title: Immobilized HIV-1 Tat protein promotes gene transfer via a transactivation-independent mechanism which requires binding of Tat to viral particles.
abstract: BACKGROUND
  Retroviral transduction of cells is improved upon virus adsorption onto immobilized fibronectin (FN) fragments. Because HIV-1 Tat possesses the same functional domains that lead to increased transduction efficiency in FN by colocalization of bound virus and cells, we hypothesized that Tat could enhance gene transfer by a similar mechanism.
  
  
  METHODS
  Single-cycle replication retro- or lentivirus carrying green fluorescent protein or cloramphenicol acetyltransferase as reporter genes were added to wells coated with Tat or Tat peptides. Wells were extensively washed to remove unbound virus and levels of transduction were detected by measuring reporter gene expression. Virus adsorption to immobilized Tat was measured using a p24 antigen capture assay.
  
  
  RESULTS
  Immobilized Tat efficiently binds retro- and lentiviral particles and mediates virus transmission at virus input doses that were otherwise unable to transduce susceptible cells. Virus adsorption to Tat is not mediated by envelope glycoprotein (Env) because immobilized Tat binds and retains vesicular stomatitis virus G (VSV-G) pseudotypes as well as envelope-free particles. HIV-1 Env or VSV-G are required for Tat-assisted transduction, which is abrogated by an antibody blocking the HIV-1 Env-CD4 interaction. Tat-assisted transduction is mediated by the cysteine-rich region of Tat, which is known to be essential for Tat transactivation activity. However, Tat transactivation is not required for Tat-assisted transduction, as indicated by the enhancement of transduction by transactivation-silent Tat mutants.
  
  
  CONCLUSIONS
  Immobilized Tat promotes virus transduction by a transactiva- tion-independent mechanism, which requires binding of virus to Tat. Recombinant Tat or Tat fragments provide a new method to increase efficiency of retro- and lentiviral based gene transfer and gene therapy.
authors: National AIDS Center, Istituto Superiore Sanità, Rome, Italy.
authors: Filomena
authors: F
authors: Nappi
authors: Chiara
authors: C
authors: Chiozzini
authors: Valentina
authors: V
authors: Bordignon
authors: Alessandra
authors: A
authors: Borsetti
authors: Stefania
authors: S
authors: Bellino
authors: Marco
authors: M
authors: Cippitelli
authors: Giovanni
authors: G
authors: Barillari
authors: Antonella
authors: A
authors: Caputo
authors: Mudit
authors: M
authors: Tyagi
authors: Mauro
authors: M
authors: Giacca
authors: Barbara
authors: B
authors: Ensoli
mesh_terms: D001665:Binding Sites; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015533:Transcriptional Activation; D014161:Transduction, Genetic; D014771:Virion / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus