The expression and incorporation of retroviral enzymes into virions in the form of Gag/Pol precursor polyproteins is believed to be important for the assembly of infectious viral particles. HIV-1 encodes a 160 kDa Gag/Pol precursor that includes Gag, protease (PR), reverse transcriptase (RT) and integrase (IN). We have developed the use of HIV accessory proteins (Vpr and Vpx) as vehicles to incorporate protein of both viral and non-viral origin into virions by expression in trans as heterologous fusion proteins. To analyze the role of Gag/Pol in the formation of infectious virions, we incorporated RT and IN into HIV-1 particles in trans, as fusion partners of viral protein R (Vpr). Virions derived from an RT and IN minus proviral clone were infectious and replicated through a complete cycle of infection when RT and IN proteins were provided in trans. These results demonstrate that functional RT and IN proteins can be provided in trans, and that their expression and incorporation into virions as components of Gag/Pol are not required for the formation of infectious virions. Thus, for the first time, we have demonstrated for a human pathogenic retrovirus that processes of assembly and the function of critical viral enzymes can be unlinked. This finding will provide unique opportunities to explore retroviral RT/IN function and the role of Gag/Pol in the formation of infectious virions in the context of a replicating virus (in vivo).