Binding of HIV-1 capsid (CA) to cleavage and polyadenylation specificity factor 6 (CPSF6) is hypothesized to provide a significant fitness advantage to in vivo viral replication, explaining why CA-CPSF6 interactions are strictly conserved in primate lentiviruses. We recently identified a Q4R mutation in CA after propagation of an interferon (IFN)-β-hypersensitive CA mutant, RGDA/Q112D (H87R, A88G, P90D, P93A and Q112D) virus, in IFN-β-treated cells. The Q4R substitution conferred significant IFN-β resistance to the RGDA/Q112D virus by affecting several properties of the virus, including the sensitivity to myxovirus resistance protein B (MxB), the kinetics of reverse transcription, and the initiation of uncoating. Notably, the Q4R substitution restored the CPSF6 interaction of the RGDA/Q112D virus. To better understand how the Q4R substitution modulated the CA-CPSF6 interaction, we generated a series of CA mutants harboring substitutions at the 4th and 112th residues. In contrast to the effect in the RGDA/Q112D background, the Q4R substitution diminished CA-CPSF6 interaction in an otherwise wild-type virus. Our genetic and structural analyses revealed that while either the Q4R or Q112D substitution impaired CA-CPSF6 interaction, the combination of these substitutions restored this interaction. These results suggest that the 4th and 112th residues in HIV-1 CA cooperatively modulate CA-CPSF6 interactions, further highlighting the tremendous levels of plasticity in primate lentivirus CA, which is one of the barriers to antiretroviral therapy in HIV-1-infected individuals.