National Center for Biodefense and Infectious Diseases, Department of Molecular and Microbiology, George Mason University
Guo, J., Enos, C., Wu, Y. Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein. J. Vis. Exp. (43), e2198, doi:10.3791/2198 (2010).
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.
1. Transfection of Plasmids for Production of the Rev-dependent Lentiviral Particles
Set up: The Rev-dependent GFP lentiviral vector, pNL-GFP-RRE-SA, was described previously4,5,6. To assemble into a viral particle, the plasmid was contransfected into HEK293 T cells with an HIV-1 packaging construct, pCMVΔ8.2 (kindly provided by Dr. Dider Trono), and a plasmid carrying the VSV-G glycoprotein (pHCMV-G). The transfection was carried out using the calcium phosphate method.
Preparation of buffers: 10 X HBS (Hepes Buffered Saline) was prepared by dissolving 5 g of Hepes, 8 g of NaCl, 0.37 g of KCl, 1 g of Dextrose, 0.103 g of Na2HPO4 (anhydrous) in 100 mL of H2O. Aliquot the10 X HBS buffer into 1 mL aliquots and store at -20°C. At the time of transfection, convert the 10 X HBS to 2 X HBS with H2O (1: 5 dilution), and adjust the pH to between 7.05 - 7.12. (for 10 mL 2 X HBS, it usually requires approximately 50 μL of 1M NaOH). Filter sterilize the 2X HBS buffer by passing through a 0.22 μM Millipore filter. CaCl2 buffer was made by dissolving CaCl2 in 10 mM Hepes to a final concentration 2M. Adjust pH to 5.8, filter sterilize the buffer and store at 4°C.
2. Concentrate Viral Particles and Determine Viral Titer
3. Marking HIV-1 Positive Cells with the Lentiviral Particle, vNL-GFP-RRE-SA
Set up: a human CD4 T cell line, CEM-SS, was first infected with HIV-1. At 48 hours post infection, cells were superinfected with the lentiviral particles, vNL-GFP-RRE-SA. Following superinfection for another two days, GFP-positive cells were analyzed by flow cytometry. As a control, HIV-1 uninfected CEM-SS cells were identically infected with vNL-GFP-RRE-SA. Only the HIV-1-infected CEM-SS cells gave rise to GFP positive cells but not the HIV-1 uninfected CEM-SS cells.
4. Representative Results
If the experiments are successfully performed, a sizable GFP population will be detected in HIV-1-infected CEM-SS cells by the flow cytometer, whereas, in the control, GFP positive cells will not be detected in HIV-1 uninfected CEM-SS cells.
If the experiments are successfully performed, the Rev-dependent lentiviral vector, vNL-GFP-RRE-SA, will permit the highly specific detection of replicating HIV in living cell populations, through measurement of green fluorescence protein (GFP) expression. In this example, harvested CEM-SS cells were stained with a PE-labeled rat monoclonal antibody against mouse CD24, HSA, and then analyzed on a flow cytometer for both HSA and GFP expression.
Figure 1. As shown here, a sizable GFP population was detected in HIV-1-infected cells superinfected with vNL-GFP-RRE-SA (Figure 1c). In contrast, GFP positive cells were not detected in either HIV-1 uninfected CEM-SS cells without lentiviral superinfection (Figure 1a) or HIV-1 uninfected cells with lentiviral superinfection (Figure 1b).
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.
No conflicts of interest declared.
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.
|37°C 5%CO2 incubator|
|Centrifuge||Beckman Coulter Inc.||Allega„˘6KR|
|Cell counter||Nexcelom Bioscience||Cellometer„˘ AutoT4|
|HEK293T cell line||National Institutes of Health|
|CEM-SS cell line||National Institutes of Health|
|Jurkat cell line J1.1||National Institutes of Health|
|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|
|pCMVÎ”8.2||provided by Dr. Dider Trono|
|10 x HBS (Hepes Buffered Saline)||Invitrogen||11344-041|
|2M CaCl2 buffer||Invitrogen||S-013-154-10|
|50ml Falcon tubes||BD Biosciences||358206|
|5ml round bottom tubes||BD Biosciences||352063|
|0.45µM Millipore filter||EMD Millipore||SLHA033SS|
|0.20µM Millipore filter||EMD Millipore||SLFG85000|
|Size-exclusion column (100,000MW cut off)||Sartorius AG||VS2041|
|2ml frozen tube||Axygen Scientific||SCT-200|
|96-well plate||BD Biosciences||353227|
|1.7mL Microcentrifuge Tube||Axygen Scientific||MCT- 175-C|