Identification of a generalised packaging sequence for D-type retroviruses and generation of a D-type retroviral vector.

In order to construct vectors based upon D-type, rather than C-type, retroviruses, we have identified a 624-bp fragment of Mason-Pfizer monkey virus (MPMV) which constitutes a packaging sequence for at least two D-type retroviruses. When this fragment was included in an extensively deleted D-type vector genome, the D-type viruses MPMV and SRV-5, but not the C-type viruses MLV-A or MLV-E, rescued the vector RNA from HeLa cells. The recombinant virus stocks have the host range of the rescuing D-type virus as shown by expression of an internal (SV40-puromycin) cassette replacing the retroviral structural genes. The recombinant MPMV was specifically neutralized by anti-MPMV serum and receptor interference was demonstrated when it was plated on cells productively infected with wild type MPMV. When the putative D-type packaging sequence was removed from the vector genome, even though the other sequence elements required for efficient reverse transcription remained, the vector was no longer rescued from HeLa cells. These results complement the recent demonstration of broad specificity of rescue of a C-type vector (carrying only the packaging sequence of Mo-MLV) by several different C-type, but not D-type, viruses. Replacement of the D-type packaging sequence by most of the extended packaging sequence of Mo-MLV prevented the otherwise D-type vector from being rescued by D-type viruses and did not allow it to be rescued by C-type viruses. This was probably because of the incompatibility of the D-type vector sequences with the C-type retroviral proteins involved in viral reverse transcription and integration. Hence, we have localized a packaging sequence that is recognized by D-type, but not by C-type, retroviruses and have constructed a D-type vector which may be useful in gene transfer experiments.