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Dr. Mackow proposes to investigate interactions of the rotavirus VP5 protein with membranes and define requirements for VP5-induced pore formation. These studies address an essential step in the rotavirus entry process and basic mechanisms by which non-enveloped viral proteins permeabilize cellular membranes during entry.
Rotaviruses are icosahedral viruses with a triple-layered protein capsid. The outer capsid is comprised of a calcium binding glycoprotein, VP7, and a spike protein, VP4. Rotaviruses bind to cells by sialic acid (VP4) or integrin binding domains in VP4 and VP4. Rotaviruses enter cells at neutral pH by direct membrane penetration. Proteolytic cleavage of the VP4 spike into VP8 and VP5 proteins is required for infectivity and for virus permeabilization of membranes. However, little is known about the interactions of rotavirus proteins with membranes during entry. Dr. Mackow has found that purified recombinant VP5 from rhesus rotavirus (RRV) permeabilizes liposomes and that membrane permeabilization is inhibited by VP5-specific neutralizing monoclonal antibodies. He has also shown that intracellularly expressed VP5 permeabilizes cells and that VP5 forms size selective pores (~10 angstroms) within lipid bilayers. These findings suggest that VP5 permeabilization of plasma membranes is required for rotavirus entry. The mechanism by which rotaviruses and other non-enveloped viruses cross plasma membranes and enter cells is poorly understood. Dr. Mackow's findings demonstrate that purified VP5 and expressed VP5 N-terminal fragments are capable of permeabilizing membranes and cells in the absence of other viral proteins. VP5 forms pores in membranes which permit the translocation of carboxyfluorescein (CF) but not 4kDa dextrans. Permeabilizing VP5 polypeptides contain one long hydrophobic domain (HD) which shares homology with the fusion region of the alphavirus E1 protein. Residues required for E1 membrane fusion are shared by the VP5-HD and are conserved in all rotavirus strains. Further, VP5-induced CF release is blocked by neutralizing mAbs suggesting that preventing VP5 membrane permeability is a viable mechanism for neutralizing rotavirus. Dr. Mackow hypothesizes that VP5 induces pores in early endosomes which permit Ca efflux and the transition from a triple-layered particle to a transcriptionally active double-layered particle.