Fig. 3: Design and activities of MVV IN mutants.

a Locations of targeted IN residues of the CTD-CTD interfaces (left) and configuration of the CCD-CTDs linkers within eight structurally distinct IN chains of the intasome (right; amino acid sequence of the linker is shown above the superposition). Colors of protein chains are preserved from Fig. 1. Intra-tetramer and inter-tetramer CTD-CTD interfaces, the CCD-CTD linker, and specific amino acid residues targeted by mutagenesis (shown as sticks) are indicated. Due to C2 symmetry, the two CTD tetrads (Fig. 1b) are equivalent within the intasome. b Strand transfer activities of indicated MVV IN mutant proteins relative to that of WT (set to 100%), measured by real-time PCR. The bar plot displays mean values with standard deviations from n=3 independent experiments for each condition; open circles indicate values for individual repeat measurements. For clarity, bar plots are color-coded: blue bars show property of WT IN; red, control IN mutants; purple and green, mutants of the intra- and inter-tetramer CTD-CTD interfaces, respectively; yellow, mutants of the CCD-CTD linker. The gray dotted line represents the level of background (LoB), determined from three IN-omit reactions and defined as mean background +1 standard deviation. Qualitative analysis of the strand transfer products by agarose gel electrophoresis is shown in Supplementary Fig. 5b. c Average molar masses (kDa) of MVV IN variants determined by SEC-MALLS upon injections of the proteins at 8, 4, 2, and/or 1 mg/mL (indicated with upward triangles, circles, diamonds, and downward triangles, respectively). Bars represent values obtained with IN injected at 2 mg/mL. Molecular masses of MVV IN monomer (32.3 kDa), dimer, tetramer, and octamer are indicated with gray dotted lines. Statistical significance (WT vs mutant) was estimated using two-tailed paired Student’s t-test, and the results are reported as highly significant (**p < 0.01), significant (*p < 0.05), or non-significant (n.s.); exact P values are provided in Source Data file. d Size exclusion chromatography elution profiles of CSC intasomes assembled with Cy3-labeled vDNA and WT or mutant MVV INs. The curves report Cy3 absorbance at 550 nm to distinguish nucleoprotein complexes from protein aggregates. Only elution volumes 7–10 mL are shown here; the complete elution profiles (0–20 mL), including results of the intasome assemblies with the remaining MVV IN mutants, are shown in Supplementary Fig. 5d. e Infectivity of single-cycle MVV-derived vectors produced using Gag-Pol constructs incorporating WT or indicated mutant IN. Luciferase expression was measured 7 day post-infection. The bars indicate mean values with standard deviations from n = 6 biological replicates for each condition; open circles indicate values for individual measurements. f Quantification of late reverse transcription products in cells infected with WT or IN mutant MVV vectors at 8 h post-infection. The bars show mean values with standard deviations from n = 3 biological replicates for each condition; open circles indicate values for individual measurements; two-tailed paired Student’s t-test was used to estimate WT-vs-mutant statistical significance (**p = 2 × 10⁻⁵; *p = 0.02). Source data are provided as a Source Data file.