Identification of protein regions involved in the interaction of human respiratory syncytial virus phosphoprotein and nucleoprotein: significance for nucleocapsid assembly and formation of cytoplasmic inclusions.

We have reported previously that the nucleoprotein (N), the phosphoprotein (P), and the 22-kDa protein of human respiratory syncytial virus (HRSV) are components of the cytoplasmic inclusion bodies observed in HEp-2-infected cells. In addition, coexpression of N and P was sufficient to induce the formation of N-P complexes detectable by either coimmunoprecipitation with anti-P antibodies or generation of cytoplasmic inclusions. We now report the identification of protein regions required for these interactions. Deletion mutant analysis of the P protein gene indicated that its C-terminal end was essential for interacting with N. This conclusion was strengthened by the finding that an anti-P monoclonal antibody (021/12P), reacting with a 21-residue P protein C-terminal peptide, apparently displaced N from N-P complexes. The same effect was observed with high concentrations of the C-terminal peptide. However, sequence requirements for the P protein C-terminal end were not absolute, and mutants with the substitution Ser-237-->Ala or Ser-237-->Thr were as efficient as the wild type in interacting with N. In addition, P and N proteins from strains of different HRSV antigenic groups, with sequence differences in the P protein C-terminal end, were able to coimmunoprecipitate and formed cytoplasmic inclusions. Deletion mutant analysis of the N gene indicated that large segments of this polypeptide were required for interacting with P. The relevance of these interactions for HRSV is discussed in comparison with those of analogous proteins from related viruses.     

Production of polyclonal antibodies against the human respiratory syncytial virus nucleoprotein and phosphoprotein expressed in Escherichia coli

The nucleoprotein (N) and the phosphoprotein (P) of the human respiratory syncytial virus (HRSV), A2 strain, were cloned into pMAL-c2e vector. The proteins were expressed fused with the maltose-binding protein (MBP) and were preferentially found in the soluble fraction of the bacterial lysate. After their purification using amylose resin, almost no other protein was detected in SDS-PAGE. The fused proteins were cleaved by digestion with enterokinase and then used as antigens for BALB/c mice immunization. The obtained polyclonal antibodies were tested against HRSV infected cells in immunofluorescence assays. The results indicate that the antibodies generated against the recombinant proteins were able to recognize the virus proteins. We now intend to purify the cleaved N and P proteins and use them in structural studies. The recombinant proteins will also be tested as potential inducers of a protective immunity against the HRSV.     

Identification of the virus-specific proteins of respiratory syncytial virus temperature-sensitive mutants by immunoprecipitation

The proteins of Long strain RSV and three temperature-sensitive (ts) mutants of the A2 strain were compared by pulse labeling virus-infected cells with [35S]methionine and [3H]glucosamine followed by analysis of the cell lysates by polyacrylamide gel electrophoresis. At the permissive temperature (30 degrees) proteins ranging in molecular weight from 24,000 to 50,000 (VP24, VP27, VP33, VP44) could be identified. Immunoprecipitation of viral lysates by immune rabbit serum demonstrated antigenic similarity with VP27, VP44, VP50, and VP67 in all ts mutants and Long strain RSV. [3H]Glucosamine labeling demonstrated glycoproteins of 90,000 (GP90) and 50,000 (GP50) in Long strain and GP90 in the ts mutants.     

The major phosphorylation sites of the respiratory syncytial virus phosphoprotein are dispensable for virus replication in vitro

The phosphoprotein (P protein) of respiratory syncytial virus (RSV) is a key component of the viral RNA-dependent RNA polymerase complex. The protein is constitutively phosphorylated at the two clusters of serine residues (116, 117, and 119 [116/117/119] and 232 and 237 [232/237]). To examine the role of phosphorylation of the RSV P protein in virus replication, these five serine residues were altered to eliminate their phosphorylation potential, and the mutant proteins were analyzed for their functions with a minigenome assay. The reporter gene expression was reduced by 20% when all five phosphorylation sites were eliminated. Mutants with knockout mutations at two phosphorylation sites (S232A/S237A [PP2]) and at five phosphorylation sites (S116L/S117R/S119L/S232A/S237A [PP5]) were introduced into the infectious RSV A2 strain. Immunoprecipitation of (33)P(i)-labeled infected cells showed that P protein phosphorylation was reduced by 80% for rA2-PP2 and 95% for rA2-PP5. The interaction between the nucleocapsid (N) protein and P protein was reduced in rA2-PP2- and rA2-PP5-infected cells by 30 and 60%, respectively. Although the two recombinant viruses replicated well in Vero cells, rA2-PP2 and, to a greater extent, rA2-PP5, replicated poorly in HEp-2 cells. Virus budding from the infected HEp-2 cells was affected by dephosphorylation of P protein, because the majority of rA2-PP5 remained cell associated. In addition, rA2-PP5 was also more attenuated than rA2-PP2 in replication in the respiratory tracts of mice and cotton rats. Thus, our data suggest that although the major phosphorylation sites of RSV P protein are dispensable for virus replication in vitro, phosphorylation of P protein is required for efficient virus replication in vitro and in vivo.     

