Bsabi

For expression of HIV-1 RTp51 in cells for co-IP, a destination mammalian expression vector containing a codon-optimized RT sequence with a C-terminal V5 and 6×His tag was used as previously described (18 (link)). For NanoBRET (Promega), the pFC32K-RTp66-NanoLuc expression vector was used to express RTp66 with a NanoLuc fusion at the C terminus, and a pFN21-HaloTag-eEF1A vector was used to express eEF1A with a HaloTag fusion at the N terminus. The pSicor-mCherry-T2A-Pro-TRIMCypA vector used in the CsA washout assay was constructed with a modified pSicor-mCherry-T2A backbone (Addgene no. 31845), and proline-TRIMCypA was PCR amplified from a pLHA-TRIMCypA-SN vector (27 (link)) and inserted into the pSicor vector downstream of T2A by using the XmaI and EcoRI restriction enzymes (New England Biolabs). To construct the HIV-1_NL4.₃-Δenv-eGFP proviral plasmid, eGFP was inserted into the env open reading frame of WT or mutant HIV-1_NL4.3 (59 (link)) with the BsaBI and NheI restriction enzymes (New England Biolabs).

To determine the distribution of nascent leading-strand initiation sites, deproteinized replication products from chromatin reactions were cleaved with the restriction enzymes XbaI, NotI, SacI, PsiI, EcoRV or BsaBI (NEB) in Cut Smart buffer, for 30 minutes at 37°C. Digests were stopped by adding EDTA to final concentration of 50 mM, followed by deprotinization with proteinase K - SDS treatment and phenol-chloroform extraction as described above. Sample aliquots were analyzed on 1% alkaline and 0.8% native agarose gels, where required. The remaining digested products were ethanol precipitated, washed with 70% ethanol, air-dried and resuspended in 10 mM Tris-HCl, pH 8; 1 mM EDTA. For the RNase HII experiments, digestion products were further treated with RNase HII enzyme (NEB) for 1 hour at 37°C. The reactions were stopped with 50 mM EDTA and processed as described above for the restriction digests. For polyacrylamide gel analysis an equal volume of 2x loading dye (80% formamide; 0.05% SDS; 10 mM EDTA; 100 mM NaCl; 0.04% xylene cyanol; 0.04% bromophenol blue) was added to the samples. Samples were incubated for 3 minutes at 95°C, promptly transferred to ice, before being applied to a 40 cm x 20 cm denaturing 4% polyacrylamide (Bis-Acrylamide 19:1 – Fisher scientific), 7 M Urea, in 1x Tris-Borate-EDTA buffer (TBE) gel. Gels were run for 170 minutes at constant 40 Watt.

A volume of 17 µl of the PCR amplicons was digested using the respective restriction enzymes (AccI, AciI, AleI, AlwNI, ApoI, AvaI, BbvI, BccI, BceAI, BclI, BsaAI, BsaBI, BsaI, BsiEI, BsmI, BspMI, BsrI, BstAPI, HaeII, HaeIII, HgaI, HinfI, HpaII, HphI, NcoI, NspI, PleI, PstI, PvuII, RsaI, Tth111I, XcmI; all from New England Biolabs) and recommended concentrations. PCR products were digested for 4h or overnight to avoid partial digestion and loaded on a 1% agarose gel. Genotypes were determined by visual inspection of gels and gel photographs (Figures S3 and S4). Sometimes, despite long digestion time, we observed incomplete digestion. Genotypes were assigned as follows: (1) A lack of a smaller fragment was always interpreted as a homozygous genotype. (2) A band at the position of the uncut amplicon together with a second band was interpreted as a heterozygous genotype, but only if the uncut band was stronger in intensity (as the same number of longer sized DNA molecules results in a stronger signal). (3) Complete lack of a band at the size of the uncut fragment or a band that had less intensity than the digested fragments were interpreted as a homozygous genotype for the alternative allele.