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Co-packaging of non-vector RNAs generates replication-defective retroviral vector particles: a novel approach for blocking retrovirus replication.

A Moloney murine leukemia virus (MoMuLV)-derived packaging retroviral vector, pUCMoTN-PR3, was previously developed in which the packaging (psi) signal was cloned within the 5'-long terminal repeat (LTR) U3-r and U5 sequences. The MoTN-PR3 vector particles released from a transfected packaging cell line contain RNAs with r-psi-U5 sequences at the 5'-end and U3-r sequences at the 3'-end. Upon infection, these vector particles can efficiently transduce the neomycin phosphotransferase (neo) gene to the target cells. The structure of the proviral DNA synthesized in these cells was shown to contain modified 5'- and 3'-LTRs with U3-r-psi-U5 sequences, indicating that this vector can undergo reverse transcription and integration. Analysis of psi signal-containing RNAs revealed that in addition to vector RNA transcribed from the MoMuLV 5'-LTR promoter, readthrough neo RNA transcribed from the internal herpes simplex virus (HSV) thymidine kinase (tk) promoter and cellular RNAs transcribed from the MoMuLV 3'-LTR promoter are produced. Of these, the downstream cellular RNAs are also packaged within the vector particles. These vector particles containing the vector and non-vector RNAs carrying the MoMuLV psi signal are non-infectious. It is proposed that intracellular expression of packageable non-viral RNAs may represent an effective strategy for inhibiting animal and plant virus replication.