Although the mutagenesis studies strongly suggested that the changes in V5 at 5-months were Nab escape mutations, these specific residues were not maintained in the subsequent Nab resistant Envs (Fig. S3) . However, the sequence of the V5 domain continued to evolve over time, suggesting ongoing selective pressure from Nab. At the last time point analyzed, 28-months, genetically distinct lineages of Nab escape variants were circulating (Fig. S1A ). The chimera-mapping approach revealed that these different Env variants had acquired resistance through at least two distinct mutational pathways (Fig. S4 , see bottom 3 panels). More detailed mapping revealed that for 28-month EnvPL5.1, the V5 domain continued to contribute to Nab resistance (Fig. 4A) , retaining the major escape pathway operative at 5-months. By contrast, 28-month EnvPL3.1 achieved a similar level of resistance through an escape pathway that required the gp41 ectodomain in addition to the cognate V1V5 domain (Fig. 4B ). For this Env, the V1V5 region from the 28-month Env independently conferred only partial escape onto the 0-month Env, while insertion of the 28-month ectodomain alone had no effect on Nab sensitivity. Further dissection revealed somewhat surprisingly that the V1V2 domain was a major contributor to Nab resistance in the context of the gp41 ectodomain, while the V3V5 region in this context did not appear to contribute to escape. Thus, a cooperative interaction between the gp41 ectodomain and V1V2 appeared to be conferring Nab resistance in this Env.