Fundamental property of viruses is to rapidly adapt themselves under changing conditions of virus replication. Using HIV-1 derivatives that poorly replicate in macaque cells as model viruses, we studied here mechanisms for promoting viral replication in non-natural host cells. We found that the HIV-1s could evolve to grow better in both macaque and human cells by the continuous culture in macaque lymphocyte cell lines. Notably, only several mutations at defined sites of the Pol-integrase and/or the Env-gp120 reproducibly appeared in repeated adaptation experiments and were sufficient to cause the phenotypic change. Meanwhile, no amino acid changes to enhance viral replication in macaque cells were found in interaction sites for the known anti-retroviral proteins. These findings disclose a hitherto unappreciated evolutionary pathway to augment HIV-1 replication in primate cells, where tuning of viral interactions with positive rather than negative factors for replication can play a dominant role. © 2013 Institut Pasteur.