Stable RNA Secondary Structure in a Retroviral Vector Insert Terminates Reverse Transcriptase ElongationIn Vitrobut Not in Cultured Cells

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Abstract
We wished to test whether an RNA signal that causes termination of elongation by reverse transcriptase in vitro would affect retroviral vector function. A synthetic oligonucleotide containing a sequence capable of forming a very stable RNA secondary structure was subcloned into the retrovirus vector N2. The integration of this sequence into N2 causes termination of elongation by reverse transcriptase in vitro at the precise positions previously reported in a different sequence context. However, no premature termination of DNA synthesis was observed in the unintegrated DNA of vector transduced cells. Likewise, there was no deleterious effect of the sequence insert on vector titer. These results indicate that termination signals defined in in vitro systems cannot be used as predictors of in vivo function and suggest that viral proteins in addition to reverse transcriptase play an important role in transcript initiation and elongation. The performance of individual retroviral vectors is somewhat unpredictable because of the complexity of the vector life cycle. An important concern for future refinement of retroviral vectors is the identification of structural elements that might inhibit the efficient production of the vector. Alford and Belmont investigate the effect of an in vitro reverse transcriptase termination signal on DNA synthesis and titer in a prototype vector.