Monoclonal antibody neutralization escape mutants of respiratory syncytial virus with unique alterations in the attachment (G) protein.

Walsh, Edward E.; Falsey, Ann R.; Sullender, Wayne M.

doi:10.1099/0022-1317-79-3-479

Cited by 50 publications

(46 citation statements)

References 40 publications

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“…Some of these mutations presented by field isolates were similar to some of those already described for in vitro HRSV antibody escape mutants (45,74). However, this work constitutes the first evidence that natural in vivo infections can occur with mutants lacking the four cysteines involved in the two disulfide bridges.…”

Section: Discussionmentioning

confidence: 69%

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Evolution of Bovine Respiratory Syncytial Virus

Valarcher

Schelcher

Bourhy

2000

J Virol

164

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Until now, the analysis of the genetic diversity of bovine respiratory syncytial virus (BRSV) has been based on small numbers of field isolates. In this report, we determined the nucleotide and deduced amino acid sequences of regions of the nucleoprotein (N protein), fusion protein (F protein), and glycoprotein (G protein) of 54 European and North American isolates and compared them with the sequences of 33 isolates of BRSV obtained from the databases, together with those of 2 human respiratory syncytial viruses and 1 ovine respiratory syncytial virus. A clustering of BRSV sequences according to geographical origin was observed. We also set out to show that a continuous evolution of the sequences of the N, G, and F proteins of BRSV has been occurring in isolates since 1967 in countries where vaccination was widely used. The exertion of a strong positive selective pressure on the mucin-like region of the G protein and on particular sites of the N and F proteins is also demonstrated. Furthermore, mutations which are located in the conserved central hydrophobic part of the ectodomain of the G protein and which result in the loss of four Cys residues and in the suppression of two disulfide bridges and an ␣ helix critical to the three-dimensional structure of the G protein have been detected in some recent French BRSV isolates. This conserved central region, which is immunodominant in BRSV G protein, thus has been modified in recent isolates. This work demonstrates that the evolution of BRSV should be taken into account in the rational development of future vaccines. (38,39,40,49). These two related viruses share common epidemiological, clinical, and pathological characteristics. The respiratory syncytial viruses (RSV) are the most common and important cause of lower respiratory tract illness in cattle and young infants (71). More than 70% of calves exhibit a positive serological response against BRSV by the age of 12 months. Bovine respiratory syncytial virus (BRSV) and human respiratory syncytial virus (HRSV) are members of the genus Pneumovirus, subfamily Pneumovirinae, and family ParamyxoviridaeNeutralization tests (9) and reaction patterns with specific monoclonal antibodies (2, 48) have revealed that HRSV contains two major groups, A and B. The main differences between these two groups are located in the glycoprotein (G protein), while others are located in the fusion protein (F protein) and nucleoprotein (N protein) (48, 51). Subgroups within the G and F proteins of BRSV have been characterized more recently by serological analysis of a limited number of isolates and confirmed by phylogenetic analysis (20,54,60). Further studies of BRSV variability have focused on the G protein, which was shown to be the most variable protein and, with the F protein, one of the targets for neutralizing antibodies. A low percentage of sequence divergence between and within the G proteins of BRSV subgroups has been reported, suggesting that BRSV has the same extent of diversity as HRSV (15,21,36,44,66).The G protein is responsib...

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Section: Discussionmentioning

confidence: 69%

“…74,2000 EVOLUTION OF BRSVvaccination was widely practiced in Europe. This analysis could not be performed in the absence of selective pressure due to cattle vaccination, as the N and F protein gene sequences of isolates from Denmark and Sweden were not available from GenBank.…”

Section: Four Highly Conserved Cys Residues Have Been Replaced By Argmentioning

confidence: 99%

Evolution of Bovine Respiratory Syncytial Virus

Valarcher

Schelcher

Bourhy

2000

J Virol

164

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“…A portion of these virus preparations was further purified through a sucrose gradient by centrifugation in a TH-641 rotor (Thermo Fisher Scientific) and Beckman ultracentrifuge at 287,000 ϫ g for 20 h. Gradient fractions were examined by immunoblotting and staining with an MAb to the N protein (32). Fractions containing virus were separated by SDS-PAGE, and G protein was detected by immunoblotting using an MAb, L9 (33).…”

Section: Methodsmentioning

confidence: 99%

Preventing Cleavage of the Respiratory Syncytial Virus Attachment Protein in Vero Cells Rescues the Infectivity of Progeny Virus for Primary Human Airway Cultures

