Molecular evidence of inefficient transduction of proliferating human B lymphocytes by VSV-pseudotyped HIV-1-derived lentivectors.
Lentiviral vectors are attractive tools to transduce dividing and nondividing cells. Human tonsillar B lymphocytes have been purified and induced to proliferate by the addition of anti-CD40 + IL-4 or anti-CD40 + anti-micro signals and transduced at high MOI with a VSV pseudotyped lentivector carrying the eGFP gene under the control of the PGK promoter. Parallel cultures of PHA-stimulated T lymphocytes containing a comparable amount of cycling cells during the infection reached over 70% eGFP transduction. By contrast, only less than 3% B lymphocytes became eGFP positive after 7 days from transduction. Molecular analysis of the viral life cycle shows that cytoplasmic retrotranscribed cDNA and nuclear 2LTR circles are detectable at lower levels and for a shorter period of time in proliferating B cells with respect to proliferating T lymphocytes. Moreover, FACS-sorted eGFP-positive and negative B cell populations were both positive for the presence of retrotranscribed cDNA and 2LTR circles nuclear forms. By contrast, nested Alu-LTR PCR allowed us to detect an integrated provirus in FACS-sorted eGFP-positive cells only. Together with the demonstration that infection in saturation conditions led to an increase in the percentage of transduced cells (reaching 9%), these findings suggest that in proliferating B lymphocytes, lentiviral transduction is an inefficient process blocked at the early steps of the viral life cycle possibly involving partially saturable restriction factors.
Serafini
M
M
Istituto di Ricerche Farmacologiche "Mario Negri", 20157, Milan, Italy.
Naldini
L
L
Introna
M
M
D021581:Active Transport, Cell Nucleus; D001402:B-Lymphocytes / Q000166:cytology / Q000821:virology*; D001483:Base Sequence; D002453:Cell Cycle; D002455:Cell Division; D004279:DNA, Viral / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000254:growth & development; D008164:Luminescent Proteins / Q000235:genetics; D011533:Proviruses / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D014158:Transcription, Genetic; D014161:Transduction, Genetic; D014721:Vesicular stomatitis Indiana virus / Q000145:classification / Q000235:genetics*; D016662:Virus Integration
D004279:DNA, Viral; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins