Functional Analysis of Recombinant Respiratory Syncytial Virus Deletion Mutants Lacking the Small Hydrophobic and/or Attachment Glycoprotein Gene

Techaarpornkul, Sunee; Barretto, Naina; Peeples, Mark E.

doi:10.1128/jvi.75.15.6825-6834.2001

Cited by 245 publications

(276 citation statements)

References 42 publications

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“…Studies demonstrated that an rRSV strain in which 26 amino acids containing the highly conserved cystine noose were deleted could be rescued without adverse effects on replication (52). Indeed, an rRSV strain with the G protein completely deleted was successfully rescued (51,53). However, the complete absence of G protein severely restricted replication in vivo and thus limited the potential of the rRSV strain as a vaccine.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Recombinant Respiratory Syncytial Viruses with the Region Responsible for Type 2 T-Cell Responses and Pulmonary Eosinophilia Deleted from the Attachment (G) Protein

Elliott¹,

Pryharski²,

et al. 2004

J Virol

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It is essential that preventative vaccines for respiratory syncytial virus (RSV) elicit balanced T-cell responses. Immune responses dominated by type 2 T cells against RSV antigens are believed to cause exaggerated respiratory tract disease and may also contribute to unwanted inflammation in the airways that predisposes infants to wheeze through adolescence. Here we report on the construction and characterization of recombinant RSV (rRSV) strains with amino acids 151 to 221 or 178 to 219 of the attachment (G) glycoprotein deleted (rA2cp⌬G150-222 or rA2cp⌬G177-220, respectively). The central ectodomain was chosen for modification because a peptide spanning amino acids 149 to 200 of G protein has recently been shown to prime several strains of naïve inbred mice for polarized type 2 T-cell responses, and peripheral blood T cells from most human donors recognize epitopes within this region. Quantitative PCR demonstrated that synthesis of nascent rRSV genomes in human lung epithelial cell lines was similar to that for the parent virus (cp-RSV). Plaque assays further indicated that rRSV replication was not sensitive to 37°C, but pinpoint morphology was observed at 39°C. Both rRSV strains replicated in the respiratory tracts of BALB/c mice and elicited serum neutralization and anti-F-protein immunoglobulin G titers that were equivalent to those elicited by cp-RSV and contributed to a 3.9-log 10 -unit reduction in RSV A2 levels 4 days after challenge. Importantly, pulmonary eosinophilia was significantly diminished in BALB/c mice primed with native G protein and challenged with either rA2cp⌬G150-222 or rA2cp⌬G177-220. These findings are important for the development of attenuated RSV vaccines.

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Section: Discussionmentioning

confidence: 99%

Characterization of Recombinant Respiratory Syncytial Viruses with the Region Responsible for Type 2 T-Cell Responses and Pulmonary Eosinophilia Deleted from the Attachment (G) Protein

Elliott¹,

Pryharski²,

et al. 2004

J Virol

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“…That the recombinant rgRSVΔG and rgRSVΔGΔSH viruses were similar to the A2 virions indicated that G and SH did not play a role in defining overall particle size and morphology. Although not essential under tissue-culture conditions, these proteins have been reported to elevate infectivity (25).…”

Section: Discussionmentioning

confidence: 99%

Architecture of respiratory syncytial virus revealed by electron cryotomography

Liljeroos

Krzyzaniak²,

Helenius³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

177

173

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Human respiratory syncytial virus is a human pathogen that causes severe infection of the respiratory tract. Current information about the structure of the virus and its interaction with host cells is limited. We carried out an electron cryotomographic characterization of cell culture-grown human respiratory syncytial virus to determine the architecture of the virion. The particles ranged from 100 nm to 1,000 nm in diameter and were spherical, filamentous, or a combination of the two. The filamentous morphology correlated with the presence of a cylindrical matrix protein layer linked to the inner leaflet of the viral envelope and with local ordering of the glycoprotein spikes. Recombinant viruses with only the fusion protein in their envelope showed that these glycoproteins were predominantly in the postfusion conformation, but some were also in the prefusion form. The ribonucleocapsids were left-handed, randomly oriented, and curved inside the virions. In filamentous particles, they were often adjacent to an intermediate layer of protein assigned to M2-1 (an envelopeassociated protein known to mediate association of ribonucleocapsids with the matrix protein). Our results indicate important differences in structure between the Paramyxovirinae and Pneumovirinae subfamilies within the Paramyxoviridae, and provide fresh insights into host cell exit of a serious pathogen.cryo-ET | paramyxovirus | virus structure H uman respiratory syncytial virus (HRSV) causes severe disease, especially in children and the elderly (1, 2). In a study covering data from 2005, it was found that HRSV was responsible for 22% of acute lower respiratory infections worldwide and 66,000-199,000 deaths in children under the age of 5 y (2). Treatment is currently limited to purine analogs (Ribavirin) and passive immune-prophylaxis with humanized monoclonal antibodies (Palivizumab) (3).RSV belongs to the Paramyxoviridae, a large family of enveloped, negative-sense RNA viruses with many members from humans and animals. Together with human metapneumovirus, it constitutes the Pneumovirinae subfamily. Like other paramyxoviruses, it has a plasma-membrane-derived lipid envelope and a helical ribonucleocapsid (RNP) that contains a single viral RNA molecule associated with nucleoproteins (N), and an RNA-dependent RNA polymerase. The virion contains, in addition, a matrix protein (M) (4-7) and a transcription antiterminator, M2-1 (an envelopeassociated protein known to mediate association of RNPs with the M protein) (8-11). The lipid envelope contains two transmembrane glycoproteins: the fusion protein, F, and a glycoprotein, G, used for attachment to host cells. In addition, HRSV contains a small hydrophobic protein, SH, of unclear function and proteins of cellular origin, such as actin and chaperones (12). The RNP and F have been analyzed using 3D-EM and X-ray crystallography (13,14).HRSV particles are pleomorphic with both spherical and filamentous particles of different sizes (15). Early electron microscopy studies showed the presence of a helical p...

