Rotavirus protein expression is important for virus assembly and pathogenesis
Rotaviruses have a unique morphogenesis in which particles obtain a transient membrane-envelope as newly made subviral particles bud into the endoplasmic reticulum (ER). This process is mediated by a viral nonstructural glycoprotein, NSP4. We have found that NSP4 has pleiotropic properties that became evident following expression of this protein in eukaryotic cells. NSP4 expressed in insect cells bound double-layered rotavirus particles in a manner similar to receptor-ligand interactions and this interaction is thought to trigger the particle budding process. Expression of NSP4 in insect cells also increases intracellular calcium ([Ca2+]i) levels and this effect may explain the toxicity of this protein in eukaryotic cells. Increases in [Ca2+]i levels in insect cells also are observed following exogenous addition to cells of purified NSP4 or of a synthetic peptide of NSP4. Experiments to determine the mechanism by which NSP4 causes an increase in [Ca2+]i showed that Ca2+ is released from a subset of the thapsigargin-sensitive store [endoplasmic reticulum (ER)]. However, exogenously added and endogenously expressed NSP4 use different mechanisms to alter the Ca2+ permeability of the ER membrane. We hypothesize that NSP4-mediated changes in ER membrane permeability trigger viral budding into the lumen of the ER, and eventually induce cell death and release of virus particles from infected cells. We also propose that release of NSP4 following cell lysis and the concomitant stimulation of a Ca2+ signal transduction pathway in neighboring cells contributes to altered ion transport in intestinal epithelium resulting in diarrheal disease.
KeywordsInsect Cell Baculovirus Recombinant Outer Capsid Protein Subviral Particle Mature Viral Particle
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- 4.Au K-S, Chan W-K, Estes MK (1988) Rotavirus morphogenesis involves an endoplasmic reticulum transmembrane glycoprotein. In: Compans R, Helenius A, Oldston M (eds) Cell Biology of Virus Entry, Replication and Pathogenesis. Alan R Lill, New YorkGoogle Scholar
- 9.Holmes IH (1993) Rotaviruses. In: Joklik WK (ed) The Reoviridae. Plenum Publishing, New York, pp 359–423Google Scholar
- 10.Petrie BL (1983) Biologic activity of rotavirus particles lacking glycosylated proteins. In: Compans RW, Bishop DHL (eds) Double-Stranded RNA Viruses. Elsevier, New York, pp 146–156Google Scholar
- 22.Schilling WP, Chen X, Sinkins WG, Rajan L (1994) Expression of a human bradykinin B2 receptor in Sf9 insect cells using baculovirus expression vector. FASEB J 8: A352 (Abstract)Google Scholar
- 23.Chen X, Schilling WP (1994) Desensitization of recombinant human thrombin receptor: evidence for active tethered ligand. FASEB J 8:A89 (Abstract)Google Scholar
- 25.Mattion NM, Cohen J, Estes MK (1994) The Rotavirus Proteins. In: Kapikian A (ed) Viral Infections of the Gastrointestinal Tract, 2nd edn. Marcel Dekker, New YorkGoogle Scholar
- 26.Tian P (1994) Pleiotropic properties of rotavirus nonstructural glycoprotein NSP4 in viral morphogenesis and pathogenesis. Baylor College of Medicine, PhD Thesis, p 119Google Scholar