Human Respiratory Syncytial Virus Glycoproteins Are Not Required for Apical Targeting and Release from Polarized Epithelial Cells (original) (raw)
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Proceedings of the National Academy of Sciences, 1986
A cDNA copy of the G glycoprotein gene of human respiratory syncytial virus (RSV) was placed under control of a vaccinia virus promoter and inserted into the thymidine kinase locus of the vaccinia virus genome. The recombinant vaccinia virus retained infectivity and expressed a 93-kDa protein that migrated with the authentic RSV G glycoprotein upon polyacrylamide gel electrophoresis. Glycosylation of the expressed protein and transport to the cell surface were demonstrated in the absence of other RSV proteins. Cotton rats that were inoculated intradermally with the infectious recombinant virus produced serum antibody to the G glycoprotein that neutralized RSV in vitro. Furthermore, the vaccinated animals were resistant to lower respiratory tract infection upon intranasal inoculation with RSV and had reduced titers of RSV in the nose.
Journal of Virology, 1986
Human respiratory syncytial virus (HRSV) is released from the apical membrane of polarized epithelial cells. However, little is known about the processes of assembly and release of HRSV and which viral gene products are involved in the directional maturation of the virus. Based on previous studies showing that the fusion (F) glycoprotein contained an intrinsic apical sorting signal and that N-and O-linked glycans can act as apical targeting signals, we investigated whether the glycoproteins of HRSV were involved in its directional targeting and release. We generated recombinant viruses with each of the three glycoprotein genes deleted individually or in groups. Each deleted gene was replaced with a reporter gene to maintain wild-type levels of gene expression. The effects of deleting the glycoprotein genes on apical maturation and on targeting of individual proteins in polarized epithelial cells were examined by using biological, biochemical, and microscopic assays. The results of these studies showed that the HRSV glycoproteins are not required for apical maturation or release of the virus. Further, deletion of one or more of the glycoprotein genes did not affect the intracellular targeting of the remaining viral glycoproteins or the nucleocapsid protein to the apical membrane.
Journal of …, 1987
Previous reports have established that vaccinia virus (VV) recombinants expressing G, F, or N protein of respiratory syncytial (RS) virus protect small animals against intranasal challenge with live RS virus. This work demonstrates that a variety of parameters affect the protection induced by recombinant viruses. The route of vaccination, the subtype of challenge virus, and the species used influenced the antibody titers and extent of protection. During these studies, observations were also made on the subclass of antibody generated, and pulmonary histopathological changes induced by challenge after vaccination were noted. The effect of route of inoculation on host response was examined by vaccinating mice intranasally, intraperitoneally, or by scarification with a recombinant VV expressing the RS virus G glycoprotein. Intranasal vaccination induced 25-fold-higher titers of antibody to RS virus in the lung than the intraperitoneal route did, but both routes resulted in complete suppression of virus replication after intranasal challenge 21 days after vaccination. Scarification was a less effective method of vaccination. The antibody induced by recombinant VV in mice was mostly immunoglobulin G2a (IgG2a) with some IgG2b. No antibody to RS virus was detected in the IgA, IgM, IgGl, or IgG3 subclass irrespective of the vaccination route. The G and F glycoproteins were shown to elicit similar subclasses of antibody. However, animals vaccinated with the G and F vectors differed strikingly in their response to challenge by heterologous virus. Mice or cotton rats vaccinated with recombinant VV carrying the G gene of RS virus were protected against challenge only with homologous subtype A virus. Vaccination with a recombinant VV expressing the F glycoprotein induced protection against both homologous and heterologous subtype B virus challenge. The protection induced in mice was greater than that detected in cotton rats, indicating that the host may also affect immunity. Finally, this report describes histological examination of mouse lungs after vaccination and challenge. Vaccinated mice that were subsequently challenged had significantly greater lung lesion scores than unvaccinated challenged mice. The lesions were primarily peribronchiolar and perivascular infiltrations of polymorphonuclear cells and lymphocytes. Further work will establish whether these pulmonary changes are a desirable immune response to virus invasion or a potential immunopathogenic hazard. The results have important implications for planning a strategy of vaccination against RS virus and emphasize potential dangers that may attend the use of recombinant VV as vaccines.
1987
Vaccinia virus (W) recombinants were constructed that contained full-length cDNA copies of the fusion (F) protein gene of human respiratory syncytial (RS) virus. The F protein gene was placed next to the strong early-late VV 7.5-kilodalton promoter and was located within the VV thymidine kinase (tk) gene. Full-length recombinant transcripts that initiated at both the tk and the 7.5-kilodalton promoters accumulated in cells early in infection, and one or more of these transcripts was translated to yield a glycoprotein which comigrated with F., the fusion protein precursor. This precursor was processed by proteolytic cleavage to produce the two disulfide-linked subunits F, and F2, which were both glycosylated and of the same electrophoretic mobility as authentic F, and F2. Immunofluorescence studies demonstrated that the mature F protein was transported to and expressed on the surface of recombinant W-infected cells. Inoculation of rabbits with a recombinant vector expressing F resulted in the production of antiserum specific for the RS virus F protein. This antiserum neutralized virus infectivity and was capable of preventing fusion in RS virus-infected cells. Mice were vaccinated with recombinants expressing the F protein. At 3 weeks postinoculation, these animals had serum antibody against RS virus F protein. At 5 days after intranasal challenge with RS virus, the lungs of the mice previously vaccinated with recombinants expressing F protein were free of detectable RS virus, whereas the lungs of unvaccinated mice contained 104.2 PFU of virus per g.
