How Mutations in the Respiratory Syncytial Virus Fusion Protein Affect Fusion Activity and Antibody Binding

Monoclonal antibodies are important therapeutics for viral infections; however viruses can mutate to avoid being bound by these antibodies. This project focusses on the monoclonal antibody motavizumab and its interaction with the Respiratory Syncytial Virus Fusion (RSV-F) glycoprotein. RSV-F mediates fusion of the virus lipid bilayer with that of the host cell during entry of the virus into a cell. This causes infected cells to fuse with other cells, causing syncytia to form. Motavizumab blocks these functions of RSV-F. The current literature has identified one mutation in the RSV-F motavizumab binding pocket that allows the virus to escape the neutralizing effects of motavizumab. Our group performed molecular modeling to identify additional potential escape variants. Some of the predicted escape mutants not only evaded neutralization by motavizumab, but also had varying growth rates and levels of syncytia formation. This project expands on those observations by studying RSV-F in isolation rather than looking at the entire virus. The RSV-F variant genes were isolated and placed in a backbone plasmid to be used in transient transfections. The RSV-F expressing transfected cells were imaged to determine the variant\u27s relative fusion activity. After imaging, the cells were lysed to test for amount of RSV-F protein present and the ability of those RSV-F variants to bind to motavizumab. Through these methods, it was found that the mutants expressed a wide range of fusion activity as well as motavizumab binding. These studies will be important in understanding how RSV can evolve to avoid antibody binding while retaining fusion activity and will also provide rapid screens for evaluating antibody binding and fusion of potential escape variants

Application of the Open qPCR Instrument for the in Vitro Selection of DNA Aptamers against Epidermal Growth Factor Receptor and <i>Drosophila</i> C Virus

The low-cost Open qPCR instrument can be used for different tasks in the aptamer selection process: quantification of DNA, cycle course optimization, screening, and final binding characterization. We have selected aptamers against whole <i>Drosophila</i> C virus (DCV) particles and recombinant epidermal growth factor receptor (EGFR). We performed systematic evolution of ligands by exponential enrichment (SELEX) using the Open qPCR to optimize each amplification step. The Open qPCR instrument identified the best aptamer candidate. The Open qPCR has the capacity to perform melt curves, and we used this function to perform thermofluorimetric analysis (TFA) to quantify target-aptamer binding. We confirmed target-aptamer binding using flow cytometry. A sandwich type luminescence bioassay based on our anti-DCV aptamer was sensitive to DCV and did not respond to a related virus, demonstrating that our selected anti-DCV aptamer can be used to specifically detect DCV

Lytic Cell Death Mechanisms in Human Respiratory Syncytial Virus-Infected Macrophages: Roles of Pyroptosis and Necroptosis

Human respiratory syncytial virus (RSV) is the most common cause of viral bronchiolitis and pneumonia in infants and children worldwide. Inflammation induced by RSV infection is responsible for its hallmark manifestation of bronchiolitis and pneumonia. The cellular debris created through lytic cell death of infected cells is a potent initiator of this inflammation. Macrophages are known to play a pivotal role in the early innate immune and inflammatory response to viral pathogens. However, the lytic cell death mechanisms associated with RSV infection in macrophages remains unknown. Two distinct mechanisms involved in lytic cell death are pyroptosis and necroptosis. Our studies revealed that RSV induces lytic cell death in macrophages via both of these mechanisms, specifically through the ASC (Apoptosis-associated speck like protein containing a caspase recruitment domain)-NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) inflammasome activation of both caspase-1 dependent pyroptosis and receptor-interacting serine/threonine-protein kinase 3 (RIPK3), as well as a mixed lineage kinase domain like pseudokinase (MLKL)-dependent necroptosis. In addition, we demonstrated an important role of reactive oxygen species (ROS) during lytic cell death of RSV-infected macrophages

New Perspectives on Ebola Virus Evolution.

Since the recent devastating outbreak of Ebola virus disease in western Africa, there has been significant effort to understand the evolution of the deadly virus that caused the outbreak. There has been a considerable investment in sequencing Ebola virus (EBOV) isolates, and the results paint an important picture of how the virus has spread in western Africa. EBOV evolution cannot be understood outside the context of previous outbreaks, however. We have focused this study on the evolution of the EBOV glycoprotein gene (GP) because one of its products, the spike glycoprotein (GP1,2), is central to the host immune response and because it contains a large amount of the phylogenetic signal for this virus. We inferred the maximum likelihood phylogeny of 96 nonredundant GP gene sequences representing each of the outbreaks since 1976 up to the end of 2014. We tested for positive selection and considered the placement of adaptive amino acid substitutions along the phylogeny and within the protein structure of GP1,2. We conclude that: 1) the common practice of rooting the phylogeny of EBOV between the first known outbreak in 1976 and the next outbreak in 1995 provides a misleading view of EBOV evolution that ignores the fact that there is a non-human EBOV host between outbreaks; 2) the N-terminus of GP1 may be constrained from evolving in response to the host immune system by the highly expressed, secreted glycoprotein, which is encoded by the same region of the GP gene; 3) although the mucin-like domain of GP1 is essential for EBOV in vivo, it evolves rapidly without losing its twin functions: providing O-linked glycosylation sites and a flexible surface