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“…The A2 strain of RSV was kindly provided by Robert Channock. Recombinant green fluorescent protein-expressing viruses were a gift of Mark Peeples and have been described in detail elsewhere (50). The nomenclature used for these viruses is based on the surface glycoproteins they express: rgRSV-F expresses only the F glycoprotein, rgRSV-GF expresses G and F, and rgRSV-SGF expresses S, G, and F. All viruses were propagated in HEp-2 cells, and working stocks of virus were prepared as previously described (19).…”

Section: Methodsmentioning

confidence: 99%

“…Data from the antigen reduction assay and the flow-based binding assay were used to calculate 50% effective concentration (EC 50 Assessment of postattachment inhibition of viral infectivity. HEp-2 cells in 96-well plates were chilled to 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…This process is mediated by the RSV F glycoprotein (53), a type 1 viral fusion protein (9). The two additional viral surface glycoproteins, G and SH, appear to play a role in augmenting F-mediated fusion (24) and are important for viral viability in vivo, but they have been shown to be dispensable for the replication of RSV in cultured cells (6,10,27,50). Although G was originally identified as the attachment protein for RSV (31), the viability of viruses lacking G indicates that F alone can mediate both attachment and fusion.…”

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confidence: 99%

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RhoA-Derived Peptide Dimers Share Mechanistic Properties with Other Polyanionic Inhibitors of Respiratory Syncytial Virus (RSV), Including Disruption of Viral Attachment and Dependence on RSV G

Budge

Beeler

et al. 2004

J Virol

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Large polyanionic molecules, such as sulfated polysaccharides (including soluble heparin and dextran sulfate), synthetic polyanionic polymers, and negatively charged proteins, have been shown to broadly inhibit several enveloped viruses. We recently reported the antiviral activity of a peptide derived from amino acids 77 to 95 of a potential binding partner of respiratory syncytial virus F protein (RSV F), the GTPase RhoA. A subsequent study with a truncated peptide (amino acids 80 to 94) revealed that optimal antiviral activity required dimerization via intermolecular disulfide bonds. We report here that the net negative charge of this peptide is also a determining factor for its antiviral activity and that it, like other polyanions, inhibits virus attachment. In a flow cytometry-based binding assay, peptide 80-94, heparin, and dextran sulfate inhibited the attachment of virus to cells at 4°C at the same effective concentrations at which they prevent viral infectivity. Interestingly, time-of-addition experiments revealed that peptide 80-94 and soluble heparin were also able to inhibit the infectivity of a virus that had been prebound to cells at 4°C, as had previously been shown for dextran sulfate, suggesting a potential role for postattachment effects of polyanions on RSV entry. Neutralization experiments with recombinant viruses showed that the antiviral activities of peptide 80-94 and dextran sulfate were diminished in the absence of the RSV attachment glycoprotein (G). Taken together, these data indicate that the antiviral activity of RhoA-derived peptides is functionally similar to that of other polyanions, is dependent on RSV G, and does not specifically relate to a protein-protein interaction between F and RhoA.

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Functional Analysis of Recombinant Respiratory Syncytial Virus Deletion Mutants Lacking the Small Hydrophobic and/or Attachment Glycoprotein Gene

Cited by 245 publications

References 42 publications

Characterization of Recombinant Respiratory Syncytial Viruses with the Region Responsible for Type 2 T-Cell Responses and Pulmonary Eosinophilia Deleted from the Attachment (G) Protein

Characterization of Recombinant Respiratory Syncytial Viruses with the Region Responsible for Type 2 T-Cell Responses and Pulmonary Eosinophilia Deleted from the Attachment (G) Protein

Architecture of respiratory syncytial virus revealed by electron cryotomography

RhoA-Derived Peptide Dimers Share Mechanistic Properties with Other Polyanionic Inhibitors of Respiratory Syncytial Virus (RSV), Including Disruption of Viral Attachment and Dependence on RSV G

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