293T cells and U87.CD4.CXCR4 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, penicillin, and streptomycin (from Invitrogen) and maintained at 37°C with 10% CO2. CD4 and CXCR4 expression in the U87 cell cultures was selected with 300 mg/mL Geneticin (G418) and 1 mg/mL puromycin (Life Technologies, Inc.), respectively. MT4 cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics at 37°C with 5% CO2.
Five non-syncytium inducing (NSI) HIV-1 isolates were isolated from HIV-infected Ugandans, characterized for co-receptor usage, and subtyped based on phylogenetic sequence analyses as previously described . Two primary subtype A HIV-1 isolates (115-A and 120-A) and three subtype D strains (89-D, 122-D, and 126-D) were selected. Due to previous confusion in strain nomenclature, we have modified the virus names from that previously published (A14 is now 115-A, A15 = 120-A, D13 = 122-D, D14 = 126-D, and D15 = 89-D) . All HIV-1 isolates were syncytium-inducing (SI) and utilized the CXCR4-coreceptor (X4) for entry . All viral stocks were previously propagated and expanded in PHA-stimulated, IL-2 treated peripheral blood mononuclear cells as described . Tissue culture dose for 50% infectivity (TCID50) was determined for each isolate using the Reed and Muench method , and titers were expressed as infectious units per milliliter (IU/ml) . All the env genes of these HIV-1 isolates have been previously sequenced , aligned, and have the following accession numbers: 115-A, 120-A, 89-D, 122-D, and 126-D.
Single cycle tissue culture assay
Single cycle assays were completed using an assay previously developed by our laboratory [30, 32]. HIV-1 envelope gene fragments from subtypes A and D (HXB2 nt 6420–7520) were PCR amplified from viral DNA and cloned in a pKS-derived Knr/ORI plasmid following standard cloning techniques [32, 47]. As previously described, we have designed two types of plasmids, pLac+ and pLac- plasmids carry, as genetic markers, either a functional LacZ ' gene or a sequence complementary to the mRNA coding for Escherichia coli malT gene, respectively. These two genetic markers, lac+ and lac- in their respective plasmids are schematically represented in Figure 2B. All constructions were verified by sequencing. [48, 49]Defective retrovirus particles were produced as described . The medium was replaced 8 h after transfection, and the vector supernatants were recovered 36 h later. Non-internalized DNA was removed by treatment of the vector supernatants with DNaseI (1 μg/ml in the presence of 1 μM MgCl2) for 30 min at 37°C. Amount of p24 present in supernatants was determined by using the HIV-1 p24 enzyme-linked immunosorbent assay kit (PerkinElmer Life Sciences). When necessary, vector supernatants were concentrated by using Centricon® YM-50 centrifugal filter devices (Amicon-Millipore) before transduction.
MT4 cells were transduced with 200 ng of p24 antigen per 106 cells (an approximate multiplicity of infection of 20) in 35-mm dishes in a 500-μl volume. Two hours post-transduction, the cells were diluted up to a 4-ml volume with supplemented RPMI medium and maintained at 37°C in a 5% CO2 incubator for 40 h. The reverse transcription products were purified by Hirt  as described . The purified double stranded DNA was digested with DpnI for 2 h at 37°C (in order to eliminate possible contaminating DNA of bacterial origin) prior to PCR amplification (20 cycles) with primers BH and SH (Figure 2). The amplified product was purified after electrophoresis on agarose gel, digested with PstI and BamHI, ligated into an appropriate plasmid vector and transformed in E.coli. Plating on IPTG/X-Gal containing dishes allowed blue/white screening of recombinant and parental colonies, respectively .
