Determination of the disulfide bond arrangement of human respiratory syncytial virus attachment (G) protein by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Gorman, Jeffrey J.; Ferguson, Bettina L.; Speelman, Dan J.; Mills, John

doi:10.1002/pro.5560060619

Cited by 67 publications

(53 citation statements)

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“…The central conserved region of the G protein contains four evolutionarily conserved cysteines in a cysteine noose structure, within which lies a CX3C chemokine motif (9, 29, 34). The G protein CX3C motif is also immunoactive, as suggested by studies with the mouse model that show that G protein CX3C motif interaction with CX3CR1 alters pulmonary inflammation (41), RSV-specific T-cell responses (12), FI-RSV vaccine-enhanced disease, and expression of the neurokinin substance P (14) and also depresses respiratory rates (32).…”

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confidence: 99%

Prophylactic Treatment with a G Glycoprotein Monoclonal Antibody Reduces Pulmonary Inflammation in Respiratory Syncytial Virus (RSV)-Challenged Naïve and Formalin-Inactivated RSV-Immunized BALB/c Mice

Radu

Caidi

Miao

et al. 2010

J Virol

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We examined whether prophylactically administered anti-respiratory syncytial virus (anti-RSV) G monoclonal antibody (MAb) would decrease the pulmonary inflammation associated with primary RSV infection and formalin-inactivated RSV (FI-RSV)-enhanced disease in mice. MAb 131-2G administration 1 day prior to primary infection reduced the pulmonary inflammatory response and the level of RSV replication. Further, intact or F(ab) 2 forms of MAb 131-2G administered 1 day prior to infection in FI-RSV-vaccinated mice reduced enhanced inflammation and disease. This study shows that an anti-RSV G protein MAb might provide prophylaxis against both primary infection and FI-RSV-associated enhanced disease. It is possible that antibodies with similar reactivities might prevent enhanced disease and improve the safety of nonlive virus vaccines.Respiratory syncytial virus (RSV) infection in infants and young children causes substantial bronchiolitis and pneumonia (11, 27, 28, 40) resulting in 40,000 to 125,000 hospitalizations in the United States each year (27). RSV is also a prominent cause of respiratory illness in older children; those of any age with compromised cardiac, pulmonary, or immune systems; and the elderly (6,7,11,17,18, 39). Despite extensive efforts toward vaccine development (3,5,8,20, 30, 38), none is yet available. Currently, only preventive measures are available that focus on infection control to decrease transmission and prophylactic administration of a humanized IgG monoclonal antibody (MAb) directed against the F protein of RSV (palivizumab) that is recommended for high-risk infants and young children (4,7,17). To date, no treatment has been highly effective for active RSV infection (17, 21).The first candidate vaccine, a formalin-inactivated RSV (FI-RSV) vaccine developed in the 1960s, not only failed to protect against disease but led to severe RSV-associated lower respiratory tract infection in young vaccine recipients upon subsequent natural infection (8, 16). The experience with FI-RSV has limited nonlive RSV vaccine development for the RSVnaïve infant and young child. Understanding the factors contributing to disease pathogenesis and FI-RSV vaccine-enhanced disease may identify ways to prevent such a response and to help achieve a safe and effective vaccine.The RSV G, or attachment, protein has been implicated in the pathogenesis of disease after primary infection and FI-RSV-enhanced disease (2, 26, 31). The central conserved region of the G protein contains four evolutionarily conserved cysteines in a cysteine noose structure, within which lies a CX3C chemokine motif (9, 29, 34). The G protein CX3C motif is also immunoactive, as suggested by studies with the mouse model that show that G protein CX3C motif interaction with CX3CR1 alters pulmonary inflammation (41), RSV-specific T-cell responses (12), FI-RSV vaccine-enhanced disease, and expression of the neurokinin substance P (14) and also depresses respiratory rates (32). Recent studies demonstrated that therapeutic treatment with a murine anti-R...

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confidence: 99%

Prophylactic Treatment with a G Glycoprotein Monoclonal Antibody Reduces Pulmonary Inflammation in Respiratory Syncytial Virus (RSV)-Challenged Naïve and Formalin-Inactivated RSV-Immunized BALB/c Mice

Radu

Caidi

Miao

et al. 2010

J Virol

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“…In combination with enzymatic digestions, chemical modifications, and separation by high-performance liquid chromatography (HPLC), MALDI and ESI have been widely used in protein sequencing and disulfide mapping [7][8][9][10][11].…”

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Gas-phase scrambling of disulfide bonds during matrix-assisted laser desorption/ionization mass spectrometry analysis

