HIV-1 Nef and the unrelated murine leukemia virus glycoGag similarly enhance the infectivity of HIV-1 virions. We now show that the effects of Nef and glycoGag are similarly determined by variable regions of HIV-1 gp120 that control Env trimer association and neutralization sensitivity. Whereas neutralization-sensitive X4-tropic Env proteins conferred high responsiveness to Nef and glycoGag, particles bearing neutralization-resistant R5-tropic Envs were considerably less affected. The profoundly different Nef/glycoGag responsiveness of a neutralization-resistant and a neutralization-sensitive R5-tropic Env could be switched by exchanging their gp120 V1/V2 regions, which also switches their neutralization sensitivity. Within V1/V2, the same determinants governed Nef/glycoGag responsiveness and neutralization sensitivity, indicating that these phenotypes are mechanistically linked. The V1/V2 and V3 regions, which form an apical trimer-association domain, together determined the Nef and glycoGag responsiveness of an X4-tropic Env. Our results suggest that Nef and glycoGag counteract the inactivation of Env spikes with relatively unstable apical trimer-association domains.