In Vitro recombination assays
In vitro recombination assays were performed using the reconstituted system previously developed in our laboratory . RNA synthesis was performed as previously described . RT purification and activity tests were carried out as described by Canard and colleagues . Constructs used for RNA synthesis were generated following standard cloning procedures. Reverse transcription was carried out on the donor RNA (100 mM) in the presence of an equimolar amount of acceptor RNA after annealing an oligonucleotide specifically onto the donor template. Reverse transcription was started by the addition of HIV-1 RT at a final concentration of 400 nM and carried out for 60 min. Synthesis of the second DNA strand, BamHI and PstI digestion, ligation, and E. coli transformation were carried out as previously described . The frequency of recombination is derived from the rate of blue colonies (recombinant) divided by the sum of blue plus white (parental) colonies.
Measuring the frequency of recombination in the in vitro reverse transcription assay and single-cycle infection assay
To determine the frequency of recombination in the single cycle assay, the total number of blue and white colonies were calculated and applied in the equation as previously described . The frequency of recombination (F) is calcuated by the equation F = b/(2/3 [N(n/48) + b]), where N and b are the total number of white and blue colonies, respectively. The recombination rates per nucleotide (f) within a given interval (i) is given by f = F (x
/X)/z, where F is as above, x
is the number of colonies analyzed where recombination was identified to have occurred within the interval considered, X is the total number of colonies on which mapping was performed, and z is the size in nucleotides of the interval. This takes into account the generation of heterozygous particles as described by the Hardy-Weinberg equation  and the bias introduced into the estimation of the frequency of recombination by cloning reverse transcription products from lac-/- (1/3) relative to lac+/- vectors . For the calculation of the frequency of recombination in heterozygous particles, the total number of colonies must be multiplied by two-thirds. To accurately estimate the frequency of recombination, another factor to take into account is the background among the white colonies derived from cloning of cellular DNA which co-purified with the reverse transcription products. Typically, 48 white colonies are analyzed in each assay and a correction factor is established, given by n/48, where n is the number of colonies resulting from cloning of reverse transcription products (very rare).
HIV-1 dual infection assay
Different pairs of two HIV-1 isolates were used to simultaneously infect PBMC as described previously . We performed four separate dual infections of U87.CXC4.CD4 cells with two HIV-1 isolates at the same multiplicity of infection (i.e. 10-2:10-2 or 10-3:10-3). Virus mixtures were added to adherent U87.CD4.CXCR4 cells (500,000/well) for 2 h at 37°C, 5% CO2 in DMEM complete medium. Supernatants and two aliquots of cells were harvested at peak virus production (typically day 10) as measured by reverse transcriptase activity in the supernatant [46, 55]. Cells were resuspended in 10% DMSO/90% fetal bovine serum, and then stored at -80°C for subsequent analysis.
PCR strategy to amplify HIV-1 recombinants in the multiple cycle assay
For all dual infection experiments, proviral DNA was extracted from lysed PBMC using the QIAamp DNA Blood Kit (Qiagen). A segment of the env genes of HIV-1 genome were PCR amplified using a set of universal primers: envB  – envN  for a ~3 Kbp fragment encoding the gp120 of env. Subtype-specific primers internal to the previous env products were then used to PCR amplify subtype A/D recombinants of using subtype- or isolate-specific primers, a 115-envC1 (TAGTGCAGAAAAGCATAATG; a 115-A-specific sense primer), d -envC4 (TGTCAATTTCTCTTTCCCAC; a subtype-D specific antisense primer), a 120-envC1 (AAGCATATGATGCAGAAGTAC; a 120-D specific sense primer), d -envC1 (TAAAACAGAGGCACATAATA; a subtype D specific sense