Roles of the K 101 E Substitution in HIV-1 Reverse Transcriptase in Resistance to 1 Rilpivirine and other Non-Nucleoside Reverse Transcriptase Inhibitors 2 3

35 Resistance against the recently approved non-nucleoside reverse transcriptase inhibitor (NNRTI) 36 rilpivirine (RPV) commonly involves substitutions at positions E138K and K101E in HIV-1 37 reverse transcriptase together with a M184I substitution that is associated with resistance against 38 co-utilized emtricitabine (FTC). Previous biochemical and virological studies have shown that 39 compensatory interactions between substitutions E138K and M184I can restore enzyme 40 processivity and viral replication capacity. Structural modeling studies have also shown that 41 disruption of the salt bridge between K101 and E138 can affect RPV binding. The current study 42 was designed to investigate the impact of K101E, alone or in combination with E138K and/or 43 M184I on drug susceptibility, viral replication capacity, and enzyme function. We show here that 44 K101E can be selected in cell culture by the NNRTIs etravirine (ETR), efavirenz (EFV), and 45 dapivirine (DPV) as well as by RPV. Recombinant RT enzymes and viruses containing K101E, 46 but not E138K were highly resistant to nevirapine (NVP) and delavirdine (DLV) as well as ETR 47 and RPV but not EFV. The addition of K101E to E138K slightly enhanced ETR and RPV 48 resistance, compared to E138K alone, but restored susceptibility to NVP and DLV. The K101E 49 substitution can compensate for deficits in viral replication capacity and enzyme processivity 50 associated with M184I, while M184I can compensate for the diminished efficiency of DNA 51 polymerization associated with K101E. The coexistence of K101E/E138K does not impair either 52 viral replication or enzyme fitness. We conclude that K101E can play a significant role in 53 resistance to RPV. 54