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. 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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI044917-01
Application #
2827242
Study Section
Virology Study Section (VR)
Project Start
1999-09-01
Project End
2003-08-31
Budget Start
1999-09-01
Budget End
2000-08-31
Support Year
1
Fiscal Year
1999
Total Cost
Indirect Cost
Name
State University New York Stony Brook
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Golantsova, Nina E; Gorbunova, Elena E; Mackow, Erich R (2004) Discrete domains within the rotavirus VP5* direct peripheral membrane association and membrane permeability. J Virol 78:2037-44
Geimonen, Erika; Fernandez, Imelyn; Gavrilovskaya, Irina N et al. (2003) Tyrosine residues direct the ubiquitination and degradation of the NY-1 hantavirus G1 cytoplasmic tail. J Virol 77:10760-868
Geimonen, Erika; LaMonica, Rachel; Springer, Karen et al. (2003) Hantavirus pulmonary syndrome-associated hantaviruses contain conserved and functional ITAM signaling elements. J Virol 77:1638-43
Geimonen, Erika; Neff, Sherry; Raymond, Tracy et al. (2002) Pathogenic and nonpathogenic hantaviruses differentially regulate endothelial cell responses. Proc Natl Acad Sci U S A 99:13837-42
Gavrilovskaya, I N; Peresleni, T; Geimonen, E et al. (2002) Pathogenic hantaviruses selectively inhibit beta3 integrin directed endothelial cell migration. Arch Virol 147:1913-31
LaMonica, R; Kocer, S S; Nazarova, J et al. (2001) VP4 differentially regulates TRAF2 signaling, disengaging JNK activation while directing NF-kappa B to effect rotavirus-specific cellular responses. J Biol Chem 276:19889-96