Supplementary Material For: novel yeast-based recombination method to clone and propagate diverse HIV-1 isolates Dawn M. Dudley 1,2,*, Yong Gao 2,*, Kenneth N. Nelson 2, Kenneth R. Henry 2, Immaculate Nankya 1, Richard M. Gibson 2, and Eric J. rts 1,2 1Department of Molecular iology and Microbiology, ase Western Reserve University, leveland, OH, US and 2Division of Infectious Diseases, Department of Medicine, ase Western Reserve University, leveland, OH, US iotechniques 46:458-467 (May 09) doi.2144/000113119 Supplementary material for this article is available at /article/113119. *D.M.D. and Y.G. contributed equally to this work. Production of cloning vectors The cloning vector pre nf l HIV-1 was produced by using yeast homologous recombination (Supplementary Figure 1). preenv contains the orotidine-5 - phosphate decarboxylase gene (UR3) for selection of homologous recombination, the yeast centromere (en6) and autonomously replicating sequence (RSH4) to maintain an episomal plasmid in yeast, and β-isopropylmalate dehydrogenase (LEU2) for maintenance of the plasmid in yeast grown in LEU drop-out media. This vector also contains an ampicillin resistance marker for selection in bacterial cells, a zeocin marker for maintenance in mammalian cells as an expression vector, and an env gene from the laboratory HIV-1 strain, HX2. To produce preenv, the UR3 selection marker was PR-amplified from the plasmid prs316 (1) and purified using a PR purification kit (Qiagen, Valencia,, US). The primers used to amplify UR3 contain 40 nucleotides of homology to promote yeast recombination into the target plasmid, 40 nucleotides of homology to the first half of the pnl4 3 HIV-1 genome (without the 5 LTR) that will be subsequently cloned into the preenv vector, and nucleotides of homology to prs316 to amplify UR3. The linearized target plasmid and the PR-amplified UR3 region were transfected into yeast using the lithium acetate method to allow for homologous recombination. Yeast was plated on MM-Leu- Ura selection media for 4 5 days at 30. olonies were grown in 2 ml MM-Leu media, lysed with 4 600 μm glass beads to extract crude DN, and transformed into electrocompetent DH cells (Invitrogen, arlsbad,, US). acterial colonies were screened for the correct recombination by colony PR. The first half of pnl4 3 was amplified from the PS region to the end of the pol gene. These primers are homologous to the 40-nucleotide region within the primers used to amplify and insert UR3 into the vector as mentioned above (Supplementary Figure 1). Not I-linearized preenv was transformed into yeast along with the PR amplified first half of pnl4 3 to allow for recombination to replace UR3 with the pnl4 3 sequence. Homologous recombination was selected on MM-Leu+5- FO plates. The insertion of the first half of pnl4 3 was verified by PR using primers that amplify a region of the pol gene (data not shown). UR3 was then amplified again using primers that contain homology to the newly produced pre env/nfl1 as well as homology to the end of the pnl4 3 genome and to the UR3 sequence. oth the digested (XhoI) pre env/ nfl1 plasmid and the UR3 fragment were transformed into yeast and plated on MM-Leu-Ura media to select for pre env/nfl1/ur3. The second half of the pnl4 3 genome was then amplified with a sense primer containing the last 40 nucleotides of the first half of the genome and with an antisense primer with homology to the sequence in the pre env/nfl1/ur3 vector flanking the first half of the genome. Once again the linearized (SacII) pre env/nfl1/ur3 plasmid and the second half of NL4 3 were transformed into yeast and selected on MM-Leu+5-FO plates. rude yeast DN was extracted, transformed into bacteria, and bacterial colonies were screened for the insertion of the second half of pnl4 3 by