Figure 1

The MSD is required for gag, pol, and env expression. (A) Structure of the FV R region. Mutations of the MSD and poly(A) signal are depicted below the scheme. MSD nucleotides complementary to the U1snRNA are in black, and mutations are underlined. Arrows indicate the positions of the promoters. (B) Western blotting analysis of BHK-21 cells transfected with pHSRV2 or pHSRV2-SDm1. Transfections were performed in duplicate. Gag was detected with a Gag-antiserum, and GAPDH served as a loading control. (C) Northern blotting analysis of total RNA from BHK-21 cells transfected with pHSRV-13 MSD derivatives showing that the MSD is required for expression of 5’LTR derived transcripts (compare lanes 1 with lanes 3 and 5) and that an additional poly(A) inactivation rescues the SDm2 phenotype. A tas/bet-specific probe was used to detect viral RNAs. The positions of the 18S (1.9 kb) and 28S (4.7 kb) rRNAs are indicated. Ratios of gag+pol/gaph transcripts are indicated below the blots. In case of pHSRV13-SDm1 (−*), the ratio could not be determined due to the lack of gag and pol transcripts. (D) Western blotting analysis of BHK-21 cells using a monoclonal Gag antibody or a polyclonal integrase-specific antiserum. The latter detects the unprocessed Pol precursor (p130) as well as integrase (p41). GAPDH served as a loading control.