA start and polyadenylation signals typical of paramyxoviruses. The single open reading frame encodes a polypeptide of 553 amino acids, with a predicted molecular weight of 59042. The F polypeptide has three regions of high hydrophobicity: an N-terminal signal peptide, the N terminus of F1 (known from protein sequencing) and a C-terminal membrane-spanning region by which the F glycoprotein is anchored to the membrane. The cleavage site of F0 is located in a highly basic region of the F polypeptide. Five potential asparagine-linked glycosylation sites are present in the amino acid sequence, of which one is in F2 and the others in F1. Comparison of the NDV F amino acid sequence to those from other paramyxoviruses reveals homology to Sendai virus, simian virus 5 and human respiratory syncytial virus. There is also limited homology between the N terminus of F1 of NDV and the N termini of HA2 of influenza viruses. Post-translational modifications of the NDV F polypeptide are discussed in the light of information provided by the amino acid sequence.

Sequence analysis of the P and C protein genes of human parainfluenza virus type 3: patterns of amino acid sequence homology among paramyxovirus proteins.

The complete nucleotide sequence of the P + C mRNA of human parainfluenza virus type 3 (PF3) was determined by sequencing cDNA, viral genomic RNA and mRNA. The P + C mRNA is 2009 nucleotides in length, exclusive of poly(A), and contains two overlapping open reading frames (ORFs). The P + C mRNA encodes two proteins, the 602 amino acid nucleocapsid phosphoprotein P and the 199 amino acid non-structural protein C. Peptide mapping confirmed that the two proteins are unrelated. Hybrid-arrest translation experiments assigned each of the two proteins to its respective ORF. These studies showed that the coding strategy of the PF3 P + C mRNA is similar to that of Sendai virus. Amino acid sequence alignment showed that the P and C proteins of PF3 and Sendai virus represent homologous pairs. However, these homologies are represented by high contents of accepted amino acid substitutions and by similarity in hydropathy profiles rather than by high contents of exact amino acid matches. Homology with the P and C proteins of measles, canine distemper and respiratory syncytial viruses was at the threshold of significance. The patterns of amino acid sequence homology among the paramyxovirus HN, F, NP, P and C proteins are compared.

Detection of respiratory syncytial virus in nasopharyngeal secretions by enzyme-linked immunosorbent assay, indirect immunofluorescence, and virus isolation: a comparative study.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of respiratory syncytial virus (RSV) antigens in nasopharyngeal secretions (NPS) from children with acute respiratory disease. Antisera against RSV nucleocapsids were used as immunoreagents for this test system. The results obtained by RSV antigen ELISA were compared to those of indirect immunofluorescence (IF) and tissue culture virus isolation (TC). Of the 404 NPS obtained, 278 were tested in parallel by ELISA and IF and 205 by ELISA and TC, and 89 were screened in parallel by all three methods. The sensitivity of ELISA in relation to IF was 86.7%, the specificity 95.7%. Sensitivity and specificity obtained by ELISA were 89.9% and 94.4%, respectively, compared to TC. False-negative results were obtained with all three test systems used.

Murine pneumonia virus: seroepidemiological evidence of widespread human infection.

More than 75% of a random sample of adult human sera exhibited moderate to high murine pneumonia virus (PVM)-neutralizing activity. There was no correlation between PVM-neutralizing activity and respiratory syncytial virus or parainfluenza type 3 virus-neutralizing activities of the same sera. In children the proportion of sera with moderate to high titres increased with age, indicating early exposure to infection. Seroconversion (i.e. greater than fourfold increase in titre) was observed in four of 108 paired samples of previously undiagnosed respiratory infections. These observations suggest that the human population is frequently exposed to infection with PVM or an antigenically related virus. The sera of patients suffering from Paget's disease of bone tended to exhibit higher than normal PVM-neutralizing titres in comparison with the sera of patients with other bone diseases. Thus, PVM (or an antigenically related virus) resembles some other parainfluenza viruses in being circumstantially associated with Paget's disease of bone.

Immunovirological studies on human respiratory syncytial virus structural proteins.

Immunovirological studies suggest that human respiratory syncytial virus may well be composed of five structural proteins as are other members of the Paramyxoviridae family: the two external membrane glycoproteins H (90 000) and Fo (F1, 49 000; F2, 20 000; disulfide linked), the internal membrane protein M (34 000), the nucleoprotein N (42 000), and a protein (78 000) designated P that could be the equivalent of the polymerase of the morbillivirus and paramyxovirus genus. Neutralizing monoclonal antibodies showed, by immunoprecipitation and immunoblotting, that the fusion protein carries neutralizing epitopes. One monoclonal antibody, which shows a high neutralizing titer, immunoblotted directly with the F1 fragment (49 000) of the fusion protein. Analysis in mice of the immunogenicity of the structural proteins separated on sodium dodecyl sulphate gels indicated that, under our conditions, only the fusion protein dimer Fo and its F1 fragment were capable of inducing neutralizing antibodies.

Rapid detection of respiratory syncytial virus in nasopharyngeal secretions by enzyme-linked immunosorbent assay.

