We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP.
Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the usefulness of LTR-based reporter to mark HIV positive cells 1,2,3. Here, we constructed an expression lentiviral vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites 4,5,6. The vector was incorporated into a lentiviral reporter virus, permitting highly specific detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of the green fluorescence protein (GFP). The application of this vector as reported here offers a novel alternative approach to existing methods, such as in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector can also express therapeutic genes for basic or clinical experimentation to target HIV-positive cells.
As with the earlier developed Tat-dependent expression vectors, the Rev system described here is an exploitation of an evolved HIV process. The inclusion of Rev-dependency renders the LTR-based expression vector highly dependent on the presence of replicating HIV. The application of this vector as reported here, an HIV-dependent reporter virus, offers a novel new approach to identify HIV-positive cells. The vector permits examination of living cells, can express any gene for basic or clinical experimentation, and as a pseudo-typed lentivirus has access to most cell types and tissues.
The authors have nothing to disclose.
The work was supported in part by Public Health Service grant 1R01AI081568 from NIAID to Y. W. and by the generous donations of the donors and riders of the 2009 NYCDC AIDS Ride organized by M. Rosen and Day2 Inc.
Material Name | Typ | Company | Catalogue Number | Comment |
---|---|---|---|---|
Biosafety Cabinet | ||||
37°C 5%CO2 incubator | ||||
Flow cytometer | FACSCalibur | |||
Centrifuge | Beckman | Allega™6KR | ||
4°C regrigerator | ||||
-20°C refrigerator | ||||
-80°C refrigerator | ||||
Cell counter | Nexcelom | Cellometer™ AutoT4 | ||
HEK293T cell line | NIH | |||
CEM-SS cell line | NIH | |||
Jurkat cell line J1.1 | NIH | |||
DMEM | Invitrogen | 11965-118 | ||
RPMI 1640 | Invitrogen | 21870 | ||
HI FBS | Invitrogen | 10438-018 | ||
HIV-1NL4-3 | prepared by tranfsection of HeLa cells with cloned proviral DNA | |||
pNL-GFP-RRE | copmlete deletion of all HIV ORFs of pNL4-3 | |||
pNL-GFP-RRE-SA | Insert HIV-1 A5 and D4 into pNL-GFP-RRE | |||
pCMVΔ8.2 | provided by Dr. Dider Trono | |||
pHCMV-G | ||||
10 x HBS (Hepes Buffered Saline) | Invitrogen | 11344-041 | ||
2M CaCl2 buffer | Invitrogen | S-013-154-10 | ||
1% paraformaldehy | Sigma Aldrich | 158127 | ||
Bleach | ||||
50ml Falcon tubes | BD Bioscience | 358206 | ||
5ml round bottom tubes | BD Bioscience | 352063 | ||
0.45μM Millipore filter | Millipore | SLHA033SS | ||
0.20μM Millipore filter | Millipore | SLFG85000 | ||
100mm Petri-dish | Sigma Aldrich | CLS430588 | ||
Size-exclusion column (100,000MW cut off) | SartoriuStedim biotech | VS2041 | ||
2ml frozen tube | Axygen | SCT-200 | ||
96-well plate | BD Bioscience | 353227 | ||
1.7mL Microcentrifuge Tube | Axygen | MCT- 175-C |