Murine leukemia virus (MLV) and MLV/HIV chimeric vector constructs were derived from the MLV vector, pLNCX . The fundamental MLV vector as described in Figure 1A was developed as follows: i) the WPRE (woodchuck hepatitis virus posttranscriptional regulatory element) was first subcloned into the EcoRI/HindIII sites of a pCLNCX self-inactivating (SIN) vector ; ii) internal trans elements were replaced with a CMV-GFP cassette inserted by subcloning a BamHI/XhoI fragment from pTK113 (HIV-1 vector in Kafri lab), also retaining the HIV-1 cPPT-CTS (labeled cPPT in Figure 1A) in this fragment; iii) the 3' SIN LTR was replaced with a complete MoMLV 3' LTR from pLNCX by subcloning the HindIII/PmeI fragment; and iv) the firefly luciferase (originally obtained from pBI-GL, Clontech) was subcloned into the BamHI site of the MLV vector generating pTK1328, the basic MLV vector in Figure 1A. All notations of plasmids beginning with pTK are for ease of reference to the plasmid library in the Kafri lab.
All subsequent MLV/HIV chimeric constructs were derived from pTK1328 by inserting various HIV-1 cis elements. All HIV-1 cis elements were derived from a standard HIV-1 vector from our lab, such as pTK113 . The MLV/HIV RRE vector (pTK1332) was derived from the basic MLV vector by subcloning a BamHI fragment (~850 bp) comprising the RRE into a BamHI site of pTK1328. The MLV/HIV RU5PS vector (pTK1442) was generated by subcloning a BamHI fragment into the BamHI site of pTK1328. The BamHI fragment was derived from a PCR amplified product originally cloned into pCR2.1-TOPO (Invitrogen) (pTK1428) and moved into the SmaI/Acc65I sites of pBlueScript (Strategene) (pTK1441). The PCR amplified product, comprising the HIV-1 R, U5, PBS, and canonical packaging signal which extends into the 5' end of Gag, was generated with the following primers: HIV RU5 For 5'-gcggccgcttaattaagggtctctctggttagaccagatctgagcc-3'; and HIV RU5Pack. Rev 5'-gcggccgcttgctgtgcgg-3'. The MLV/HIV RRE + RU5 vector (pTK1439) was constructed by subcloning a PCR amplified fragment into the NotI site of pTK1332. The PCR amplified product was formally cloned into pCR2.1-TOPO (Invitrogen) (pTK1427). The following primers were used to generate a PCR product comprising the HIV-1 R and U5 regions: HIV RU5 For primer as described above and HIV RU5 Rev 5'-gcggccgcactgctagagattttccacactgac-3'. The MLV/HIV RRE + PS vector (pTK1423) was constructed by subcloning a fragment, consisting of the canonical packaging signal and extending into the 5' end of Gag and HIV-1 RRE, into the BamHI sites of pTK1332. Upon cutting with BamHI the existing RRE in pTK1332 would not be retained. The last construct in the series, the MLV/HIV RRE + RU5PS vector (pTK1440), was generated by subcloning the NotI fragment from pTK1428 into the NotI site of pTK1332. The NotI fragment contains the HIV-1 R, U5, PBS, and canonical packaging signal extending into the 5' end of Gag.
The MLV/HIV RRE vector (pTK1086) described in Figure 7 was an earlier generation of pTK1332 above, which did not have the luciferase gene inserted. The packaging constructs supplying necessary structural/enzymatic proteins were 4XCTE Gag-Pol (kindly provided by the laboratory of Dr. Christopher Baum), or ΔNRF . Expression of structural/enzymatic proteins from 4X CTE Gag-Pol is independent of all HIV-1 accessory genes, and encodes the complete gag and pol genes. The HIV-1 Rev protein was independently expressed from the EF-1α promoter in the E2F-Rev plasmid. The ΔNRF was described previously, but gag-pol gene expression is Rev-dependent. The envelope protein was supplied from pMD.G, a VSV-G expressing construct.