restriction digest and PR amplification of the envelope gene. The full-length pre_nfl_hiv-1 vector was verified by restriction mapping with HindIII, PstI, and SacII and compared with 351 the restriction map of pnl4 3, as shown in Supplementary Figure 1. onstruction of a pmv_ cplt vector and 293T cplt stable cell line s shown in Supplementary Figure 2, the cytomegalovirus (MV) sequence was PR-amplified from pcdn3.1zeo/t (Invitrogen). This PR product was subsequently used as a primer to amplify the R, U5, and gag regions from pnl4 3; this amplicon was then cloned into the pr XL TOPO vector (Invitrogen). The TOPO vector was then subjected to digestion by and stxi to generate a MV promoter-driven R, U5 and gag fragment. The resulting fragment was cloned back into the pcdn3.1zeo/t backbone to generate the pmv_cplt vector. The pmv_cplt vector was subsequently transfected into to establish the stable cell line 293T cplt, and was maintained with mg/ml of zeocin selection (Invitrogen). Reverse-transcription PR, PR, and quantitative realtime PR Viral RN was extracted from 140 μl of transfection supernatants from 293T or 293T cplt cells and from 140 μl of U87. D4.R5/U87.D4.XR4 supernatants using the QImpViral RN mini kit (Qiagen). ellular RN was also extracted from 293T or 293T cplt cells, and U87.D4.XR4 cells using an RNeasy kit (Qiagen). cdn was produced in the pol region of nfl_hiv-1 and from the tag region flanking the 5 LTR in the pmv_ cplt vector using the following protocol:
Yeast homologous recombination recombines out full length HIV-1 due to repeated LTR regions. 5'LTR PS Env 3'LTR loning in the near full length HIV-1 genome into precenv. 5'LTR strand transfer prec HIV-1 prec 3'LTR prec prec no genome 5'LTR Env 3'LTR U3 R U5 PS Not I 1. 2. 3. 4. prs316 UR3 PS XhoI prs316 UR3 Env 3 LTR UR3 MV Pr UR3 Not I PS UR3 3 LTR Xho I PS UR 3 SacII preenv pre env/ur3 pre env/nfl 1 pre env/nfl 1/UR3 H=HindIII P=PstI S=SacI Verification of plasmid constructs by RE digests - H P S - H P S - H P S M Kbp 8 6 5 4 3.5 3 2.5 2 1.5 pnl4-3 pre nfl HIV-1 pmv cpltru5- gag/tag 1.0 0. 0.5 0. SacI (4.8Kbp) PstI (5.6) y pbs HindII (5.9) gag PstI (7.1) pol SacI (.2) HindII (.2) env HindII (12.3) ole LTR pre_nfl_hiv-1 (parental pcdn 3.1 Zeo) 170 bp SV40 p SacI (13.7) HindII (13.8) Zeocin EN6 RSH4 LEU2 Supplementary Figure 1. Schematic representation of production of pre_nfl_hiv-1. () The HIV-1 genome contains two regions of homology between the 5 LTR and 3 LTR that leads to the recombination of most of the genome out of a vector during yeast homologous recombination and strand transfer of the full length genome. () Stepwise cloning scheme to introduce the near full length HIV-1 pnl4 3 genome into the preenv shuttle vector (see Production of cloning vectors section for more details). Step one involves amplification of the UR3 gene from prs316 for insertion into a digested preenv. pre env/ur3 is then digested with NotI and a PR product containing the first half of pnl4 3 from the primer binding site to the middle of the pol gene is inserted via yeast homologous recombination (step 2). UR3 is PR amplified from prs316 and inserted into pre env/nfl 1 digested with XhoI (step 3). Finally, the second half of pnl4 3 is PR-amplified and inserted into pre env/nfl1/ur3 via recombination (step 4). () Verification of the final pre_nfl_hiv-1 vector by restriction mapping with HindIII, PstI, and SacI, along with the pnl4 3 and the final pmv_cplt (see Supplementary Figure 2) vectors. 352
