During HIV-1 viral assembly, both Pr160gag-pol and primer tRNA(Lys3) are packaged into the virus. tRNA(Lys) packaging (both tRNA(Lys3) and tRNA(Lys1,2) is dependent upon the presence of RT sequences within Pr160gag-pol. In this work, we have monitored the effect of Pr160gag-pol mutations upon incorporation of tRNA(Lys3) and Pr160gag-pol into HIV-1 produced from COS-7 cells transfected with mutant HIV-1 proviral DNAs. Mutations include carboxy deletions of Pr160gag-pol and small amino acid insertions and replacements within the various functional domains of the reverse transcriptase (RT). tRNA(Lys3) incorporation was monitored both by 2D PAGE of viral RNA, and by hybridization with tRNA(Lys3)-specific DNA probes. Our data indicates: (1) deletion of integrase sequence has a moderate effect upon select tRNA(Lys3) packaging, while carboxy terminal deletions extending further into the RNase H and connection domains more strongly reduce viral tRNA(Lys3) content; (2) tRNA(Lys3) incorporation is strongly reduced by small inframe amino acid insertions or replacements in the carboxy region of the thumb domain and the amino half of the connection domain of RT, but tRNA(Lys3) incorporation is altered little, or not at all, by similar amino acid insertional mutations within other RT domains, such as the fingers, palm, RNase H, the amino portion of the thumb, and the carboxy region of the connection domain. The inability of connection domain mutant virus to incorporate tRNA(Lys3) and to properly process precursor proteins in the virus is due to the inability of mutant Pr160gag-pol to be incorporated into the virus. These mutant precursor proteins are maintained at levels in the cytoplasm similar to wild-type.