293T cells were maintained in DMEM (Hyclone) supplemented with 10% FBS (Invitrogen). Media was also supplemented with a 100X Antibiotic-Antimycotic solution containing penicillin, streptomycin, and amphotercin B (Cellgro).
Viral Particle Production and Concentration
Vector particles were produced by transient tranfection into 293T cells as described previously . Briefly, each 10 cm dish of 293T cells was transfected with 15 μg vector, 10 μg packaging helper, 5 μg VSV-G envelope, and 5 μg Rev expressing plasmids. Plasmid amounts were compensated for in experiments in the absence of Rev with the empty plasmid construct, pCI-neo (Promega). Vector particles were harvested in conditioned media 48-60 hours post-transfection and filtered through a 0.45 μm filter. Vector titers were determined by serial dilution on 293T cells and scoring for GFP positive cells using a Leica Leitz DMIRB inverted fluorescent microscope. Vector titers are expressed as values normalized to p24 (HIV-1 viral particles), or reverse transcriptase activity (MLV viral particles). The p24 assay and the reverse transcriptase assays are described below. Concentration of vector particles was executed as done previously , with the exception that vector was purified over a single sucrose gradient, concentrated, and resuspended in 1X PBS.
HIV-1 p24 Capsid Concentration
Details of this assay were described previously . Briefly, EIA/RIA plates were coated with p24 antibody (NIH AIDS Research and Reference Reagent Program, #3537) at 1:1000 dilution and incubated overnight at 4°C. After blocking, samples/standards were treated with a 1% triton x-100 sample buffer, diluted appropriately, and added to plate for overnight incubation at 4°C. After washing, polyclonal rabbit anti-p24 antibody (NIH AIDS Research and Reference Reagent Program, #SP451T) at 1:300 was added to the plate, and incubated at 37°C for 3 hours. After washing, goat anti-rabbit IgG peroxidase (Pierce) at 1:15000 was added to the plate and incubated at 37°C for 2 hours. Assay was completed as described previously . Data used for titer normalization, and determining amount of viral particles for RNA isolation, are a mean of replicate samples.
MLV Reverse Transcriptase Assay
MLV reverse transcriptase activity was executed on vector particles (5 μl) harvested from the media by mixing with 25 μl of RT activity reagent (60 mM Tris pH8, 0.6 mM MnCl2, 90 mM KCl, 0.125 mg/ml Poly A [Roche], 6 μg/ml oligo dT16, 25 mM DTT, 0.06% Triton X-100, and 0.25 mCi/ml 3H-TTP [MP Biomedicals]). After incubation for 1 hour at 37°C the entire volume was spotted onto DE81 anion exchange chromatography paper (Whatman), and placed into 5% Na2PO4 for 5 minutes at room temperature. The samples were washed at room temperature five times, for 5 minutes each, with 5% Na2PO4 two times with water, and one time with 95% ethanol. The samples were dried, placed in scintillation fluid, and radioactivity was measured on a Beckman LS 6500 scintillation counter. Values were recorded as CPM. Data used for titer normalization, and determining amount of viral particles for RNA isolation, are a mean of replicate samples.
Details of this assay were described previously . Briefly, at the indicated times post-transduction cells were fixed, and GFP expression was assessed on a Dako CyAn flow cytometer at the University of North Carolina Flow Cytometry Core Facility. Data was analyzed with Summit v4.3.01 software (Dako).
Luciferase lysates were prepared by pelleting transfected 293T cells at the time of vector collection, and resuspending the pellet in 1X passive lysis solution (Promega). After freeze-thawing cell debris was pelleted by centrifugation for 15 minutes at 14,000 rpm and 4°C. Equivalent volumes of supernatant were assayed for firefly luciferase expression using 100 μl luciferin reagent (Promega). Luminescence was measured with a Victor3 multilabel counter and Wallac 1420 Workstation software (Perkin-Elmer). Results are expressed as relative light units (RLU)/mg protein. Protein concentrations were assayed according to manufacturer's instructions for Pierce BCA Protein Assay kit.