Supplementary Table 1. prenfl HIV-1 loning Vectors with HIV-1 Genomic Regions Replaced with UR3 Samples were quantitated based on cdn standards run alongside the samples with known copy numbers based on RN concentration. To create cdn standards, a region from pnl4 3 pol and a region of pmv_cplt tag were cloned into the pr2.1-topo vector (Invitrogen) and transcribed with T7 polymerase using a MEGscript transcription kit (mbion, ustin, TX, US). RN produced during transcription was quantitated using a spectrophotometer (iophotometer 6131; Eppendorf, Hamburg, Germany) and diluted by -fold serial dilutions to create standards just prior to reverse transcription. RN was reversepre- NFL-HIV-1 Deletions Location of NL4-3 Deletion Size of Deletion mplicon Oligos Set Location of mplicon hiv-1 \UR3 790-9533 8745 HIV pre 741-760 : 9563-9582 gag-pol-env\ur3 790-8785 7998 GG-POL-ENV pre 741-760 : 88-8834 gag-pol-env2\ur3 790-8264 7476 GG-POL-ENV2 pre 741-760 : 8294-8313 gag-pol\ur3 790-96 45 GG-POL pre 741-760 : 5126-5145 gag\ur3 790-2292 13 GG pre 741-760 : 2322-2341 gag p17\ur3 790-1185 396 GG p17 pre 741-760 : 12-1234 gag p24\ur3 1186-1878 693 GG p24 pre 11-16 : 1908-1927 gag p7\ur3 1921-2133 213 GG p7 pre 1872-1891 : 2163-2182 gag p6\ur3 2134-2292 9 GG p6 pre 85-24 : 2322-2341 pol\ur3 85-96 3012 POL pre 36-55 : 5126-5145 pol prot\ur3 23-49 297 POL PROT pre 24-2223 : 79-98 pol rt\ur3-3869 13 POL RT pre 01- : 3899-3918 pol prot-rt\ur3 23-3869 1617 POL PROT- RT pre 24-2223 : 3899-3918 pol rnase H\UR3 3870-4229 360 POL Rnase H pre 3821-3840 : 49-4278 pol-env\ur3 85-8785 6701 POL-ENV pre 36-55 : 88-8834 pol-env-s\ur3 85-8264 6180 POL-ENV2 pre 36-55 : 8294-8313 pol int \UR3 4230-96 867 POL INT pre 4181-40 : 5126-5145 vif-vpr-tat-rev vpu-env-nef\ur3 41-9407 4367 VIF-NEF pre 4992-11 : 94-9456 vif\ur3 41-5619 579 VIF pre 4992-11 : 5649-5668 vpr\ur3 5559-5849 291 VPR pre 55-5529 : 5879-5898 tat\ur3 5830-8459 2630 TT pre 5781-5800 : 8489-88 tat-ex1\ur3 5830-6044 2 TT ex1 pre 5781-5800 : 6074-6093 tat-ex2\ur3 8369-8459 91 TT ex2 pre 83-8339 : 8489-88 rev\ur3 5969-8643 26 REV pre 59-5939 : 8673-8692 rev-ex1\ur3 5969-6044 76 REV ex1 pre 59-5939 : 6074-6093 rev-ex2\ur3 80-8643 274 REV ex2 pre 8321-8340 : 8673-8692 vpu\ur3 6061-6306 246 VPU pre 6012-6031 : 6336-6355 env\ur3 6221-8785 65 ENV pre 6172-6191 : 88-8834 env-s\ur3 6221-8264 43 ENV2 pre 6172-6191 : 8294-8313 env gp1\ur3 6221-7747 27 ENV gp1 pre 6172-6191 : 7777-7796 env gp1 v1/v2\ur3 6611-6802 192 ENV gp1 v1/v2 pre 6562-6581 : 6832-6851 env gp1 v3\ur3 7-77 8 ENV gp1 v3 pre 7051-7070 : 72-76 env gp1 v4/v5\ur3 7368-7627 260 ENV gp1 v4/v5 pre 7319-7338 : 7657-7676 env gp41\ur3 7748-8785 38 ENV gp41 pre 7699-7718 : 88-8834 env gp41-s\ur3 7748-8264 517 ENV gp41-s pre 7699-7718 : 8294-8313 rre\ur3 7716-8069 354 RRE pre 7667-7686 : 8099-8118 nef\ur3 8787-9407 621 NEF pre 8738-8807 : 94-9456 3'-ltr U3\UR3 9076-9533 458 3' LTR U3 pre 9027-9046 : 9563-9582 5 μl of extracted RN was added to 2 μl of antisense primer ( pmol/μl) and cycled for 88 for 2 min, 70 for min, 55 for min, for min, and 4 hold. Next, 5 first strand buffer (Invitrogen), 0.1 M dtt (Invitrogen), and mm dntps were added to each reaction and cycled at for min, 42 for 2 min, and a 4 hold. Finally, MMLV RT (Invitrogen) was added to the reaction and cycled at 42 for 1 h, 70 for min, and a 4 hold. β-globin was also reverse-transcribed from cellular RN using random primers. Taqman real-time PR was performed on the cdn that was described in the paragraph above. riefly, 5 μl of a 1:5 dilution of cdn was added to 1 Taqman Universal PR Master Mix (pplied iosystems, Foster ity,, US) and the appropriate primers (300 nm/reaction) and probes ( nm/reaction) to specifically detect either the pol or tag region. Probes were labeled with FM/MGNFQ (pplied iosystems) and were designed in part using Primer Express software (pplied iosystems). Samples were run on an I PRISM 7700 sequence detection system in a 96-well format ( 2 min, 95 min, and 40 cycles of 95 for s. and 58 for 1 min) and analyzed using SDS 1.9.1 software (pplied iosystems). 353
