Effects of an internal transcription unit and its orientation on retrovirus titre and expression

Using the retroviral vector pMsp, constructs were produced with different coding sequences under the control of the Herpes simplex virus type 1 (HSV) thymidine kinase (tk) promoter, with the internal coding sequence in the same or reverse orientations with respect to long terminal repeat (LTR)-driven transcription and with or without an internal tk polyadenylation (polyA) signal. Following introduction of these constructs into ecotropic or amphotropic packaging cell lines by transfection or infection, it was found that, most consistently, those constructs in which the internal coding sequence/polyA signal component was inserted in the same orientation as LTR-driven transcription produced lower titres of virus than those in which this component was inserted in the reverse orientation. Also, in a construct containing a coding sequence in the same orientation as LTR-driven transcription, but lacking an internal polyA signal, virus titre was much greater than in the corresponding construct possessing the internal polyA signal. Additionally, although functional assays have previously demonstrated expression of the inserted sequences, transcription from the internal tk promoter was inefficient in all these constructs.