Downregulation of virus receptors on the cell surface is considered to be important in preventing superinfection. HIV-1 encodes multiple gene products, Env, Vpu, and Nef, involved in downregulation of CD4, a major HIV-1 receptor. We found that simultaneous mutations in both vpu and nef severely impaired virus replication. We examined the involvement of CD4 downregulation mediated by Vpu and Nef in the modification of virus infectivity. The mutation in vpu increased CD4 incorporation into virions without affecting the Env content in it, inhibiting the attachment step of virions to the CD4-positive cell surface. Although a single mutation in nef suppresses virus infectivity via a CD4-independent mechanism, it could augment CD4 incorporation in virions in combination with a vpu mutation. These results indicated that CD4 downregulation was necessary for maintenance of Env function in the virion.