We are investigating the molecular mechanisms of infection of epithelia by enveloped viruses. Fusion proteins of enveloped viruses play an essential role in the early stages of infection, enabling fusion of the viral envelope with a cellular membrane and release of the internal viral nucleoprotein complexes into cells to initiate the infection process. At later stages of infection the fusion glycoproteins of many viruses are expressed on plasma membranes, and those with a neutral pH optimum induce cell-to-cell fusion, a prominent cytopathic effect with many viruses. Although major advances have been made in our knowledge of the structure of viral fusion proteins, many aspects of the fusion process remain unknown. We are extending our novel recent observations concerning the cytoplasmic domains of the fusion (F) glycoproteins of paramyxoviruses, which contain specific amino acid sequences that regulate membrane fusion activity. We are investigating the mechanisms by which the cytoplasmic domain regulates cell fusion, and the consequences for virus-cell interaction. Many of our studies focus on a recently characterized paramyxovirus, designated SER virus, which exhibits no observable cell fusion activity in several cell types; the lack of fusion activity is the result of an extended cytoplasmic domain in the SER F protein. We are pursuing the following aims: (1.) To investigate the possible mechanism involved in suppression of fusion activity, we will test the hypothesis that the extended cytoplasmic domain stabilizes the metastable "pre-fusion" state of the SER F protein, thus increasing the activation energy required for conversion to a fusion-active state. (2.) We will determine if the marked differences in fusion phenotypes of SER virus result in differences in virus-cell interaction, including whether SER virus entry into cells may differ from that of other paramyxoviruses by involving a low pH-dependent process. We will also determine whether serial passage of SER virus results in the appearance of fusogenic variants with a truncated cytoplasmic tail, and whether such viruses possess a replicative advantage. (3.) We will determine the effects of specific changes in the cytoplasmic domain on virus replication in the context of otherwise isogenic recombinant viruses. We will introduce SER F genes with defined sequence changes to replace the F gene of SV5 in an infectious DNA clone, and determine their effects on virus replication in cell culture as well as in a mouse model.

[compans@microbio.emory.edu]