MxB restricts HIV-1 by targeting the tri-hexamer interface of the viral capsid

Sarah Sierra Smaga; Chaoyi Xu; Brady James Summers; Katherine Marie Digianantonio; Juan Roberto Perilla; Yong Xiong

The CA mutations G208R and T210K drastically reduced MxB binding ( Fig 3B) . MxB 1-187 35 -MBPdi binding to CA G208R was indistinguishable from the MxB 1-35-AAA -MBPdi background 188 signal. Binding to CA T210K was also substantially reduced. By neutralizing the strong negative 189 charge at the tri-hexamer interface, these mutations likely disrupt charge-charge interactions that 190 are required for MxB binding. It is also possible that the bulkiness of these residues occludes the 191 well at the tri-hexamer interface and sterically blocks MxB access. The relatively modest 192 mutation P207S had a minor effect on MxB interaction ( Fig 3B) . These data lend strong support 193 to our mapping results that identify the MxB targeting site to the tri-hexamer interface on capsid. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/444067 doi: bioRxiv preprint 233 Results from our MD analysis show that MxB 1-35 binds persistently in the tri-hexamer 234 region throughout the entirety of the simulation, with extensive molecular contacts between MxB 235 N-terminal region and CA residues (Table 2) . Importantly, CA residues in the tri-hexamer 236 interface, E71, E75 and E213, interact with the 11 RRR 13 motif of MxB with high occupancies (Fig 237 4D and Table 2 ), which is in good agreement with our co-pelleting assay data (Fig 2C and 2D ). In 238 fact, there is a remarkable agreement between the MD results with the experimental binding data 239 obtained independently. For example, contacts between E212 and the MxB N-terminus were 240 observed less frequently, which is consistent with the experimental finding that the E212A 241 mutation has a smaller disruption of MxB binding than other glutamate mutations do at the tri-242 hexamer interface ( Fig 2D) . Moreover, we observe CA residues T210, G208, and P207 243 interacting with the 11 RRR 13 motif of MxB, which accounts for the importance of these sites in 244 MxB binding ( Fig 3B) and sensitivity [9] . In addition, we found contacts between CA and 245 residues outside of the 11 RRR 13 motif ( Table 2, The MxB-binding pocket revealed by the simulation is primarily formed by key CA 253 residues located in adjacent CA hexamers (Table 2 and Fig 4D and SI Appendix 3) . The MxB 254 11 RRR 13 motif is anchored to the bottom of the pocket, primarily interacting with the negatively-255 charged CA E213 residues, while the rest of the MxB 1-35 peptide exhibits significantly higher 256 flexibility (Fig 4D and SI Video 1) . This observation is supported by the experimental finding 257 that the E213A mutation shows the largest effect on MxB binding ( Fig 2D) . Notably, the 258 significant difference in effect between E212A and E213A on MxB binding is consistent 259 experimentally and computationally. This is likely due to their relative positions at the interface: 260 E213 is closer to the 3-fold symmetry axis (Fig 2b) and other E213 residues from adjacent 261 hexamers, such that MxB can bind E213 from multiple CA monomers simultaneously ( Fig 4D) . The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/444067 doi: bioRxiv preprint 301 interface, which is likely the binding site for the positively charged 11 RRR 13 motif in MxB that is 302 critical for capsid interaction and HIV-1 restriction [14] . Consistently, we found that the tri-303 hexamer interface mutations that escape