SV40 p Research Reports LTR gag pnl4-3 HIV-1 genome pol env LTR MV1 HivR-MV PMV pcdn3.1zeo/t PS Ψ gag ole Hiv-gag4 Zeoc in PR amplify with DN as primer ut with Mlu I and st XI PMV R U5 PS Ψ gag stxi stxi ut with Mlu I and st XI pcdn3.1zeo/t PMV R U5 PS Ψ gag stxi ole SV40 p Zeoc in Ligate R U5 pbs Y Dgag tag pmv_cplt Zeocin E ol SV40 p Supplementary Figure 2. Schematic representation of the production of pmv_cplt. () The MV promoter was amplified from pcdn 3.1zeo/T and subsequently used as a primer to amplify the R-U5-gag region of pnl4 3. () This product was then digested with and stxi along with pcdn3.1 zeo/t. () The R-U5-gag region of pnl4 3 was then ligated into pcdn3.1zeo/t to produce the pmv_cplt vector. 354
trn Lys,3 R U5 pbs Ψ gag pol env U3 R - strong-stop DN RNase H activity INTRSTRND TEMPLTE SWITHING MODEL + HIV-1 RN MODEL FOR OMPLEMENTTION Initiating RT ion from cpltru5gag HIV-1 RN - strong-stop DN Ψ gag 1st template switch nfl HIV-1 RN from prenfl HIV-1 is the same as the HIV-1 RN remaining after the 1st template switch - HIV-1 DN 5 RNase H activity 2nd template switch mediated by the PS complementarily ppt selection and priming + HIV-1 DN 5 + strong-stop DN ds proviral HIV-1 DN 5 Supplementary Figure 3. trnlys3 primes off the primer-binding site (PS) in the LTR region to initiate reverse transcription that results in double-stranded HIV proviral DN following the rolling circle viral replication scheme. Reverse transcription scheme for the intrastrand template switching model () and for the complementation model herein presented (). The complementation model predicts initiation of HIV-1 reverse transcription from a trn Lys,3 isoacceptor species annealed to the primer binding sequence of the cplt RN. Following the first strand switch in the intrastrand or complementation model, minus-strand and plus-strand DN synthesis would follow the intrastrand model of reverse transcription (). 355
Transfection ells (α-p24) Infection ells (α-p24) pre_nfl 1.0 -- 0. 0.1 0.5 -- pmv_cplt -- -- 0. 0.9 0.5 1.0 pnl4-3 -- 1.0 -- -- -- -- 0 1 pre_nfl -- 0. 0.1 0.5 -- pmv_cplt -- 0. 0.9 0.5 1.0 pnl4-3 1.0 -- -- -- -- 0 1 Transfection Sups (α-p24) D Infection Sups (α-p24) pre_nfl 1.0 -- 0. 0.1 0.5 -- pmv_cplt -- -- 0. 0.9 0.5 1.0 pnl4-3 -- 1.0 -- -- -- -- 0 1 pre_nfl -- 0. 0.1 0.5 -- pmv_cplt -- 0. 0.9 0.5 1.0 pnl4-3 1.0 -- -- -- -- 0 1 E α-ctin F α-gpdh pre_nfl 1.0 -- 0. 0.1 0.5 -- pmv_cplt -- -- 0. 0.9 0.5 1.0 pnl4-3 -- 1.0 -- -- -- -- 0 1 pre_nfl -- 0. 0.1 0.5 -- pmv_cplt -- 0. 0.9 0.5 1.0 pnl4-3 1.0 -- -- -- -- 0 1 Supplementary Figure 4. Full-length images of Western blots shown in main-text Figure 1. (,, E) Full-length Western blots detecting p24 or actin, corresponding to the cropped images shown in main-text Figure 1D. (, D, F) Full-length Western blots detecting p24 or GPDH, corresponding to the cropped images shown in main-text Figure 1G. Representative ladders show span of ladder based on the actual run of each individual gel shown. 356
nfl HIV-1 leu ole pre_nfl_hiv (parental pcdn 3.1 Zeo) 170 bp SV40 p Zeocin or 293T cplt cells mp HIV-1 pnl4-3 Transfection cells (panels & ) Transfection supernatants (panel ) Infect U87.D4.R5 (or XR4) cells complementation of reverse transcription (Figure 2) Infection supernatants (panel D) D ells Mock pnl4-3 pre_nfl_hiv-1 293T cell line Tag Env 1 2 3 4 1 2 3 4 1 2 3 4 1= 2=pNL4-3 3=pRE_nfl_HIV-1 4=293T 293TcpltRU5gag cell line Tag Env 1 2 3 4 β-globin 1 2 3 4 1 2 3 4 β-globin 293Tcplt 293T 0 0 0 00 00 3000 Reverse transcriptase activity (cpm x 2 /ml) 293T Transfected ells 1= 2=pNL4-3 3=pRE_nfl_HIV-1 4=293T cplt α-β-ctin 293T Transfected Supernatants Transfection supernatant extracts (p24) 1 1:5 1: 1:1 pnl4-3 + 293Tcplt pnl4-3 + 293T α-p24 mb pre_nfl_hiv-1 + 293Tcplt pre_nfl_hiv-1 + 293T 1 1:5 1: 1:1 E TID (log infectious units/ml) pnl4-3 + 293T pnl4-3 + 293Tcplt pre_nfl_hiv-1 + 293T pre_nfl_hiv-1+ 293Tcplt Mock 293T Mock 293Tcplt U87 infection supernatants 5 4 3 2 1 0 pre pnl4-3 Mock nfl_hiv-1 Supplementary Figure 5. Transfection of pre_nfl_hiv-1 into 293T cplt stable cells. () Schematic representation to show which part of the complementation system is represented by each panel in this figure. () PR amplification of reverse transcribed RN extracted from 293T cplt or transfected with pre_nfl_hiv-1. Primers were designed to specifically amplify the tag region of pmv cplt, the pol region of nfl HIV-1, or the env region of nfl HIV-1. β-globin RN was reverse transcribed with random primers and then amplified with specific β-globin primers. () p24 Western blot analysis from lysed 293T or 293T cplt cells transfected with pre_nfl_hiv-1 or pnl4 3 plasmids. Lysates were diluted 1:5, loaded onto gels, and probed with either anti-p24 24 4 (or anti-p24-11) or anti β-actin antibodies. (D) Reverse transcription (cpm) measured from supernatants collected after transfection of either pnl4 3 or pre_nfl_hiv-1 into 293T or 293T cplt cells. Supernatants from untransfected and transfected cells were also measured. The right side of the panel shows Western blot analysis of p24 protein found in the transfection supernatants, using serial 5-fold dilutions of the viral lysates. (E) TID values (representing the amount of functional virus) of viruses made by passaging cell supernatants created by transfections of pre_nfl_hiv- 1, pnl4 3, or transfections through U87.D4.XR4 cells. 357
transcribed using the same protocol to make cdn from transfected cells, described earlier in this section. cdn produced from reverse transcription was then used as standards in real-time PR alongside the samples. β-globin was also amplified by real-time PR with β-globin specific primers from cdn of cell lysates as a way to standardize cdn input into the real-time PR reactions. Nonquantitative PR was also applied to detect pol, env, and tag RN with the same primer sets. for efficient manipulation of DN in Saccharomyces cerevisiae. Genetics 122:19-27. Western lots 293T or U87.D4.XR4 cells were pelleted and lysed in SDS lysis buffer (0 μl or μl, respectively). One milliliter of the viral supernatants collected off the or U87.D4.XR4 cells were pelleted at 32,000 g for 1 h at 4. Virus pellets were resuspended in 80 μl SDS lysis buffer. Samples were loaded into % SDS PGE gels with 4% stacking gels and run at constant volts for 1 h. Protein was transferred onto nitrocellulose (Whatman, Dassel, Germany) at constant volts for 1 h. Nitrocellulose membranes were blocked in TENG 0.1% triton overnight and probed with mouse anti-p24 (#24 4) (at. no. 6521; IDS Research and Reference Reagent Program, Germantown, MD, US) (1:,000 dilution) or goat anti-actin (-11) (at. no. sc-16; Santa ruz iotechnology, Inc, Santa ruz,, US) (1:00 dilution). Membranes were then probed with either a goat anti-mouse HRP-labeled secondary antibody (at. no. 31430; Pierce, Rockford, IL, US) or a rabbit anti-goat HRP-labeled secondary antibody (at. no. 31402; Pierce). lots were developed using EL-plus chemiluminescence (at. no. RPN2133; GE Healthcare, Piscataway, NJ, US). Notes The real-time PRs, Western blots, and reverse transcription assays of the pnl4 3 control were performed in a separate experiment from the conditions involving pre_nfl_hiv-1 in the cell infection studies. This was because the pnl4 3 virus replicated extremely fast and destroyed the cells by day 7, when peak infection was seen for the cotransfection system. Thus, the cells and viral supernatants were collected earlier for this virus (day 5) to preserve intact cells from which to extract RN and protein. References 1. Sikorski, R.S. and P. Hieter. 1989. system of shuttle vectors and yeast host strains designed 358