At 48-60 hours post-transfection vectors were harvested from the media, and cells were divided for luciferase assay, total protein, and cytoplasmic RNA/protein fractionation. Cells were removed from the plate by trypsinizing, and pelleted. The cytoplasmic fraction was separated by treating the pelleted cells with a MES (2-(N-morpholino)ethanesulfonic acid) buffered 0.1% Triton X-100 solution (10 mM MES pH 6.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 250 mM sucrose, 0.1% Triton X-100) + protease inhibitor minicocktail (Roche) + 200 units/ml RNase Inhibitor (Fermentas). Pellets were incubated 2 minutes on ice, and nuclei were pelleted at 1500 rpm and 4°C. The cytoplasmic supernatant fraction was collected and separated for RNA isolation and protein analysis. Cytoplasmic fraction was monitored by western blot analysis for the absence of nucleolin protein, compared to total cellular proteins. RNA was purified from the cytoplasmic supernatant using the PARIS protein and RNA isolation kit (Ambion). Manufacturer's instructions were followed starting with addition of the 2X lysis/binding solution to an equal volume of cytoplasmic supernatant. Purified RNA was treated with DNase I Turbo (Ambion) for 1.5 hours at 37°C, and inactivated according to manufacturer's instructions. RNA integrity and concentration were regularly assessed by agarose gel electrophoresis. Cytoplasmic RNA was then utilized for analysis in qRT-PCR and northern blot.
Isolation of RNA from vector particles was carefully executed so that all vectors packaged into HIV-1 viral particles were normalized for equivalent amounts of p24 prior to isolation. For each sample at the time of RNA isolation p24 equivalents of vector particles and 5 × 106 293T cells were concomitantly added to the RLT lysis solution in preparation for column purification of the RNA with the RNeasy Plus Mini kit (Qiagen). Spiking the vector particles with 293T cells at time of purification has multiple advantages: i) 293T cell RNA is a carrier during RNA purification; ii) RNA can be easily quantified after purification; and iii) quantity and integrity of purified RNA can be monitored by agarose gel electrophoresis. For purposes of homogenization, the lysed particles/293T cell RNA were passed through a QIAshredder column (Qiagen) and subsequently through a genomic DNA eliminator column provided with the RNeasy plus mini kit. Lysate was then purified through the RNA isolation column provided with the RNeasy plus kit. Purified RNA was treated with DNase I Turbo (Ambion) as described for cytoplasmic RNA. Vector particle RNA was then utilized for analysis in qRT-PCR and northern blot. RNA was isolated from MLV viral particles in a similar fashion, except that viral particles were normalized for equivalent amounts of MLV reverse transcriptase. The reverse transcriptase assay is described above.
Purified cytoplasmic and vector RNA were heated to 65°C for 5 minutes prior to preparing each reverse transcription (RT) reaction. Each RT reaction was prepared at final concentrations of 1X RT Buffer (Qiagen OmniScript kit), 0.5 mM each dNTP, 10 μM random prime nonamer, 10 units RNase Inhibitor (Fermentas), 500 ng RNA, and 4 units OmniScript reverse transcriptase (Qiagen). As controls for the presence of contaminating DNA each reaction was also performed in the absence of reverse transcriptase. Reactions were executed for 1 cycle on a BioRad MyCycler at 37°C for 1 hour. Equivalent amounts of cDNA were used in the subsequent quantitative PCR reaction. Each qPCR reaction was prepared at final concentrations of 1X ABI Taqman mix (2X Taqman Gene Expression Master Mix, Applied Biosystems), 0.9 μM forward primer, 0.9 μM reverse primer, and 0.1 μM probe. Reactions were performed at 1 cycle of 50°C/2 minutes, 1 cycle of 95°C/10 minutes, and 40 cycles of 95°C/15 seconds + 60°C/30 seconds on a 7300 real time PCR system (Applied Biosystems). Vector RNAs were detected with a primer/probe set to the luciferase gene: For Luc 5'-aggtcttcccgacgatga-3', Rev Luc 5'-gtctttccgtgctccaaaac-3', and probe #70 (Roche Universal Probe Library). All quantitative PCR reactions were normalized to an endogenous control reaction for TATA Binding Protein (TBP) using the following primer/probe set in independent reactions: For TBP 5'-gaaccacggcactgattttc-3', Rev TBP 5'-tgccagtctggactgttcttc-3', and probe #92 (Roche Universal Probe Library).
The levels of vector RNA (VRNA) and cytoplasmic RNA (CRNA) derived from the qRT-PCR data are expressed as arbitrary units (AU). The values were determined by normalizing the cycle threshold (Ct) for luciferase to that obtained for TBP, from each reaction. The calculation was as follows: 2-ΔCt, where the ΔCt is (Luc Ct - TBP Ct). Independent reactions were performed for each VRNA and CRNA sample. All isolated VRNA could be normalized to TBP since each sample was copurified with 293T cell RNA, as described above. Calculation of the encapsidation efficiency, where VRNA relative to CRNA is expressed as AU, was determined for each independent sample. The calculation was as follow: 2-ΔΔCt, where the ΔΔCt is (VRNA Luc Ct - VRNA TBP Ct) - (CRNA Luc Ct - CRNA TBP Ct). All relative encapsidation efficiencies are expressed as an average of at least three independent experiments with corresponding standard deviations (S.D.).
Northern Blot Analysis
Cytoplasmic RNA was isolated as described, and vector RNA was isolated from concentrated vector particles. Vector RNA was prepared from equivalent levels of p24, and at the time of isolation the samples were copurified with 293T cells as described above. Prior to resolving RNA on a denaturing formaldehyde agarose gel, RNA was denatured at 65°C for 10 minutes. Equivalent amounts of cytoplasmic RNA, and vector RNAs, were resolved on the gel. Northern blot analysis was performed under standard conditions. Probes were random prime labeled with α32P-dCTP (Easy Tide Deoxycytidine 5'-triphosphate, Perkin-Elmer) at 37°C for 1 hour. Probes were either a BstEII fragment from pTK1440 generated to the 5' end of the vector RNA, or to the GFP gene located in the 3' end of the RNA. Images were obtained on BioMax MR film (Kodak), or by phosphorimager (Molecular Dynamics Storm System).
Western Blot Analysis
Cytoplasmic and total cell lysates were resolved by standard denaturing SDS-PAGE analysis on 10% gels and blotted to Hybond P membrane. Membranes were probed with rabbit anti-nucleolin (1 ug/ml; Abcam, cat# ab22758-100) and rabbit anti-GAPDH (1:500; Santa Cruz Biotechnology, cat# FL-335). Detection was achieved by probing blots with goat anti-rabbit IgG peroxidase (1:40000; Pierce). All antibody incubations and washes were done in 1X PBST. Signals were detected using ECL (GE Healthcare).
Southern Blot Analysis
293T cells were transduced with MLV/HIV RRE + RU5PS vector packaged into HIV-1 or MLV viral particles, at a MOI of 5. Total cellular DNA was isolated at 5 days post-transduction (referred to as P0, or no cell passages, in figures), and after five passages (P5) of the transduced cells. To collect total DNA transduced cells were lysed in a proteinase K solution (10 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS, 0.4 M NaCl, and 200 μg/ml proteinase K) for 48 hours at 55°C. Total DNA was extracted with v/v phenol (Invitrogen) and v/v phenol/chloroform/isoamyl alcohol (Invitrogen) and then treated with RNase A (20 μg; Fermentas) at 37°C for 2 hours. A second round of extractions was performed and total DNA was precipitated using standard ethanol precipitation.
