Functional replacement of the R region of simian immunodeficiency virus-based vectors by heterologous elements.
Substitution of lentiviral cis-acting elements by heterologous sequences might allow the safety of lentiviral vectors to be enhanced by reducing the risk of homologous recombination and vector mobilization. Therefore, a substitution and deletion analysis of the R region of simian immunodeficiency virus (SIV)-based vectors was performed and the effect of the modifications on packaging and transfer by SIV and human immunodeficiency virus type 1 (HIV-1) particles was analysed. Deletion of the first 7 nt of R reduced vector titres by 10- to 20-fold, whilst deletion of the entire R region led to vector titres that were 1500-fold lower. Replacement of the R region of SIV-based vectors by HIV-1 or Moloney murine sarcoma virus R regions partially restored vector titres. A non-retroviral cellular sequence was also functional, although to a lesser extent. In the absence of tat, modification of the R region had only minor effects on cytoplasmic RNA stability, steady-state levels of vector RNA and packaging, consistent with the known primary function of R during reverse transcription. Although the SIV R region of SIV-based vectors could be replaced functionally by heterologous sequences, the same modifications of R led to a severe replication defect in the context of a replication-competent SIV. As SIV-based vectors containing the HIV-1 R region were transferred less efficiently by HIV-1 particles than wild-type SIV vectors, a match between R and cis-acting elements of the vector construct seems to be more important than a match between R and the Gag or Pol proteins of the vector particle.
Brandt
Sabine
S
Department of Molecular and Medical Virology, Ruhr University Bochum, D-44780 Bochum, Germany.
Grunwald
Thomas
T
Department of Molecular and Medical Virology, Ruhr University Bochum, D-44780 Bochum, Germany.
Lucke
Susann
S
Department of Molecular and Medical Virology, Ruhr University Bochum, D-44780 Bochum, Germany.
Stang
Alexander
A
Department of Molecular and Medical Virology, Ruhr University Bochum, D-44780 Bochum, Germany.
Überla
Klaus
K
Department of Molecular and Medical Virology, Ruhr University Bochum, D-44780 Bochum, Germany.
D004252:DNA Mutational Analysis; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics; D008979:Moloney murine leukemia virus / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic; D015302:Simian Immunodeficiency Virus / Q000235:genetics* / Q000254:growth & development; D019065:Virus Assembly / Q000235:genetics; D014779:Virus Replication / Q000235:genetics
D012367:RNA, Viral