Oral Presentation Abstracts

We are currently investigating two aspects of the paramyxovirus fusion (F): i) the relevance of proteolytic cleavage for protein activation and ii) the significance of specific amino acid changes for resistance to peptides that correspond to heptad repeat B sequences. On the one hand, whereas most paramyxovirus F proteins are cleaved only once during maturation, the respiratory syncytial virus (RSV) F protein is cleaved twice, at two furin sites separated by 27 amino acids. In addition, most paramyxovirus F proteins require the cooperation of the receptor binding protein (HN or H) for membrane fusion; however, RSV F is independent of the attachment (G) glycoprotein for fusion activity. To investigate whether or not these two differences are interconnected, a series of mutations were introduced in a cDNA copy of the Sendai virus (SeV) F gene to reproduce partially or completely the RSV cleavage sites and the intervening sequence between them. The mutants were subsequently tested in two different cell-cell fusion assays. The results obtained indicate that the double cleavage of RSV F (and loss of the intervening peptide) somehow substitutes for the participation of the receptor binding protein in controlling the activation and activity of the SeV F protein. On the other hand, we have confirmed that peptides of a certain length, which reproduce sequences of the RSV F protein heptad repeat B (HRB), neutralize virus infectivity. To investigate their mechanism of action, RSV escape mutants were isolated after repeated passage of the virus in the presence of HRB peptides. Ten independent resistant viruses were selected containing single amino acid substitutions that changed four different residues of the RSV F protein. None of the changes occurred in HRB. However, in a 3-D model of RSV F, these residues were located near amino acid changes reported for mutants resistant to small synthetic drugs. Interestingly, some of the peptide escape mutants have lost the capacity to form large syncytia. In addition, the amino acid change in one of these mutants is identical to that described in a virus resistant to a neutralizing antibody. Hence, the peptide escape mutants identify residues that influence the fusogenic capacity of RSV F without ablating its activity. # 1 Section: Viral Structure, Entry, Replication and Cell Biology Title: Role of Human Respiratory Syncytial Virus glycoproteins in apical targeting during viral maturation in polarized epithelial cells. Authors: Melissa Batonick, Antonius G.P. Oomens, Gail W. Wertz Affiliations: Department of Pathology, University of Virginia, Charlottesville, VA 22908 Abstract: Human Respiratory Syncytial Virus (HRSV), an enveloped negative sense single-stranded RNA virus, encodes eleven known gene products. Of these, three are viral transmembrane glycoproteins, the small hydrophobic protein, SH, the attachment protein, G, and the fusion protein, F. During the course of infection of both polarized epithelial cell lines and ciliated human airway epithelial cells, the virus matures and buds from the apical surface. The viral gene products responsible for this directional release are largely unexplored, although one previous study, using overexpressed fusion glycoprotein in the absence of other viral proteins, implicated the transmembrane domain of F as containing intrinsic apical targeting signals. With some viruses, such as VSV, Measles, and Marburg, directional targeting is affected by multiple viral proteins. Therefore, we chose to examine the contribution of each of the HRSV glycoproteins to apical targeting and release, in the context of infectious virus. We generated HRSV viruses engineered to have the three glycoprotein genes deleted either individually or in groups. These viruses were used to infect polarized epithelial cells, grown on porous substrates. Prior to viral budding, HRSV proteins traffic from the site of synthesis to the site of release at the apical membrane. To study the impact of each glycoprotein on viral protein sorting to the apical plasma membrane, the cell surface localization of F and/or G glycoproteins as well as the intracellular localization of the nucleocapsid N protein were monitored by confocal microscopy. In conjunction, a cell-based ELISA quantified the amount of transmembrane glycoproteins that reached the apical and basolateral cell surfaces after infection. We also analyzed the site of directional release of the engineered viruses using an anti-N ELISA on the apical and basolateral supernatants. Our data suggest that the three viral glycoproteins, individually or in combination, are not the major Human Respiratory Syncytial Virus (HRSV), an enveloped negative sense single-stranded RNA virus, encodes eleven known gene products. Of these, three are viral transmembrane glycoproteins, the small hydrophobic protein, SH, the attachment protein, G, and the fusion protein, F. During the course of infection of both polarized epithelial cell lines and ciliated human airway epithelial cells, the virus matures and buds from the apical surface. The viral gene products responsible for this directional release are largely unexplored, although one previous study, using overexpressed fusion glycoprotein in the absence of other viral proteins, implicated the transmembrane domain of F as containing intrinsic apical targeting signals. With some viruses, such as VSV, Measles, and Marburg, directional targeting is affected by multiple viral proteins. Therefore, we chose to examine the contribution of each of the HRSV glycoproteins to apical targeting and release, in the context of infectious virus. We generated HRSV viruses engineered to have the three glycoprotein genes deleted either individually or in groups. These viruses were used to infect polarized epithelial cells, grown on porous substrates. Prior to viral budding, HRSV proteins traffic from the site of synthesis to the site of release at the apical membrane. To study the impact of each glycoprotein on viral protein sorting to the apical plasma membrane, the cell surface localization of F and/or G glycoproteins as well as the intracellular localization of the nucleocapsid N protein were monitored by confocal microscopy. In conjunction, a cell-based ELISA quantified the amount of transmembrane glycoproteins that reached the apical and basolateral cell surfaces after infection. We also analyzed the site of directional release of the engineered viruses using an anti-N ELISA on the apical and basolateral supernatants. Our data suggest that the three viral glycoproteins, individually or in combination, are not the major determinants of apical sorting or release of HRSV when in the presence of all other viral proteins. # 2 Section: Viral Structure, Entry, Replication and Cell Biology Title: Persistent of Respiratory Syncytial Virus In Human Dendritic Cells and Influence of Nitric Oxide Authors: Lynsey Hobson and Mark L Everard Affiliations: Dept. of Respiratory Medicine Sheffield Children’s Hospital, Western Bank, Sheffield, S10 2TH, UK Abstract: The annual epidemics of respiratory syncytial virus (RSV) infection are probably explained by poor herd immunity and the existence of a dormant reservoir of virus that is activated by an unknown trigger. The virus causes particular problems in infants, the elderly and patients with COPD. During two consecutive winters, human monocyte derived dendritic cells [DCs] were exposed on a single occasion to one of two forms of RSV labeled with a fluorescent expresser genes [rgRSV or rrRSV] during the epidemic season. The cultures were maintained for many months with fresh DCs being added at monthly intervals. The cultures were variously exposed to 600ppb nitric oxide for 15 minutes, nitric oxide [NO] donors and NO inhibitors outside of the RSV epidemic season. The pattern of productive infection of DCs in vitro appeared to parallel the natural epidemics in that DCs only exhibited evidence of viral replication and productive infection as manifest by intra cellular fluorescence and infection of HeLa cells during the RSV epidemic season. When the long term cultures were exposed to the above agents outside of the RSV epidemic season there was again evidence of vigorous replication and productive infection as evidenced by the reappearance of fluorescence and productive infection of HeLa cells. The results indicate that RSV may remain dormant in dendritic cells for prolonged periods and that replication appears to be activated by suppression of endogenous NO production. These observations may be key to our understanding of the mechanisms contributing to the annual epidemics of RSV infection. The annual epidemics of respiratory syncytial virus (RSV) infection are probably explained by poor herd immunity and the existence of a dormant reservoir of virus that is activated by an unknown trigger. The virus causes particular problems in infants, the elderly and patients with COPD. During two consecutive winters, human monocyte derived dendritic cells [DCs] were exposed on a single occasion to one of two forms of RSV labeled with a fluorescent expresser genes [rgRSV or rrRSV] during the epidemic season. The cultures were maintained for many months with fresh DCs being added at monthly intervals. The cultures were variously exposed to 600ppb nitric oxide for 15 minutes, nitric oxide [NO] donors and NO inhibitors outside of the RSV epidemic season. The pattern of productive infection of DCs in vitro appeared to parallel the natural epidemics in that DCs only exhibited evidence of viral replication and productive infection as manifest by intra cellular fluorescence and infection of HeLa cells during the RSV epidemic season. When the long term cultures were exposed to the above agents outside of the RSV epidemic season there was again evidence of vigorous replication and productive infection as evidenced by the reappearance of fluorescence and productive infection of HeLa cells. The results indicate that RSV may remain dormant in dendritic cells for prolonged periods and that replication appears to be activated by suppression of endogenous NO production. These observations may be key to our understanding of the mechanisms contributing to the annual epidemics of RSV infection.