Sugrue, R. | Search bioRxiv

bioRxiv2022

Evidence for a functional interaction between the respiratory syncytial virus fusion and attachment proteins in the envelope of infectious virus particles.

We have examined the interaction between the respiratory syncytial virus (RSV) F and G proteins on the surface of infected cells during multiple cycle infection using a low multiplicity of infection (MOI) model, and on the surface of virus particles that were isolated from infected cells. A combination of the proximity ligation assay (PLA) and confocal microscopy was used to demonstrate the interaction between the F and G proteins within the virus filaments on infected cells. Co-precipitation of the F and G proteins was confirmed using detergent extracts prepared from infected cells and in detergent extracts prepared from purified virus particles. The influence of the G protein in mediating virus spread in the low MOI model was further examined using the recombinant virus isolates rg224RSV (that expresses all virus proteins) and rg224RSV-{Delta}G (which does not express the G protein). While cells could be initially infected by both viruses, the rg224RSV-{Delta}G virus exhibited severely impaired localised virus transmission in the multiple cycle infection assay. Collectively these data provide evidence that the F and G proteins interact within the envelope of RSV particles, and suggests that this interaction may promote virus transmission. The interaction between these proteins in a single protein complex represents a potential new target for the development of antivirus strategies and in the development of RSV vaccine candidates.

microbiology↗

bioRxiv2021

The cellular characterisation of SARS-CoV-2 spike protein in virus-infected cells using Receptor Binding Domain-binding specific human monoclonal antibodies.

A human monoclonal antibody panel (PD4, PD5, PD7, SC23 and SC29) was isolated from the B cells of convalescent patients and used to examine the S protein in SARS-CoV-2- infected cells. While all five antibodies bound conformational-specific epitopes within SARS-CoV-2 Spike (S) protein, only PD5, PD7, and SC23 were able to bind to the Receptor Binding Domain (RBD). Immunofluorescence microscopy was used to examine the S protein RBD in cells infected with the Singapore isolates SARS-CoV-2/0334 and SARS-CoV-2/1302. The RBD-binders exhibited a distinct cytoplasmic staining pattern that was primarily localised within the Golgi complex and was distinct from the diffuse cytoplasmic staining pattern exhibited by the non-RBD binders (PD4 and SC29). These data indicated that the S protein adopted a conformation in the Golgi complex that enabled the RBD recognition by the RBD-binders. The RBD-binders also recognised the uncleaved S protein indicating that S protein cleavage was not required for RBD recognition. Electron microscopy indicated high levels of cell-associated virus particles, and multiple cycle virus infection using RBD-binder staining provided evidence for direct cell-to-cell transmission for both isolates. Although similar levels of RBD-binder staining was demonstrated for each isolate, the SARS-CoV-2/1302 exhibited slower rates of cell-to-cell transmission. These data suggest that a conformational change in the S protein occurs during its transit through the Golgi complex that enables RBD recognition by the RBD-binders, and suggests that these antibodies can be used to monitor S protein RBD formation during the early stages of infection. ImportanceThe SARS CoV-2 spike (S) protein receptor binding domain (RBD) mediates the attachment of SARS CoV-2 to the host cell. This interaction plays an essential role in initiating virus infection and the S protein RBD is therefore a focus of therapeutic and vaccine interventions. However, new virus variants have emerged with altered biological properties in the RBD that can potentially negate these interventions. Therefore an improved understanding of the biological properties of the RBD in virus-infected cells may offer future therapeutic strategies to mitigate SARS CoV-2 infection. We used physiologically relevant antibodies that were isolated from the B cells of convalescent COVID19 patients to monitor the RBD in cells infected with SARS CoV-2 clinical isolates. These immunological reagents specifically recognise the correctly folded RBD and were used to monitor the appearance of the RBD in SARS CoV-2-infected cells and identified the site where the RDB first appears.

microbiology↗