Characterization of the Interaction Between the Attachment and Fusion Glycoproteins Required for Paramyxovirus Fusion: a Dissertation

The first step of viral infection requires the binding of the viral attachment protein to cell surface receptors. Following binding, viruses penetrate the cellular membrane to deliver their genome into the host cell. For enveloped viruses , which have a lipid bilayer that surrounds their nucleocapsids , entry into the host cell requires the fusion of viral and cellular membranes. This process is mediated by viral glycoproteins located on the surface of the virus. F or many enveloped viruses, such as influenza, Ebola, and human immunodeficiency virus, the fusion protein is responsible for mediating both attachment to cellular receptors and membrane fusion. However, paramyxoviruses are unique among fusion promoting viruses because their receptor binding and fusion activities reside on two separate proteins. This unique distribution functions necessitates a mechanism by which the two proteins can transmit the juxtaposition of the viral and host cell membranes , mediated by the attachment protein (HN/H), into membrane fusion, mediated by the fusion (F) protein. This mechanism allows for paramyxoviruses to gain entry into and spread between cells , and therefore , is an important aspect of virus infection and disease progression. Despite the conservation of receptor binding activity among members of the Paramyxovirinae subfamily, for most of these viruses , including Newcastle disease virus (NDV), heterologous HN proteins cannot complement F in the promotion of fusion; both the HN and F proteins must originate from the same virus. This is consistent with the existence of a virusspecific interaction between the two glycoproteins. Thus , one or more domains on the HN and F proteins is thought to mediate a specific interaction between them that is an integral part of the fusion process. Therefore , the primary focus of this thesis is the identification of the site(s) on HN that directly contacts F in the HN F interaction. The ectodomain of the HN protein consists of a stalk and a terminal globular head. Analysis of the fusion activity of chimeric paramyxovirus HN proteins indicates that the stalk region ofHN determines its F protein specificity. The first goal of this research was to address the question of whether the stalk not only determines Fspecificity, but does so by directly mediating the interaction with F. To establish a correlation between the amount of fusion and the extent of the HN F interaction, a specific and quantitative co-immunoprecipitation assay was used that detects the HN-F complex at the cell surface. As an initial probe of the role of the HN stalk in mediating the interaction with F, Nglycans were individually added at several positions in the region. glycan addition at positions 69 and 77 in the stalk specifically and completely block both fusion and the HNinteraction without affecting either HN structure or its other activities. However, though they also prevent fusion, N-glycans added at other positions in the stalk also modulate activities that reside in the globular head of HN. This correlates with an alteration of the tetrameric structure of the protein as indicated by sucrose gradient sedimentation analyses. These additional N-glycans likely indirectly affect fusion, perhaps by interfering with changes in the conformation of HN that link receptor binding to the fusion activation of F. To address the issue of whether N-glycan addition at any position in HN would abolish fusion, an N-glycan was added in another region at the base of the globular head ofHN (residues 124152), which was previously predicted by a peptide-based analysis to mediate the interaction with F. HN carring this additional N-glycan exhibits significant fusion promoting activity, arguing against this site being part of the F-interactive domain in HN. These data support the idea that the F-interactive site on HN is defined by the stalk region of the protein. Site-directed mutagenesis was used to begin to explore the role of individual residues in the stalk in the interaction with F. The characteristics of the F interactive domain in the stalk of r-" HN are that it is a conserved motif with enough sequence heterogeneity to account for the specificity of the interaction. One such region that meets these requirements is the intervening region (IR) (residues 89-95), a non-helical domain situated between two conserved heptad repeats. Several amino acid substitutions for a completely conserved proline residue in this region impair not only fusion and the HN-F interaction, but also decrease neuraminidase activity in the globular domain and alter the structure of the protein, suggesting that the substitutions indirectly affect the HN-F interaction. Substitutions for L94 also interfere with fusion, but have no significant effect on any other HN function or its structure. Amino acid substitutions at two other positions in the IR (A89 and L90) also modulate only fusion. In all cases , diminished fusion correlates with a decreased ability of the mutated HN protein to interact with F at the cell surface. These findings indicate that the IR is critical to the role of HN in the promotion of fusion and are consistent with its direct involvement in the interaction with the homologous F protein. These are the first point mutations in the HN protein for which a correlation has been demonstrated between the extent of the HN-F interaction and the amount of fusion. This argues strongly that the co-IP assay is an accurate reflection of the HN-F interaction. The second goal of this research was to address the HNinteraction from the perspective of the F protein by investigating the relationship between receptor binding, the HNinteraction, and fusion using a highly fusogenic form of the F protein. It has previously been shown that an L289A substitution in NDV F eliminates the requirement for HN in the promotion of fusion and enhances HN-dependent fusion above wild-type (wt) levels. Here, it was shown that the HN-independent fusion exhibited by L289A-F in Cos-7 cells cannot be duplicated in BHK cells. However, when L289A-F is co-expressed with wt HN , enhanced fusion above wt levels is observed in BHK cells. Additionally, when L289A-F is co-expressed with IR-mutated HN proteins previously shown to promote low levels of fusion with wt F , a 2. fold increase in fusion was observed. However, similar to wt F , an interaction between L289A-F and the IR-