Extended Data Fig. 5. Comparative enzymology of ORF2p RT with HIV-1 and HERV-K.

a, Single nucleotide incorporation kinetic curves and parameters of dATP with 36-nt RNA or DNA template and 20-nt DNA primer with ORF2p core (33 nM), HIV-1 RT (4 nM) and HERV-K RT (12 nM). For each enzyme, Michaelis-Menten parameter k_cat/K_M is nearly identical on both templates (n = 3 (DNA template) and n = 4 (RNA template) independent samples over 2 independent experiments; data represented as mean ± SD). b, Comparison of HIV-1 RT and ORF2p in extension of very short (5–10 nt) primers, pre-annealed to DNA and RNA templates. ORF2p extends all DNA and RNA primer lengths, with somewhat reduced efficiency at 5 nt and, to a lesser extent, 6 nt. In contrast, HIV-1 does not extend the same DNA:DNA template:primer mixes of these lengths and does not extend 5 nt and has reduced activity with 6 nt DNA primers on RNA templates. ORF2p also makes NTA (+) and template jumping/switching (##) larger products; more visible on longer exposure. Notably, neither of these larger products are detectable with HIV-1 RT; quantification represents n = 3 (DNA) and n = 4 (RNA) samples from two independent experiments. c, Heparin trap processivity assay for ORF2p vs HIV-1 RT; heparin sulfate is a negatively charged sugar polymer that competes for nucleic acid binding sites. The indicated RNA or DNA primers and templates were pre-annealed and reactions were prepared and preincubated as indicated, then initiated with Mg²⁺ as a control, with heparin and Mg²⁺ together, or with a two-step “Trap control” procedure in which heparin and Mg²⁺ are added sequentially. Reactions are quenched after 5 seconds. At this very short time point, ORF2p produces full template length product (FTL, 3–9% of total signal in the lane in all conditions) and is unaffected by the heparin trap; in contrast, HIV-1 RT produces 0–3% FTL product without trap and no detectable FTL product with trap. When all products are quantified, HIV-1 extends 21–37% of primers, and this is roughly halved by the heparin trap; ORF2p extends ~10–18% of primers and is unaffected by the trap. In the trap control (TC) RNA template:DNA primer lanes, HIV- 1 performs a small amount of residual RT, consistent with a distributive pattern of synthesis, whereas ORF2p is inhibited, bound to the heparin trap. These are all consistent with high processivity for ORF2p and low- processivity distributive pattern synthesis for HIV-1 RT. Asterisk (*) Cy5-5’-label on primer. n = 1 quantified samples shown representative of two independent experiments.