GENOME PACKAGING AND HOST-PATHOGEN INTERACTIONS IN PARAMYXOVIRUS ASSEMBLY AND BUDDING

Open Access
- Author:
- Ray, Greeshma Vivekananda
- Graduate Program:
- Pathobiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 10, 2016
- Committee Members:
- Anthony Paul Schmitt, Dissertation Advisor/Co-Advisor
- Keywords:
- paramyxovirus
assembly
budding
virology - Abstract:
- Paramyxoviruses possess non-segmented, negative-sense RNA genomes that are encapsidated by viral nucleocapsid protein (NP), and are packaged into budding virus particles via NP protein interaction with viral matrix (M) proteins, thereby creating infectious viruses. We had previously identified a DLD sequence near the C- terminal end of parainfluenza virus 5 (PIV5) NP protein that was important for interaction with PIV5 M protein, and for enhancing PIV5 virus-like particle (VLP) production. We have shown here that a DLD-containing 15 amino acid sequence derived from PIV5 NP or Nipah virus N proteins is sufficient to direct a foreign protein, Renilla luciferase, into virus-like particles. This short DLD-containing sequence was also able to replace the requirement for NP protein in PIV5 VLP production. Mumps virus NP protein contains a DWD sequence instead of DLD, and consequently, PIV5 NP protein cannot interact efficiently with mumps virus M protein, in spite of the two viruses being very closely related. Altering PIV5 NP DLD sequence to mumps virus NP DWD sequence creates a new PIV5 NP protein that is now compatible with M proteins from both PIV5 and mumps virus. We hypothesize that DLD-like sequences define compatibilities between paramyxovirus M and NP proteins that can be manipulated to drive production of virus-like particles containing therapeutics for delivery to cells. A hallmark of enveloped virus infection is the hijacking of host cellular pathways during viral egress. To define host factors involved in paramyxovirus budding, a yeast two-hybrid screening approach was used previously, which identified angiomotin-like 1 (AmotL1) as a host factor that binds to the matrix (M) proteins of parainfluenza virus 5 (PIV5) and mumps virus. AmotL1 belongs to a family of proteins that also includes angiomotin (Amot) and angiomotin-like 2 (AmotL2). All three angiomotins harbor PPXY motifs and interact with WW-domain containing proteins such as Nedd4 and YAP. We found that the PIV5 and mumps virus M proteins bind to AmotL1, but not to Amot or AmotL2. Binding was mapped to a 83- amino acid region from the C-terminal portion of AmotL1. Overexpression of M- binding AmotL1-derived polypeptides inhibited production of PIV5 and mumps virus-like particles (VLPs), while overexpression of the corresponding regions of Amot and AmotL2 had little effect on VLP production. Co-immunoprecipitation studies support the presence of a three-way interaction between Nedd4, AmotL1, and M. Efficient pulldown of M with Nedd4 was observed only when AmotL1 was expressed to bridge the gap. Our findings support a model in which paramyxoviruses indirectly recruit the same WW domain-containing proteins to virus assembly sites through AmotL1 that other enveloped viruses recruit directly via PPXY late domains.