Respiratory Syncytial Virus Attachment Glycoprotein Contribution to Infection Depends on the Specific Fusion Protein

Abstract

Human respiratory syncytial virus (RSV) is an important pathogen causing acute lower respiratory tract disease in children. The RSV attachment glycoprotein (G) is not required for infection, as G-null RSV replicates efficiently in several cell lines. Our laboratory previously reported that the viral fusion (F) protein is a determinant of strain-dependent pathogenesis. Here, we hypothesized that virus dependence on G is determined by the strain specificity of F. We generated recombinant viruses expressing G and F, or null for G, from the laboratory A2 strain (Katushka RSV-A2GA2F [kRSV-A2GA2F] and kRSV-GstopA2F) or the clinical isolate A2001/2-20 (kRSV-2-20G2-20F and kRSV-Gstop2-20F). We quantified the virus cell binding, entry kinetics, infectivity, and growth kinetics of these four recombinant viruses in vitro. RSV expressing the 2-20 G protein exhibited the greatest binding activity. Compared to the parental viruses expressing G and F, removal of 2-20 G had more deleterious effects on binding, entry, infectivity, and growth than removal of A2 G. Overall, RSV expressing 2-20 F had a high dependence on G for binding, entry, and infection. IMPORTANCERSV is the leading cause of childhood acute respiratory disease requiring hospitalization. As with other paramyxoviruses, two major RSV surface viral glycoproteins, the G attachment protein and the F fusion protein, mediate virus binding and subsequent membrane fusion, respectively. Previous work on the RSV A2 prototypical strain demonstrated that the G protein is functionally dispensable for in vitro replication. This is in contrast to other paramyxoviruses that require attachment protein function as a prerequisite for fusion. We reevaluated this requirement for RSV using G and F proteins from clinical isolate 2-20. Compared to the laboratory A2 strain, the G protein from 2-20 had greater contributions to virus binding, entry, infectivity, and in vitro growth kinetics. Thus, the clinical isolate 2-20 F protein function depended more on its G protein, suggesting that RSV has a higher dependence on G than previously thought. H uman respiratory syncytial virus (hRSV or RSV) causes an annual global 3.4 million estimated severe acute lower respiratory tract infections (ALRI) in children younger than 5 years of age (1). In the United States, about 132,000 to 172,000 children younger than 5 years of age are hospitalized due to RSV every year (2). Thus far, there are no licensed vaccines, although there are multiple vaccine candidates undergoing clinical trials (3). Development of antivirals against RSV is also an active field of research and clinical development (4-6).RSV is a member of the Paramyxoviridae family, Pneumovirus genus. Members of the paramyxovirus family encode two major glycoproteins important early during infection for attachment to the host cell and the subsequent entry process. Paramyxovirus fusion mediated by the viral fusion (F) protein is generally initiated by interaction with the homologous attachment protein upon receptor engagement (reviewed ...

A live RSV vaccine with engineered thermostability is immunogenic in cotton rats despite high attenuation

“…The codon deoptimization of G in OE4 contributed significantly to the level of attenuation compared with OE4 expressing wild-type G (OE4+wtG) in NHBE-ALI (Fig. 6c), likely due to the previously described attachment role of G in primary cells30.…”

“…Last, we codon-deoptimized the RSV attachment (G) glycoprotein gene and ablated the secreted form of G by a point mutation. RSV expresses a membrane-bound form (G m ) and a secreted form (G s ) of G, which are not required for viral replication in immortalized cell lines29303132. RSV G is capable of eliciting protective neutralizing antibodies33.…”

A Recombinant Respiratory Syncytial Virus Vaccine Candidate Attenuated by a Low-Fusion F Protein Is Immunogenic and Protective against Challenge in Cotton Rats

“…We used these restriction sites to clone the corresponding G and F genes into pSynkRSV-A2-line19F. We also generated a recombinant RSV strain without G protein expression (kRSV-A2-220F-G-stop) as previously described (20,21).…”

“…These strains were chosen to represent phylogenetically distant RSV strains from both subgroups and multiple clades. We additionally generated a recombinant virus, kRSV-A2-2-20F/G-stop, which contained a premature stop codon in the G glycoprotein as previously described, in order to measure the relative effects of anti-G antibodies on neutralization (21). We also included kRSV-A2 in the panel and rescued the recombinant viruses.…”

Alternative conformations of a major antigenic site on RSV F

“…However, the mechanism and underlying cause of RSV F triggering is not well understood. Recombinant virus expressing only the RSV F protein on its surface is sufficient for infection of immortalized cell lines in vitro, suggesting that RSV F can facilitate attachment and mediate fusion in the absence of the attachment glycoprotein [7,[20][21][22]. Potential RSV F receptors include nucleolin, EGFR, and heparan sulfate proteoglycans, among others [7,[23][24][25][26][27], but the specific role each may play in the setting of natural infection remains to be defined.…”