A minimal bovine leukemia virus (BLV) RNA packaging sequence (E) required for heterologous RNAs to be packaged into BLV particles was analyzed. The BLV E was inserted into a non-viral vector, pLacZ, in order to determine if packaging of the non-viral vector RNA would occur. The construct was transfected into cells chronically infected with BLV in order to produce virus particles. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of viral RNA from virus particles revealed that non-viral RNA containing the BLV E was packaged into BLV particles, indicating that the BLV E is necessary and sufficient to allow for packaging of a non-viral vector RNA. We also analyzed the ability of a chimeric murine leukemia virus (MLV) retroviral vector (pLN) containing BLV E to be packaged into BLV particles. Interestingly, it was found that pLNA (which does not possess ψ+) could be packaged into BLV particles. This indicates that a MLV RNA region outside of ψ+ allows for packaging of the MLV RNA into BLV particles.