A new enzyme-linked immunosorbent assay (ELISA) respiratory syncytial virus antigen detection kit (Ortho Diagnostics, Inc., Raritan, N.J.) was compared with virus culture and with the indirect fluorescent antibody test (FAT) by using fresh nasal washings from children with suspected respiratory syncytial virus infection. Both uncentrifuged nasal washings and pellets from centrifuged split specimens were examined by ELISA. The ELISA was considered positive when the optical density was greater than the mean background optical density plus 0.15. Specimens positive by ELISA but negative by culture and FAT were reexamined in an ELISA blocking assay. Of 337 uncentrifuged specimens, 124 (37%) were positive by culture, 107 (32%) were positive by FAT, and 166 (49%) were positive by ELISA. Blocking assays showed that 21 of 30 (70%) of the seemingly false-positive ELISA tests were, in fact, true-positives and that the cultures and FATs were falsely negative. A patient specimen was considered positive when it was positive by virus culture, FAT, or blocking assay. The sensitivity, specificity, and positive predictive value of the ELISA test were 88, 94, and 95%, respectively. Centrifugation of nasal washings raised the sensitivity from 88 to 92% but reduced the specificity from 94 to 81%. We conclude that the Ortho ELISA test of uncentrifuged nasal washings is more sensitive than virus culture or indirect FAT and is highly specific.

Purification and characterization of the respiratory syncytial virus fusion protein.

The fusion protein of respiratory syncytial virus was purified by affinity chromatography using a monoclonal antibody. Under various conditions the protein was recovered as a 145K dimer or a 70K monomer. The 70K monomer was composed of disulphide-linked fragments of 48K and 23K. Polyclonal rabbit serum produced to the dimerized fusion protein neutralized virus but did not inhibit fusion, while rabbit serum to the 2-mercaptoethanol-treated dimerized protein neutralized virus and inhibited fusion of infected cells. Only the latter serum strongly recognized the 23K fragment when studied by Western blot analysis.

Respiratory syncytial virus polypeptides. IV. The oligosaccharides of the glycoproteins.

The cell-associated glycoproteins of respiratory syncytial (RS) virus included GP1 (90K), VP70 (70K), VGP48 (48K) and GP26 (26K). Although present in infected cells, there was no VP70 in purified virus. Trypsin treatment of infected cells removed 80 to 90% of VP70 as well as its products VGP48 and GP26. This suggested that most of the VP70 in the cell is located on the plasma membrane. The glycoproteins of purified RS virus (GP1, VGP48 and GP26) contain mannose, galactose and fucose as well as glucosamine, but the quantity of mannose in GP1 is low when compared to that of the other three sugars. The effects that follow the treatment of infected cells with the glycosylation inhibitors tunicamycin and monensin, and the treatment of the immunoprecipitated product of pulse-chase experiments with endonuclease H demonstrated that VP70 and its products contained N-linked oligosaccharides, and that the oligosaccharides of the mature VGP48 subunit were of the complex type, while GP1 contained both N- and O-linked oligosaccharides. The non-glycosylated forms of VP70 and GP1 have estimated mol. wt. of 50K and 33K respectively. Therefore, the carbohydrate contribution to the mol. wt. of VP70 and GP1, as determined by PAGE, was equivalent to 20K for the former and 57K for the latter. The majority of the GP1 oligosaccharides were O-linked, a form of sugar linkage not previously found among paramyxoviruses.

Characterization of the 10 proteins of human respiratory syncytial virus: identification of a fourth envelope-associated protein.

A total of 13 respiratory syncytial (RS) virus specific polypeptides were identified by pulse-chase metabolic labeling of infected HEp-2 cells. Ten of the 13 proteins were shown to be unique. They were the L, G, F (F1, F2), N, P, M, 24K, 14K, 11K and 9.5K proteins. These conclusions were based on peptide mapping and on previous work showing that each of 10 polypeptides are coded for by a unique mRNA. The seven largest proteins, L, G, F (F1, F2), N, P, M and 24K were identified clearly as virion structural proteins. The 24K protein was characterized by detergent and salt dissociation studies as an envelope-associated protein, bringing to four (G, F (F1, F2), M and 24K) the number of membrane associated proteins for RS virus. A fourth membrane-associated protein has not been described previously for any other paramyxovirus. Of the three smallest proteins, the 14K and 11K were characterized as non-structural proteins. The 9.5K protein was detected in low amounts in highly purified preparations of virions.

Antigenic and structural variation in the major nucleocapsid protein of respiratory syncytial virus.

Antigenic and structural variation in the major nucleocapsid protein, VPN41, from different strains of respiratory syncytial (RS) virus was observed using a combination of monoclonal antibodies and two-dimensional peptide mapping. Limited trypsin treatment of intact nucleocapsids produced two peptide fragments Mr 27K and Mr 14K. Two monoclonal antibodies, N1 and N2, reactive with primary sequence epitopes located on intact nucleocapsids also reacted with either the 27K fragment (N2) or the 14K fragment (N1). Competitive radioimmunoassay studies using N1 and N2 antibodies revealed two discrete antigenic groups among the seven human strains of RS virus examined. A bovine strain of RS virus, although antigenically similar to the human strain of RS virus, was placed in a separate group. Two-dimensional peptide mapping of 125I-labelled VPN41 purified by SDS-PAGE revealed extensive structural homology between all strains. However, several unique tryptic/chymotryptic peptides supported the grouping obtained with the monoclonal antibodies.