Sequence analysis of the respiratory syncytial virus phosphoprotein gene.

A recombinant cDNA plasmid (pRSA3) containing an almost full-length copy of the mRNA encoding respiratory syncytial virus phosphoprotein was identified in a cDNA library prepared with mRNA from respiratory syncytial virus-infected cells. The cDNA insert was sequenced, and a protein of 27,150 daltons was deduced from the DNA sequence. The protein is relatively acidic, containing two clusters of acidic amino acids, one in the middle of the molecule and the other at the C-terminus. It is devoid of both cysteine and tryptophan. There was no other potential reading frame within the phosphoprotein gene of respiratory syncytial virus. This situation is unlike that with Sendai virus, a paramyxovirus, which has a nonstructural C protein encoded by a second overlapping reading frame near the 5' end of the mRNA for phosphoprotein.     

Seven complementation groups of respiratory syncytial virus temperature-sensitive mutants.

Fifteen temperature-sensitive mutants of the RSN-2 strain of respiratory syncytial virus have been classified into six complementation groups, two of which appeared to be homologous with two of the three complementation groups of the A2 strain described by Wright et al. (P. F. Wright, M. A. Gharpure, D. S. Hodes, and R. M. Chanock, Arch. Gesamte Virusforsch, 41:238--247). Thus seven complementation groups of respiratory syncytial virus, designated A, B, C, D, E, F, and G, have been defined. The frequency and type of mutant isolated varied according to strain; group C was unique to the A2 strain, and groups D, E, F, and G were unique to the RSN-2 strain. The highest complementation indexes were obtained by preincubation for 7 h at permissive temperature, followed by incubation at restrictive temperature for 40 to 50 h in the case of A2 strain mutants or 80 to 90 h for RSN-2 strain mutants. Genetic recombination was not detected.     

Natural killer cells are involved in acute lung immune injury caused by respiratory syncytial virus infection

It is known that respiratory syncytial virus (RSV) is the main cause of bronchiolitis and pneumonia in young children. RSV infection often leads to severe acute lung immunopathology, but the underlying immune mechanisms are not yet fully elucidated. Here, we found that RSV infection induced severe acute lung immune injury and promoted the accumulation and activation of lung natural killer (NK) cells at the early stage of infection in BALB/c mice. Activated lung NK cells highly expressed activating receptors NKG2D and CD27 and became functional NK cells by producing a large amount of gamma interferon (IFN-γ), which was responsible for acute lung immune injury. NK cell depletion significantly attenuated lung immune injury and reduced infiltration of total inflammatory cells and production of IFN-γ in bronchoalveolar lavage fluid (BALF). These data show that NK cells are involved in exacerbating the lung immune injury at the early stage of RSV infection via IFN-γ secretion.     

Identification of immunodominant epitopes derived from the respiratory syncytial virus fusion protein that are recognized by human CD4 T cells

Memory CD4 T-cell responses against respiratory syncytial virus (RSV) were evaluated in peripheral blood mononuclear cells of healthy blood donors with gamma interferon enzyme-linked immunospot (Elispot) assays. RSV-specific responses were detected in every donor at levels varying between 0.05 and 0.3% of CD4 T cells. For all donors tested, a considerable component of the CD4 T-cell response was directed against the fusion (F) protein of RSV. We characterized a set of 31 immunodominant antigenic peptides targeted by CD4 T cells in the context of the most prevalent HLA class II molecules within the Caucasian population. Most antigenic peptides were HLA-DR restricted, whereas two dominant DQ peptides were also identified. The antigenic peptides identified were located across the entire sequence of the F protein. Several peptides were presented by more than one major histocompatibility complex class II molecule. Furthermore, most donors recognized several F peptides. Detailed knowledge about immunodominant antigenic peptides will facilitate the ability to monitor CD4 T-cell responses in patients and the measurement of correlates of protection in vaccinated subjects.     