Zhao¹,

Almaraz

Fan

et al. 2009

J. Am. Soc. Mass Spectrom.

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Evidence for photo-induced radical disulfide bond scrambling in the gas phase during matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is described. The phenomenon was observed during the analysis of tryptic peptides from insulin and was confirmed in the determination of disulfide bonds in the rhamnose-binding lectin SEL24K from the Chinook salmon Oncorhynchus tshawytscha. A possible mechanism for this surprising scrambling is proposed. Despite this finding, the disulfide bond pattern in SEL24K was assigned unambiguously by a multi-enzyme digestion strategy in combination with MALDI mass spectrometry. The pattern was found to be symmetrical in the tandem repeat sequence of SEL24K. To the best of our knowledge, this is the first report of disulfide bond scrambling in the gas phase during MALDI-MS analysis. This observation has important ramifications for unambiguous assignment of disulfide bonds. (J Am Soc

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“…This region overlaps four closely spaced cysteine residues (positions 173, 176, 182, and 186 in strain A2) that form disulfide bonds in the pattern 1-4 and 2-3 and create a cystine noose (Fig. 1a) (12,19,24). Monoclonal antibody-resistant mutants have been isolated in which point mutations within the 13-amino acid conserved region occurred (38) or residues 176, 182, and 186 were substituted singly or 182 and 186 were substituted together (27,31,38).…”

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The Central Conserved Cystine Noose of the Attachment G Protein of Human Respiratory Syncytial Virus Is Not Required for Efficient Viral Infection In Vitro or In Vivo

Teng

Collins

2002

J Virol

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The G glycoprotein of human respiratory syncytial virus (RSV) was identified previously as the viral attachment protein. Although we and others recently showed that G is not essential for replication in vitro, it does affect the efficiency of replication in a cell type-dependent fashion and is required for efficient replication in vivo. The ectodomain of G is composed of two heavily glycosylated domains with mucin-like characteristics that are separated by a short central region that is relatively devoid of glycosylation sites. This central region contains a 13-amino acid segment that is conserved in the same form among RSV isolates and is overlapped by a second segment containing four cysteine residues whose spacings are conserved in the same form and which create a cystine noose. The conserved nature of the cystine noose and flanking 13-amino acid segment suggested that this region likely was important for attachment activity. To test this hypothesis, we constructed recombinant RSVs from which the region containing the cysteine residues was deleted together with part or all of the conserved 13-amino acid segment. Surprisingly, each deletion had little or no effect on the intracellular synthesis and processing of the G protein, the kinetics or efficiency of virus replication in vitro, or sensitivity to neutralization by soluble heparin in vitro. In addition, neither deletion had any discernible effect on the ability of RSV to infect the upper respiratory tract of mice and both resulted in a 3-to 10-fold reduction in the lower respiratory tract. Thus, although the G protein is necessary for efficient virus replication in vivo, this activity does not require the central conserved cystine noose region.Human respiratory syncytial virus (RSV), the prototype of the Pneumovirus genus in the paramyxovirus family, is the most important viral etiologic agent of pediatric respiratory disease worldwide (6). Its genome consists of a single, negative-sense RNA of 15,190 to 15,225 nucleotides (for the three strains sequenced to date) encoding 10 major subgenomic mRNAs and 11 viral proteins. Three of these proteins, G, F, and SH, are transmembrane virion glycoproteins. The G glycoprotein has been identified as the viral attachment protein (25) although, as described below, under certain conditions it is completely dispensable for efficient replication in vitro. The F protein mediates membrane fusion and also appears to be able to serve as an auxiliary attachment protein (17,21,22,34,36). Expression of the SH protein can be eliminated with little effect on virus replication in vitro and in vivo and its function remains unknown (3, 41), although its counterpart in simian virus 5 was recently shown to function as an antagonist of apoptosis (16).RSV G is a highly glycosylated type II transmembrane protein with a single hydrophobic region near the N terminus which serves as both signal sequence and membrane anchor. The protein backbone is 292 to 299 amino acids long, depending on the virus strain (19,33), and has an M r of approxim...

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Determination of the disulfide bond arrangement of human respiratory syncytial virus attachment (G) protein by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Cited by 67 publications

References 52 publications

Prophylactic Treatment with a G Glycoprotein Monoclonal Antibody Reduces Pulmonary Inflammation in Respiratory Syncytial Virus (RSV)-Challenged Naïve and Formalin-Inactivated RSV-Immunized BALB/c Mice

Prophylactic Treatment with a G Glycoprotein Monoclonal Antibody Reduces Pulmonary Inflammation in Respiratory Syncytial Virus (RSV)-Challenged Naïve and Formalin-Inactivated RSV-Immunized BALB/c Mice

Gas-phase scrambling of disulfide bonds during matrix-assisted laser desorption/ionization mass spectrometry analysis

The Central Conserved Cystine Noose of the Attachment G Protein of Human Respiratory Syncytial Virus Is Not Required for Efficient Viral Infection In Vitro or In Vivo

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