Immunoregulatory role of secreted glycoprotein G from respiratory syncytial virus
Virus Research, 2001
The secretory glycoprotein (Gs) of respiratory syncytial virus (RSV) was enriched and investigated for its effects on T cells specific for RSV and unrelated antigens. Gs exhibited a dose dependent suppression of lymphoproliferative responses in peripheral blood mononuclear cells (PBMCs), specific for mycobacterial lysates or tetanus toxoid. However, Gs did not inhibit live RSV specific T cell responses. These results suggest that Gs may suppress immune response to unrelated antigens, but should not interfere with the overall development of RSV specific immunity.
Virology, 1990
The attachment glycoprotein G of respiratory syncytial (RS) virus is important in both the antigenic and molecular diversity of the RS viruses. Previous work has shown that the glycoprotein G of a subgroup A RS virus expressed from a recombinant vaccinia virus provides significant protection against homologous but not heterologous subgroup virus challenge. We undertook the cDNA cloning and nucleotide sequencing of the G mRNA of a subgroup B RS virus (8/60) to extend molecular comparisons of the G protein both within and between subgroups. We also tested the ability of a subgroup B G protein to provide protection against challenge by A or B subgroup viruses. Sequence analysis showed a deduced amino acid sequence having a single major open reading frame encoding a protein of 292 amino acids with an elevated serine and threonine (30%) and proline (9%) content. The 8/60 G differed from a subgroup A virus (A2) G protein with only a 56% amino acid identity while the 8/60 G shared a 98% amino acid identity with the G protein of another subgroup B virus (18537). The 8/60 G cDNA was placed in a vaccinia virus vector (vvGB) which was shown to express the 8/60 G protein. Cotton rats immunized intradermally with vvGB and later challenged intranasally with 8/60 RS virus had a significant reduction in viral titers in the lungs relative to control animals whereas similarly immunized animals were not protected against heterologous subgroup challenge. Our results indicate that a RS virus subunit vaccine containing the G protein would require both A and B subgroup G proteins to afford protection against viruses of both subgroups.
FEMS Immunology & Medical Microbiology, 2000
Plasmid vectors encoding two different variants, one cytoplasmic and one secreted version, of a candidate vaccine BBG2Na to respiratory syncytial virus (RSV), were constructed and evaluated in a nucleic acid vaccination study. The two different vectors, which employed the Semliki Forest virus gene amplification system, were found to express BBG2Na appropriately in in vitro cell cultures. Immunisation of mice with the plasmid vectors elicited significant serum anti-BBG2Na IgG responses only in the mice receiving the plasmid encoding the secreted version of BBG2Na. Consistent with antibody induction data, sterilising lung protection against RSV-A challenge was also only observed in this group. These results indicate that the targeting of antigen expression (intracellular versus secreted) would be an important factor to consider in the design of nucleic acid vaccines.
Proceedings of the National Academy of Sciences, 1986
The major glycoprotein, G, of human respiratory syncytial (RS) virus is a Mr 84,000-90,000 species that has about 60% of its mass contributed by carbohydrate, most of which is in the form of O-linked oligosaccharides. The G protein contains neither a hydrophobic N-terminal signal sequence nor a hydrophobic C-terminal anchor region. Instead, its amino acid sequence reveals only one region with significant hydrophobic character, which is between residues 38 and 66. In order to study the synthesis, processing, and functions of this unusual viral glycoprotein, full-length cDNA copies of the G protein mRNA were inserted into the DNA genome of vaccinia virus (VV) in a position that was adjacent to a strong VV promoter and within the VV gene for thymidine kinase (TK). The resulting TK- recombinant viruses were selected, plaque-purified, and characterized by Southern blot analysis of restriction enzyme digests of the viral DNA. Recombinant RNA transcripts that contained both G-specific and ...
Virology, 1999
The relative immunopathogenic potential of a recombinant fusion protein incorporating residues 130-230 of respiratory syncytial virus (RSV-A) G protein (BBG2Na), formalin-inactivated RSV-A (FI-RSV), and phosphate-buffered saline (PBS) was investigated in mice after immunization and RSV challenge. FI-RSV priming resulted in massive infiltration of B cells and activated CD4 ϩ and CD8 ϩ T lymphocytes in mediastinal lymph nodes (MLN) and lungs, where eosinophilia and elevated IFN-␥, IL-2, -4, -5, -10, and -13 mRNA transcripts were also detected. PBS-primed mice showed only elevated pulmonary IL-2 and IFN-␥ mRNAs, while an activated CD8 ϩ T cell peak was detected in MLN and lungs. Cell infiltration also occurred in MLN of BBG2Na-immunized mice. However, there was no evidence of T cell, B cell, or granulocyte infiltration or activation in lungs, while transient transcription of Th1-type cytokine genes was evident. The absence of pulmonary infiltration is unlikely due to insufficient viral antigen. Thus, this recombinant fusion RSV G fragment does not prime for adverse pulmonary immunopathologic responses.