The objective of the proposed research is to determine mechanisms by which human metapneumovirus (hMPV) virions are assembled at the host cell plasma membrane. The paramyxovirus hMPV is an emerging human pathogen that is a major cause of lower respiratory tract infection in children. Genome organization and sequence confirms its close relationship to respiratory syncytial virus (RSV). The amino acid sequence of the hMPV fusion (F) protein shares significant homology with RSV F. The paramyxovirus F proteins are type I transmembrane glycoproteins that mediate viral fusion to cell membranes. Using GFP fusion proteins encoding RSV and hMPV F transmembrane and cytoplasmic domains, F regions have been identified that are sufficient for membrane localization in cells that support infection. The central hypothesis of this research is that hMPV assembly at the plasma membrane is a highly regulated process mediated by F protein sequences contained in the transmembrane and cytoplasmic domains. Three specific aims are proposed to define the role of F domains in hMPV assembly. In Specific Aim 1 the hypothesis that specific C-terminal regions within hMPV F determine its membrane localization will be tested. Fluorescence imaging techniques will be used to evaluate membrane localization of GFP-hMPV F fusion proteins and native hMPV F in transfected and infected cells. In Specific Aim 2 the minimal determinants of hMPV F membrane localization will be identified using truncation and amino acid substitution mutants. Vesicular stomatitis virus G-hMPV F chimeric proteins will be used to characterize trafficking signals within F. In Specific Aim 3 the role of hMPV F cytoplasmic tail domain in the coordination of viral assembly will be determined by identifying hMPV protein binding partners, using yeast two-hybrid and co-immunoprecipitation analysis. These experiments will contribute important information about the role of the F protein in regulated cellular protein trafficking and viral assembly. This work will be conducted in the context of a scientific environment and training program designed to establish Dr. Peters as an independent investigator focused on hMPV cellular virology.

K08AI058006

2004-04-01

2007-12-31