title: The study of the intercellular trafficking of the fusion proteins of herpes simplex virus protein VP22.
abstract: BACKGROUND
  Genetic modifications can improve the therapeutic efficacy of mesenchymal stem cell (MSC) transplantation in myocardial infarction. However, so far, the efficiency of MSC modification is very low. Seeking for a more efficient way of MSC modification, we investigated the possibility of employing the intercellular trafficking capacity of the herpes simplex virus type-1 tegument protein VP22 on the enhancement of MSC modification.
  
  
  METHODS
  Plasmids pVP22-myc, pVP22-EGFP, pEGFP-VP22, pVP22-hBcl-xL and phBcl-xL-VP22 were constructed for the expressions of the myc-tagged VP22 and the fusion proteins VP22-EGFP, EGFP-VP22, VP22-hBcl-xL and hBcl-xL-VP22. MSCs were isolated from rat bone marrow and the surface markers were identified by Flowcytometry. COS-1 cells were transfected with the above plasmids and co-cultured with untransfected MSCs, the intercellular transportations of the constructed proteins were studied by immunofluorescence. The solubility of VP22-hBcl-xL and hBcl-xL-VP22 was analyzed by Western blot.
  
  
  RESULTS
  VP22-myc could be expressed in and spread between COS-1 cells, which indicates the validity of our VP22 expression construct. Flowcytometry analysis revealed that the isolated MSCs were CD29, CD44, and CD90 positive and were negative for the hematopoietic markers, CD34 and CD45. The co-culturing and immunofluorescence assay showed that VP22-myc, VP22-EGFP and EGFP-VP22 could traffic between COS-1 cells and MSCs, while the evidence of intercellular transportation of VP22-hBcl-xL and hBcl-xL-VP22 was not detected. Western blot analysis showed that VP22-hBcl-xL and hBcl-xL-VP22 were both insoluble in the cell lysate suggesting interactions of the fusion proteins with other cellular components.
  
  
  CONCLUSIONS
  The intercellular trafficking of VP22-myc, VP22-EGFP and EGFP-VP22 between COS-1 cells and MSCs presents an intriguing prospect in the therapeutic application of VP22 as a delivery vehicle which enhances genetic modifications of MSCs. However, VP22-hBcl-xL and hBcl-xL-VP22 failed to spread between cells, which are due to the insolubility of the fusion protein incurred by interactions with other cellular components.
authors: Department of Cardiovascular Surgery, General Hospital of Shenyang Military Area Command, Shenyang, Liaoning, China.
authors: Xiaodong
authors: X
authors: Xue
authors: Department of Cardiothoracic Surgery, Ningxia People's Hospital, Yinchuan, Ningxia, China.
authors: Jianhua
authors: J
authors: Huang
authors: Department of Cardiovascular Surgery, General Hospital of Shenyang Military Area Command, Shenyang, Liaoning, China.
authors: Huishan
authors: H
authors: Wang
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology; D001973:Bromodeoxyuridine / Q000378:metabolism; D019556:COS Cells; D002457:Cell Extracts; D002458:Cell Fractionation; D002469:Cell Separation; D002522:Chlorocebus aethiops; D005110:Extracellular Space / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000378:metabolism; D021381:Protein Transport; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012995:Solubility; D015678:Viral Structural Proteins / Q000378:metabolism*; D051020:bcl-X Protein / Q000378:metabolism
chemical_list: D015415:Biomarkers; D002457:Cell Extracts; D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins; D051020:bcl-X Protein; C485184:enhanced green fluorescent protein; C073413:herpes simplex virus type 1 protein VP22; D049452:Green Fluorescent Proteins; D001973:Bromodeoxyuridine