Extended Data Fig. 1: Purification and activity of RTIC.

a, Initial anion-exchange purification of the RTIC away from free RT and vRNA–tRNA. This purification was repeated for each sample (>10) used in the manuscript, with only slight variations in the chromatogram. b, Polishing step using size-exclusion chromatography purification of the RTIC after anion exchange. This purification was repeated for each sample used in the manuscript (>10), with only slight variations in the chromatogram. c, A final 10% native TBE gel on the purified components. RT barely enters the gel under these running conditions. The RTIC runs as a single band, but trace amounts of free vRNA and/or vRNA–tRNA complex are sometimes present. This native gel is a representative result that was repeated independently for all purified RTIC samples used in the paper (>10). d, Autoradiograph image illustrating that the RTIC is capable of incorporating an incoming α-³²P-dTTP nucleotide when extended and purified using dCTP instead of ddCTP. This gel is a representative result that was repeated independently for crosslinked and uncrosslinked samples (>6 independently prepared samples) used in dTTP incorporation assays. e, The RTIC incorporates α-³²P-dTTP at roughly 89% efficiency compared to the free components after reaching a plateau. Values are mean ± s.d. (n = 3 independent experiments) with normalization to total incorporation of free RT + vRNA–tRNA reactions. f, Autoradiograph image showing that the incorporation of dTTP is inhibited in the presence of nevirapine (NNRTI). Images have been adjusted to allow identification of the NNRTI-inhibited band. This gel is a representative result that was repeated independently for crosslinked and uncrosslinked samples (3 samples each). g, Relative activities, judged by primer usage, of wild-type, Q258C, and Q258C/E478Q reverse transcriptase mutants used in this study. Values are mean ± s.d. (n = 3 independent experiments) with normalization to the primer usage of wild-type RT. h, RTIC (triangles), RTIC with NNRTI (circles) or vRNA–tRNA + excess RT (squares) reactions were initiated by addition of α-³²P-dTTP and quenched at different time points. Data were fit using the relationship for the free vRNA–tRNA + RT reaction: \({\rm{Intensity}}=A\left(1-{e}^{-{k}_{{\rm{pol}}}t}\right)+B\left(1-{e}^{-{k}_{{\rm{slow}}}t}\right)\). Data were fit using the relationship for the RTIC (with or without NNRTI) reaction: \({\rm{Intensity}}=B\left(1-{e}^{-{k}_{{\rm{slow}}}t}\right)\) where A and B represent the amplitude of the fast and slow processes, respectively, k_pol is the apparent extension rate constant, and k_slow is the rate of the slow process. The second relationship was used for the RTIC data, as the slow process appears to dominate incorporation when the vRNA–tRNA substrate is crosslinked to RT. The best fits were obtained with: A = 0.7166 AU, k_pol = 0.1078 s⁻¹, B = 0.2754, k_slow = 0.01002 s⁻¹ for the vRNA–tRNA + excess RT; B = 0.9808, k_slow = 0.003140 s⁻¹ for the RTIC; and B = 1.095, k_slow = 0.0001714 s⁻¹ for the RTIC with NNRTI. k_slow is about 3.19 times slower for crosslinked RTIC than for un-crosslinked components. Assays were independently repeated three times to ensure reproducibility.