Corry

Johnson

Cornwell

et al. 2016

J Virol

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All live attenuated respiratory syncytial virus (RSV) vaccines that have advanced to clinical trials have been produced in Vero cells. The attachment (G) glycoprotein in virions produced in these cells is smaller than that produced in other immortalized cells due to cleavage. These virions are 5-fold less infectious for primary well-differentiated human airway epithelial (HAE) cell cultures. Because HAE cells are isolated directly from human airways, Vero cell-grown vaccine virus would very likely be similarly inefficient at initiating infection of the nasal epithelium following vaccination, and therefore, a larger inoculum would be required for effective vaccination. We hypothesized that Vero cell-derived virus containing an intact G protein would be more infectious for HAE cell cultures. Using protease inhibitors with increasing specificity, we identified cathepsin L to be the protease responsible for cleavage. Our evidence suggests that cleavage occurs in the late endosome or lysosome during endocytic recycling. Cathepsin L activity was 100-fold greater in Vero cells than in HeLa cells. In addition, cathepsin L was able to cleave the G protein in Vero cell-grown virions but not in HeLa cell-grown virions, suggesting a difference in G-protein posttranslational modification in the two cell lines. We identified by mutagenesis amino acids important for cleavage, and these amino acids included a likely cathepsin L cleavage site. Virus containing a modified, noncleavable G protein produced in Vero cells was 5-fold more infectious for HAE cells in culture, confirming our hypothesis and indicating the value of including such a mutation in future live attenuated RSV vaccines. IMPORTANCEWorldwide, RSV is the second leading infectious cause of infant death, but no vaccine is available. Experimental live attenuated RSV vaccines are grown in Vero cells, but during production the virion attachment (G) glycoprotein is cleaved. Virions containing a cleaved G protein are less infectious for primary airway epithelial cells, the natural RSV target. In the study described here we identified the protease responsible, located the cleavage site, and demonstrated that cleavage likely occurs during endocytic recycling. Moreover, we showed that the infectivity of Vero cell-derived virus for primary airway epithelial cells is increased 5-fold if the virus contains a mutation in the G protein that prevents cleavage. The blocking of cleavage should improve RSV vaccine yield, consequently reducing production costs. Posttranslational cleavage of the fusion glycoprotein of many viruses plays an essential role in activation; however, cleavage of the RSV G protein is a novel example of a detrimental effect of cleavage on virus infectivity. In adults and teenagers, most respiratory syncytial virus (RSV) infections produce upper respiratory tract infection and symptoms. However, in infants, the immunocompromised, and the elderly, RSV has the potential to cause life-threatening lower respiratory tract infection (1-4). Worldwide, in 2010 ...

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“…Besides single amino acid substitutions, some escape mutants had: (i) frame-shift mutations that altered the C-terminal one-third of the G protein (García-Barreno et al, 1990); (ii) premature stop codons that shortened the length of the G polypeptide by between 1 and 42 amino acids (Rueda et al, 1991(Rueda et al, , 1995; and (iii) ARG hypermutations that were translated into several amino acid changes, some of them involving a conserved cluster of cysteines found in the middle of the G protein ectodomain (Rueda et al, 1994;Martínez et al, 1997;Walsh et al, 1998).…”

mentioning

confidence: 99%

Major changes in the G protein of human respiratory syncytial virus isolates introduced by a duplication of 60 nucleotides

Trento

Galiano

Videla

et al. 2003

Journal of General Virology

250

264

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The entire nucleotide sequence of the G gene of three human respiratory syncytial virus (HRSV) isolates (antigenic group B) has been determined. These three viruses (named BA viruses) were isolated in Buenos Aires in 1999 from specimens collected in different hospitals and at different dates. BA viruses have an exact duplication of 60 nucleotides in the G gene, starting after residue 791. This duplication is flanked by a repeat of four nucleotides (GUGU) and can fold into a relatively stable secondary structure. These features suggest a possible mechanism for the generation of a duplicated G segment. The predicted polypeptide is lengthened by 20 amino acids (residues 260-279) and this is reflected in the slower electrophoretic mobility of the G protein precursor of BA viruses compared with related viruses. The changes reported here expand the examples of drastic genetic alterations that can be introduced into the G protein sequence of HRSV while it replicates in its natural host.

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Monoclonal antibody neutralization escape mutants of respiratory syncytial virus with unique alterations in the attachment (G) protein.

Cited by 50 publications

References 40 publications

Evolution of Bovine Respiratory Syncytial Virus

Evolution of Bovine Respiratory Syncytial Virus

Preventing Cleavage of the Respiratory Syncytial Virus Attachment Protein in Vero Cells Rescues the Infectivity of Progeny Virus for Primary Human Airway Cultures

Major changes in the G protein of human respiratory syncytial virus isolates introduced by a duplication of 60 nucleotides

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