Enhancement of retroviral production from packaging cell lines expressing the human immunodeficiency type 1 VPU gene.
The HIV-1 Vpu protein stimulates virus production by enhancing the release of viral particles from infected cells. Interestingly, Vpu was also shown to enhance the release of capsids produced by gag gene contructs of other retroviruses that lack a Vpu-like activity. To investigate the effect of Vpu expression on viral particle production in retroviral packaging cell line, we developed the Damp-VpuP cell line in which vpu expression is under the control of the tetracycline-responsive promoter. Retroviral production was measured by dosage of virion-associated reverse transcriptase activity, by capsid protein immuno-detection in cell-free supernatants and by evaluating the transfer of antibiotic resistance to target cells. Induction of the Damp-VpuP cell line caused a 40-fold increase in the titer of infectious virus-like particles when compared with control cell lines. This increase in viral titer was not the result of a clonal effect nor was it a consequence of high selective pressure but rather the effect of a Vpu-mediated enhancement of viral particle production. Similar results using the third generation psi CRIP packaging cell line confirmed these findings. Constitutive expression of vpu caused a 13-fold increase in viral titer in this packaging cell line. These results indicate that the expression of HIV-1 vpu in retroviral packaging cell lines can significantly improve the titers of infectious retroviral particles.
Kobinger
G P
GP
Département de Microbiologie et Immunologie, Faculté de Médecine, Université de Montréal, Québec, Canada.
Mouland
A J
AJ
Lalonde
J P
JP
Forget
J
J
Cohen
E A
EA
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004285:Dogs; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D054298:Human Immunodeficiency Virus Proteins; D006801:Humans; D012190:Retroviridae; D054334:Viral Regulatory and Accessory Proteins; D019065:Virus Assembly; D000086082:Viroporin Proteins
D054298:Human Immunodeficiency Virus Proteins; D054334:Viral Regulatory and Accessory Proteins; C514859:vpu protein, Human immunodeficiency virus 1; D000086082:Viroporin Proteins