Further characterization of the complementation group B temperature-sensitive mutant of respiratory syncytial virus.

ts-2, a temperature-sensitive and plaque morphology mutant of respiratory syncytial virus and sole representative of complementation group B, was compared with members of the other complementation groups of respiratory syncytial virus (group A [ts-1] and group C [ts-7]). ts-2 was found to be 10- to 1,000-fold more restricted in growth and ability to spread at restrictive temperatures (37, 38, and 39 degrees C) than at the permissive temperature (32 degrees C). In temperature shift-up experiments, the ts defect of ts-1 and other members of complementation group A was found to effect a late function that was required for at least 13 h in the replicative cycle. The ts lesion of ts-7 affected a function early in the replication cycle. In contrast, ts-2 was not temperature sensitive when studied by the shift-up technique. The discrepancy between the ts plaque property and failure to detect temperature sensitivity during the shift-up experiment was resolved when it was shown that ts-2 had a defect in adsorption or penetration or both at the restrictive temperature. Clonal analysis of revertant ts-2 showed a coordinate restoration of ts+ phenotype ans syncytium-forming capacity. It appears that ts-2 has a defect in a protein that is involved in adsorption and/or penetration of virus and is also responsible for cell fusion activity.     

Antibody effector functions are associated with protection from respiratory syncytial virus

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Identification of linear heparin-binding peptides derived from human respiratory syncytial virus fusion glycoprotein that inhibit infectivity

It has been shown previously that the fusion glycoprotein of human respiratory syncytial virus (RSV-F) interacts with cellular heparan sulfate. Synthetic overlapping peptides derived from the F-protein sequence of RSV subtype A (strain A2) were tested for their ability to bind heparin using heparin-agarose affinity chromatography (HAAC). This evaluation identified 15 peptides representing eight linear heparin-binding domains (HBDs) located within F1 and F2 and spanning the protease cleavage activation site. All peptides bound to Vero and A549 cells, and binding was inhibited by soluble heparins and diminished by either enzymatic treatment to remove cell surface glycosaminoglycans or by treatment with sodium chlorate to decrease cellular sulfation. RSV-F HBD peptides were less likely to bind to glycosaminoglycan-deficient CHO-745 cells than parental CHO-K1 cells that express these molecules. Three RSV-F HBD peptides (F16, F26, and F55) inhibited virus infectivity; two of these peptides (F16 and F55) inhibited binding of virus to Vero cells, while the third (F26) did not. These studies provided evidence that two of the linear HBDs mapped by peptides F16 and F55 may mediate one of the first steps in the attachment of virus to cells while the third, F26, inhibited infectivity at a postattachment step, suggesting that interactions with cell surface glycosaminoglycans may play a role in infectivity of some RSV strains.     

Identification of mutations causing temperature-sensitive defects in Semliki Forest virus RNA synthesis

We have sequenced the nonstructural protein coding region of Semliki Forest virus temperature-sensitive (ts) mutant strains ts1, ts6, ts9, ts10, ts11, ts13, and ts14. In each case, the individual amino acid changes uncovered were transferred to the prototype strain background and thereby identified as the underlying cause of the altered RNA synthesis phenotype. All mutations mapping to the protease domain of nonstructural protein nsP2 caused defects in nonstructural polyprotein processing and subgenomic RNA synthesis, and all mutations in the helicase domain of nsP2 affected subgenomic RNA production. These types of defects were not associated with mutations in other nonstructural proteins.     

Antibody response to respiratory syncytial virus structural proteins in children with acute respiratory syncytial virus infection.

The purified respiratory syncytial virus (RSV), Randall strain contained 10 polypeptides (72,000 molecular weight [72K], 66K, 48K, 42K, 40K, 36K, 30K, 23K, 18K, and 15K), 8 of which proved to be virus specific, and polypeptides 48K and 23K were glycosylated. In addition, a high-molecular-weight (150K), virus-specific glycopolypeptide was immunoprecipitated from RSV-infected cell lysate. The antibody response in human sera serially collected from children with primary RSV infection was mainly directed against the polypeptides 30K, 48K, and 72K. The immune response against the other viral proteins was also already detectable in the acute-phase sera. These results indicate that the immune response in RSV infection differs significantly from those for other diseases caused by paramyxoviruses.     

Sulfhydryl groups are involved in the interaction of FSH with its receptor

FSH has recently been reported to possess thioredoxin-like activity, presumably explained by the homology between a region of FSH-beta subunit and the active site of thioredoxin. The homologous sequence lies within a receptor binding region, which suggests a possible role for sulfhydryl groups in the formation of an active hormone-receptor complex and subsequent signal transduction. In order to determine the relevance of sulfhydryl groups on FSH-receptor interaction, we studied the effect of N-ethylmaleimide (NEM) and glutathione on FSH binding. The results indicate that free sulfhydryl groups, probably derived from the FSH receptor, are involved in ligand-receptor interaction.