primer), and a -envC4 (TGCTAATTTCTTTATCCCAT; a subtype A specific antisense primer). For intrasubtype dual infections, the following subtype-specific primers were used: a 115-envC4 (CCTCTTGCCAAGAATGTTC; a 115-A antisense primer), a 120-envC4 (TCTAGTGTCTGGACCGAT; a 120-D antisense primer), d 122-envC1 (GTCAGGGCGAGCATACTA; 122-D sense primer), d 122-envC4 (CCCAGTGGTTCAATCTC; a 122-D antisense primer), d 126-envC1 (ACAAGGGCAAGCATGGTA; a 126-D sense primer), and d 126-envC4 (GACCTAGTGGCTCAATTTTTAC; a 126-D antisense primer). All of these intrasubtype and intersubtype specific primer sets flanked the C1 to C4 regions of env (nt positions of approximately 6520 to 7740). Both external and nested PCR reactions were carried out in a 100-μl reaction mixture with defined cycling conditions . PCR-amplified products were separated on agarose gels and then purified using the QIAquick PCR Purification Kit (Qiagen). Control PCR amplifications were performed with subtype-specific DNA templates to rule out the possibility of Taq-generated recombinants . These isolate/subtype specific templates were generated by cloning the PCR amplified env gene of virus 115-A, 120-A, and 89-D, 122-D, and 126-D into pCR2.1 vector (Invitrogen). As an amplification/PCR quantitation control for recombination frequency calculations, plasmids containing entire env gene of the subtype A and D viruses were serially diluted and PCR amplified. Intensities of these products on agarose gels were then used as a standard curves and to calculate copy number of recombined viral RNA molecules in the dual infection based on the intensity of RT-PCR amplified products.
Calculating the frequency of recombination in the multiple cycle dual infection assay
To determine the frequency of recombination after multiple rounds of infection and replication in tissue culture, external and nested PCR amplification were used with subtype or isolate specific primers as described above. The frequency of recombination was determined using both parental sequence primer sets (eg. d -envC1 – d -envC4), and recombinant primer sets (eg. d -envC1 – a 115-envC1). The frequency of recombination was determined by quantifying the intensity of the PCR products on an agarose gel using Quantity One software (BioRad). Copy number of recombined and parental HIV-1 DNA is then derived by the intensity of the product compared to that of amplified product from known copy numbers of plasmid controls. The recombination frequency is calculated by dividing the total recombinant virus production by the total virus production in the system.
Heteroduplex tracking assay for detection of two HIV-1 env fragments
Nested PCR products of the env gene were analyzed by heteroduplex tracking analysis . The same genomic regions were PCR amplified from a subtype E HIV-1 env clone (E-pTH22)  for use as a DNA probe. For this amplification, the E80 primer was radiolabeled with T4 polynucleotide kinase and 2 μCi of [γ-32P]ATP and paired with the E125 primer to amplify the C2-C4 region of env . The same pair of cold primers were employed to PCR amplify the HIV-1 env DNA from each dual infection. Radiolabeled PCR-amplified probes were separated on 1% agarose gels and purified with the Qiaquick gel extraction kit (Qiagen). Reaction mixtures contained DNA annealing buffer (100 mM NaCl, 10 mM Tris-HCl [pH 7.8], and 2 mM EDTA), 10 μl of unlabeled PCR-amplified DNA from the competition culture, and approximately 0.1 pmol of radioactive probe DNA . The competition and probe DNA in this mixture was then denatured at 95°C for 3 min and then rapidly annealed on wet ice. After 30 min on ice, the DNA heteroduplexes were resolved on Tris-borate-EDTA buffer on 5 to 8% nondenaturing polyacrylamide gels (30:0.8 acrylamide-bisacrylamide) for 2.5 h at 200 V. The percentage of polyacrylamide in the gel matrix was dependent on the size of the amplified product employed in the heteroduplex tracking analysis. Gels were dried and exposed to X-ray film (Eastman Kodak Co., Rochester, N.Y.). Heteroduplexes representing production of each isolate in a dual infection were quantified with the Bio-Rad Phosphor-imager.
