Top10 bacteria

To investigate the interaction of APP with 37/67 kDa LR, a 37LRP-His-tag fusion protein was generated. To this end, wild-type 37LRP cDNA [56 (link)] was cloned into the pTrc-His B expression vector (Invitrogen, San Diego, CA, USA) and expressed in TOP-10 bacteria (Invitrogen) and the resulting plasmid was named pPLR2-1. According to the procedures specified by Invitrogen, TOP-10 bacteria were transformed with pPLR2-1 and pTrc-His B alone, as a control, and lysed in a denaturing lysis buffer (20 mM sodium phosphate, 500 mM sodium chloride, pH 7.8) containing 6M guanidium. Both bacterial lysates were bound to nickel-NTA agarose beads through their His-tagged N-terminus in the same denaturing buffer containing 8M urea. Beads were washed several times at pH 6.0 and 5.3, to dissociate contaminating proteins and His tagged proteins were eluted at pH 4.0. His tagged recombinant 37LRP (r37LRP) purity was than 90% pure, as assessed by SDS-PAGE and Coomassie stain, as compared to pTrc-His B eluate.
r37LRP conjugated beads and pTrc-His B-conjugated control beads, produced as described above, were washed in 50 mmol/L Tris (pH 7.5)-0.1% Triton X-100, to remove urea, and resuspended in the same buffer for pull-down assays.

Phenotypic tropism testing was performed using two methods: (1) Tropism testing in GHOST cell-lines^{27 (link)} of the viruses generated from individual clones (cPTT) and (2) replicative phenotypic tropism test rPhenotyping (pPTT) whereas described previously^{25 (link)}. In cPTT co-receptor tropism was determined by measuring Renilla luciferase activity (relative light units [RLU]) using Bright-Glo™ Luciferase Assay System (Promega, US). We consider a 10-fold shift in mean RLU of infected cells over non-infected. The pNL4-3 and pMJ4 were used as positive control for X4- and R5-tropic strains respectively. In addition to luciferase expression, in a subset of clones’ green fluorescent protein (GFP) expression was also captured using confocal microscopy (Olympus Fluoview v2.0b). The rPhenotyping was performed with 31 patients’ samples infected with HIV-1C and four QC-samples. The ligated mixture was transformed into TOP10 bacteria (Life Technologies) and inoculated directly in the LB broth supplemented with ampicillin to retain the viral diversity. The tropism was inferred by using serial dilutions of the CCR5 antagonist TAK-779 (obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, Bethesda, MD, USA) or the CXCR4 inhibitor AMD3100 (Sigma-Aldrich, St. Louis, MO, USA). rPhenotyping was compared with pGTT while cPTT were compared with cGTT.

RASs were introduced into the HCV gt3a replicon using the QuikChange II XL Site‐Directed Mutagenesis Kit (Agilent Technologies). Positions in this article refer to H77 consensus; for a full list of positions in S52‐ΔN and H77, see Supporting Table S1. All reactions were transformed into Top10 bacteria (Life Technologies), and for each mutation three clones were isolated and sequenced to confirm the viral sequence.