Lentivirus-mediated transduction of PKR into CD34(+) hematopoietic stem cells inhibits HIV-1 replication in differentiated T cell progeny.
Previous studies from this laboratory evaluated the role of p68 kinase (PKR) in the control of HIV-1 replication via retrovirus-mediated gene transfer. PKR was studied because it is a key component of the interferon (IFN)-associated innate antiviral defense pathway in mammalian cells. In this study, CD34(+) hematopoietic stem cells (HSC) were transduced with an HIV-1-based lentiviral vector encoding the PKR transgene (pHIV-PIB) and cultured under conditions that support in vitro differentiation. With high-titer pseudotyped vector stocks, the histogram suggests 100% transduction of the HSC because the cells were blasticidin resistant. Analysis of transduced cells by hybridization revealed an average proviral vector copy number of 1.8 and 2.1 copies of vector sequence per cell. Increased PKR expression and activity (phosphorylation of eukaryotic initiation factor 2alpha [eIF2alpha]) were demonstrated in PKR-transduced, differentiated HSC. There was minimal reduction in cell viability and no induction of apoptosis after transduction of PKR. HSC transduced with the pHIV-PIB lentiviral vector demonstrated normal differentiation into CD34-derived T cell progeny. Two days after HIV-1 infection, lentivirus-mediated transduction of PKR inhibited HIV-1 replication by 72% in T cell progeny compared with cells transduced with the empty vector control (pHIV-IB). By days 5 and 7 post-HIV-1 infection, the surviving PKR-transduced cells were protected from HIV-1 infection, as evidenced by a decrease in p24 antigen expression of at least two orders of magnitude. Our results demonstrate that PKR can be effectively delivered to HSC by a lentiviral vector and can protect CD34-derived T cell progeny from HIV-1 infection. These results provide support for application of the innate antiviral defense pathway in a gene therapy setting to the treatment of HIV-1 infection.
Dimitrova
Dessislava I
DI
Departments of Pharmacology, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Yang
Xiaowei
X
Reichenbach
Nancy L
NL
Karakasidis
Stephanos
S
Sutton
Richard E
RE
Henderson
Earl E
EE
Rogers
Thomas J
TJ
Suhadolnik
Robert J
RJ
D018952:Antigens, CD34 / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000502:physiology; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D005822:Genetic Vectors; D015658:HIV Infections / Q000235:genetics* / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000502:physiology* / Q000821:virology; D006801:Humans; D016176:T-Lymphocyte Subsets / Q000502:physiology; D014161:Transduction, Genetic; D014779:Virus Replication / Q000235:genetics* / Q000502:physiology; D019892:eIF-2 Kinase / Q000235:genetics*
D018952:Antigens, CD34; D019892:eIF-2 Kinase