Estimation of viral fitness
In our HIV-1 competition experiments, the final ratio of the two viruses produced from a dual infection was determined by heteroduplex tracking analysis and compared to production in the monoinfections. Production of individual HIV-1 isolates in a dual infection (f
0) was divided by the initial proportion in the inoculum (i
) and is referred to as relative fitness (w = f
0) . The ratio of the relative fitness values of each HIV-1 variant in the competition is a measure of the fitness difference (W
) between the two HIV-1 strains (W
), where w
correspond to the relative fitness of the more and less fit virus, respectively . As indicated in the text, viral fitness can also be calculated from the parental specific PCR products.
Reverse transcription/strand transfer reactions
These reconstituted in vitro reverse transcription reactions focused on template switching in the C3 region of env using 115-A or 120-A donor RNA and 89-D acceptor RNA templates. For these reactions RNA transcripts were made from PCR products derived from the subtype clones. Primer pairs (5' GATTTAGGTGACACTATAG
ATATA ATGAGGTAGTCAAACAATTA-3' and 5'-TTTATTCTGCATTTGAGAGT-3' for 115-A, 5'- GATTTAGGTGACACTATAG
ATATA AGGAGGTAGCCAAACAATTA-3' and 5'-TTTATTCTGCATTGGAGAGT-3' for 120-A, and 5'- GATTTAGGTGACACTATAG
TATAT AGAATGGAATAAAACTATAC-3' and 5'-ACCGTTTGTGTTTGTACTCT-3' for 89-D) were used in PCR reactions to amplify nts 7255-7474 (relative to HXB-2 provirus numbering) for 115-A and 120-A, and 7235-7454 for 89-D. The bolded regions of the primers are SP6 promoter sequences while italicized regions are non HIV sequences. PCR products were recovered on native polyacrylamide gels and SP6 RNA polymerase was used to produce run-off transcripts of 225 nts. A DNA primer that binds specifically to the donor RNA transcript (5'-TTTATTCTGCATTTGAGAGT-3' or 5'-TTTATTCTGCATTGGAGAGT-3' for A-115 and A-102, respectively) was32P-labeled at the 5' end with T4 polynucleotide kinase according to the manufacturer's protocol (New England Biolabs). The donor RNA was hybridized to a complementary labeled primer by mixing primer:transcript at approximately 3:1 ratio in 50 mM Tris-HCl (pH 8.0), 1 mM DTT, 80 mM KCl. The mixture was heated to 65°C for 5 min and then slowly cooled to room temperature. Donor RNA-primer DNA hybrids (2 nM final concentration of RNA) were preincubated for 3 min in the presence or absence of 10 nM acceptor RNA template and NC (as indicated) in 42 μl of buffer (see below) at 37°C. One molecule of NC per two nucleotides was used in the reactions. Wild-type and mutant NC proteins from the HIV-1 NL4-3 strain were prepared as explained previously[60, 61]. The reactions were initiated by the addition of 8 μl of HIV-RT (80 nM final in reactions) to a mixture of 50 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol, 80 mM KCl, 6 mM MgCl2, 100 μM dNTPs, 5 mM AMP (pH 7.0), 25 μM ZnCl2 and 0.4 units/μl RNase inhibitor. Reactions were allowed to incubate for 0, 30 s, 1, 2, 4, 8, 16, 32, and 64 min at 37°C prior to quenching a 6 μl aliquot of each reaction with 4 μl 25 mM EDTA (pH 8.0) and 2.5 ng of RNase-DNase free enzyme for 20 min at 37°C. Two μl of proteinase K at 2 mg/ml in 1.25% SDS, 15 mM EDTA (pH 8.0) and 10 mM Tris (pH 8.0) was then added to the above mixture, which was placed at 65°C for 1 hour. Finally, 12 μl of 2X formamide dye (90% formamide, 10 mM EDTA (pH 8.0), 0.1% xylene cyanol, 0.1% bromophenol blue) was added to the mixture and the samples were resolved on an 8% denaturing polyacrylamide gel containing 7 M urea. Extended DNA products were quantified by phosphorimager analysis using a Bio-Rad FX phosphoimager.