The rotavirus nonstructural glycoprotein NSP4 possesses membrane destabilization activity

During a unique morphogenetic process, rotaviruses obtain a transient membrane envelope when newly synthesized subviral particles bud into the endoplasmic reticulum (ER). As rotavirus particles mature, they lose their transient membrane and a layer of the glycoprotein VP7 forms the virion outer capsid shell. The nonstructural glycoprotein NSP4 functions as an intracellular receptor in the ER membrane (K. S. Au, W. K. Chan, J. W. Burns, and M. K. Estes, J. Virol. 63:4553-4562, 1989), and it has been hypothesized that NSP4 is involved in the removal of the envelope during viral morphogenesis (M. K. Estes and J. Cohen, Microbiol. Rev. 53:410-449, 1989; B. L. Petrie, M. K. Estes, and D. Y. Graham, J. Virol. 46:270-274, 1983). The purpose of the present study was to determine if NSP4 has a direct membrane destabilization activity (MDA) by using liposome leakage assays and electron microscopic visualization of liposome, microsome, and viral envelope disruption. The fluorescent marker (calcein) incorporated into liposomes was released when the liposomes were incubated with purified NSP4. A region corresponding to amino acid residues 114 to 135 of NSP4 also released calcein from liposomes. NSP4(114-135) peptide-specific antibody completely blocked the MDA of the purified NSP4 protein. These results suggest that this region contains at least part of the functional domain of NSP4. Liposomes composed of phosphatidylcholine and microsomes (to simulate ER membranes) were broken when observed by electron microscopy after incubation with NSP4 or the NSP4(114-135) peptide. In contrast, the envelope of Sendai virus, which is derived from cytoplasmic membranes, and erythrocytes were not disrupted by NSP4 and the NSP4(114-135) peptide. These results provide direct evidence that NSP4 possesses MDA and suggest that it can cause ER membrane damage. Therefore, NSP4 might play an important role in the removal of the transient envelope from budding particles during viral morphogenesis. A model for the MDA of NSP4 in viral morphogenesis is proposed.

[1]  M. Estes,et al.  The rotavirus nonstructural glycoprotein NSP4 mobilizes Ca2+ from the endoplasmic reticulum , 1995, Journal of virology.

[2]  W. Rocque,et al.  Effects of pH on bacterial porin function. , 1992, Biochemistry.

[3]  J. Thompson,et al.  Effect of temperature on receptor-activated changes in [Ca2+]i and their determination using fluorescent probes. , 1991, The Journal of biological chemistry.

[4]  J L Cornette,et al.  Prediction of immunodominant helper T cell antigenic sites from the primary sequence. , 1987, Journal of immunology.

[5]  M. Estes,et al.  Characterization of rotavirus VP2 particles. , 1994, Virology.

[6]  D. Nicholls,et al.  Intracellular calcium homeostasis. , 1986, British medical bulletin.

[7]  A. Charpilienne,et al.  Rotavirus interaction with isolated membrane vesicles , 1994, Journal of virology.

[8]  C. Rinaldo,et al.  Alterations in cell membrane permeability by the lentivirus lytic peptide (LLP-1) of HIV-1 transmembrane protein. , 1993, Virology.

[9]  M. McKinney,et al.  A simple, non-chromatographic procedure to purify immunoglobulins from serum and ascites fluid. , 1987, Journal of immunological methods.

[10]  C. Dempsey.  The actions of melittin on membranes. , 1990, Biochimica et biophysica acta.

[11]  H De Loof,et al.  Amphipathic helix motif: Classes and properties , 1990, Proteins.

[12]  A. Bellamy,et al.  Interaction of rotavirus cores with the nonstructural glycoprotein NS28. , 1989, Virology.

[13]  R. Tsien,et al.  A new generation of Ca2+ indicators with greatly improved fluorescence properties. , 1985, The Journal of biological chemistry.

[14]  M. Poruchynsky,et al.  Calcium depletion blocks the maturation of rotavirus by altering the oligomerization of virus-encoded proteins in the ER , 1991, The Journal of cell biology.

[15]  I. Pastan,et al.  pH-dependent lysis of liposomes by adenovirus. , 1986, Biochemistry.

[16]  G. Menestrina,et al.  Membrane damage by hemolytic viruses, toxins, complement, and other cytotoxic agents. A common mechanism blocked by divalent cations. , 1986, The Journal of biological chemistry.

[17]  G. Han,et al.  9-Fluorenylmethoxycarbonyl amino-protecting group , 1972 .

[18]  M. Estes,et al.  Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants , 1996, Journal of virology.

[19]  R. Schlegel,et al.  A synthetic peptide corresponding to the NH2 terminus of vesicular stomatitis virus glycoprotein is a pH-dependent hemolysin. , 1984, The Journal of biological chemistry.

[20]  P. Nandi,et al.  Interaction of rotavirus particles with liposomes , 1992, Journal of virology.

[21]  M. Estes,et al.  Age-Dependent Diarrhea Induced by a Rotaviral Nonstructural Glycoprotein , 1996, Science.

[22]  M. Estes,et al.  Rotavirus gene structure and function. , 1989, Microbiological reviews.

[23]  M. Estes,et al.  Receptor activity of rotavirus nonstructural glycoprotein NS28 , 1989, Journal of virology.

[24]  L. Smith,et al.  A novel DNA-peptide complex for efficient gene transfer and expression in mammalian cells. , 1996, Gene therapy.

[25]  R. Schlegel,et al.  Biologically active peptides of the vesicular stomatitis virus glycoprotein , 1985, Journal of virology.

[26]  R. New,et al.  Liposomes : a practical approach , 1990 .

[27]  M. Poruchynsky,et al.  Rotavirus protein rearrangements in purified membrane-enveloped intermediate particles , 1991, Journal of virology.

[28]  M. Estes,et al.  The nonstructural glycoprotein of rotavirus affects intracellular calcium levels , 1994, Journal of virology.

[29]  S. Takahashi,et al.  Modification of the N-terminus of membrane fusion-active peptides blocks the fusion activity. , 1991, Biochemical and biophysical research communications.

[30]  R. Montelaro,et al.  A versatile synthetic peptide-based ELISA for identifying antibody epitopes. , 1994, Journal of immunological methods.

[31]  J. Davies,et al.  Molecular Biology of the Cell , 1983, Bristol Medico-Chirurgical Journal.

[32]  M. Miller,et al.  A structural correlation between lentivirus transmembrane proteins and natural cytolytic peptides. , 1991, AIDS research and human retroviruses.

[33]  F. Szoka,et al.  Mechanism of leakage of phospholipid vesicle contents induced by the peptide GALA. , 1990, Biochemistry.

[34]  H. Greenberg,et al.  Rotavirus-induced fusion from without in tissue culture cells , 1995, Journal of virology.

[35]  W. DeGrado,et al.  Membrane fusion activity of the influenza virus hemagglutinin: interaction of HA2 N-terminal peptides with phospholipid vesicles. , 1991, Biochemistry.

[36]  M. Estes,et al.  A subviral particle binding domain on the rotavirus nonstructural glycoprotein NS28. , 1993, Virology.

[37]  Dr. Monique Dubois-Dalcq,et al.  Assembly of Enveloped RNA Viruses , 1984, Springer Vienna.

[38]  D. Graham,et al.  Effects of Tunicamycin on Rotavirus Morphogenesis and Infectivity , 1983, Journal of virology.