Isolated gDNA (10 μg) was digested with BsrGI and DpnI for ~24 hours at 37°C. DpnI was included to eliminate putative plasmid DNA carry-over from the transfections during vector production in 293T cells. Equivalent amounts of gDNA were resolved on 1% agarose gels, transferred to Zetaprobe membrane, and probed with a BstEII fragment from pTK1440 that recognizes the 5' end of the vector enabling a size distinction between linear, 1-LTR, 2-LTR and backbone/integrated forms. Images were captured on BioMax MR Film (Kodak), or by phosphorimager (Molecular Dynamics Storm System).
Real Time qPCR
Total cellular DNA was isolated as described for Southern blot analysis and qPCR for vector and β-globin were described previously . Briefly, vector copy number was derived from standard curve produced from FLP9 cells, which contain a single copy of HIV-1 vector per diploid genome. One nanogram of total cellular DNA was previously calculated to contain 303 copies for β-globin per diploid genome, or 151.5 copies vector per diploid genome. Primers for vector copy number were designed to the WPRE region, which is also in our MLV/HIV chimeric vectors. All primers and PCR reaction conditions were described previously .
Female C57Bl/6 mice (n = 10), 3 months old (Jackson Labs) were housed in standard conditions with 3-4 mice per cage with food and water available ad libitum. All procedures were in accordance with the Guide for the Care and Use of Laboratory Animals (DHHS Publication No. (National Institutes of Health) 85-23), and all procedures received prior approval by the NIH Institutional Animal Care and Usage Committee. All procedures were performed according to animal protocol number, 396-LNS-2014.
Stereotaxic Administration of Retroviral Vectors
Mice were anesthetized (Avertin 0.02 mg/ml, 0.5-1.0 ml injection per mouse), and MLV retrovirus (n = 5 mice), or HIV lentivirus (n = 5 mice) was injected stereotactically (2 μl using a 5 μl Hamilton microliter syringe) into the right striatum (AP = 1.0 mm anterior from bregma; lateral = 1.5 mm; ventral = 3.0 mm). Studies were executed with the MLV/HIV RRE vector (Figure 7A) packaged into MLV viral particles, or HIV-1 viral particles generated from the ΔNRF  helper construct. One month thereafter animals were given an overdose of anesthetics and perfused transcardially with cold 4% paraformaldehyde (PFA) in 0.1 M phosphate buffered saline (PBS). After 24 hr, brain tissue was equilibrated in 30% sucrose. Sequential horizontal sections (40 μm) using a sliding freezing microtome (HM450, ThermoFisher) were taken through the extent of the striatum and stored in phosphate buffered glycerol at -20°C.
Immunohistochemistry and Confocal Microscopy
Double labeling for the neuronal marker NeuN and GFP was done on a 1:6 series of 40-μm free-floating horizontal sections as described previously . Sections were washed and blocked in TBS with 3% donkey serum and 0.3% Triton X-100 (TBS-plus). Primary antibodies raised in two different species were pooled in TBS-plus and incubated for 48 h at 4°C. The neuronal marker NeuN [mouse monoclonal antibody, (Millipore) 1:100] was combined with antibody for GFP [rabbit polyclonal antibody (Millipore), 1:1000]. Corresponding secondary antibodies [donkey anti-mouse CY3 and goat anti-rabbit Alexa488 (Jackson ImmunoResearch Inc.), 1:250] were pooled, and sections were incubated for 4 h at room temperature following washing in TBS-plus. Sections were mounted and coverslipped with DABCO-PVA. Sections were imaged by confocal scanning laser microscopy (MPE1000, Olympus and Laser Scanning Zeiss 510 Meta). Brain sections with GFP positive cells were captured with the Zeiss LSM Image Browser software and used to score for GFP, NeuN, and colocalization of the two markers by scanning through the z-axis. Scoring represents positive cells from brain sections of mice transduced with the MLV/HIV RRE vector packaged into MLV (n = 5), or HIV-1 (n = 5), derived viral particles.