The U3 region is not necessary for 3' end formation of spleen necrosis virus RNA.

Primary transcripts of retroviruses contain two poly(A) sites, one near the 5' and one near the 3' end of the transcript, but only the 3' poly(A) site is used for 3' end formation of viral RNA. It was hypothesized on the basis of experiments with U3-deleted vectors of spleen necrosis virus that the U3 region contains sequences required for this RNA 3' end formation: the titer of a U3-deleted vector was 150 times lower than that of the parental vector, and the addition of the simian virus 40 poly(A) signal sequence increased the titer of the U3-deleted vector (J. P. Dougherty and H. M. Temin, Proc. Natl. Acad. Sci. USA 84:1197-1201, 1987). However, we now show that the U3 region transcribed from the 3' long terminal repeat is not required for RNA 3' end formation and that the experiments of Dougherty and Temin led to an erroneous conclusion. We show here that the deletion of the U3 region did not decrease the steady-state level of viral RNA or shift the site of poly(A) addition. The added simian virus 40 poly(A) signal sequence was used preferentially over the poly(A) signal of spleen necrosis virus, and it increased the levels of RNA transcribed from vectors with and without deletion of the U3 region. Our results indicate that alteration of regulatory sequences in retroviral vectors can change the steady-state RNA levels and titers of the vectors in an unpredictable manner.