Figure 1
Inefficient inclusion of HIV-1 exon 2 is dependent upon a suboptimal signal at 5'ss D2. (A) Map of HIV-1 genome (NL4-3) showing the locations of 5' and 3' splice sites. The positions of Exon 2, Exon 3, and ESSV are indicated above the viral genome. Probes used to analyze HIV-1 splicing are shown above and below the viral genome and splice sites. Oligonucleotide primers used for RT-PCR analysis of viral splicing are shown above the viral genome. The BSS/SJ4.7A primer pair were used to detect the 1.8 kb, completely spliced viral mRNA species. The BSS/KPNA primer pair were used to detect the 4.0 kb incompletely spliced viral mRNA species. The probe complementary to the 3'-end of the viral mRNAs used for Northern analysis is indicated by NB. The probes used for the RNase protection assays (DPHV, A1D2, A2D3, and 601c) are represented by lines and are complementary to the splice sites to which they overlap. (B) 5'ss D2 within pNL4-3 was mutagenized as shown resulting in a consensus 5'ss signal in the infectious molecular clone NLD2UP. The previously described plasmid NEVM [15] was used as a control for increased splicing at 3'ss A2. Total RNA samples from Hela cells 48 hours post transfection with the indicated plasmids were analyzed by RT-PCR using primers specific for completely spliced viral mRNA (C) or incompletely spliced viral mRNA (D). HIV-1 RNA species are indicated on the right side of the gel by exon content, the mRNA to which they encode, and mRNA spliced at 3'ss A1 are indicated by plus signs and 3'ss A2 by asterisks. (E) Total cellular RNA from 293T cells 24 hours post transfection with the indicated plasmids was subjected to Northern blot analysis with a radiolabeled probe (NB) complementary to all HIV-1 mRNAs. (F) Northern blots were quantitated and the values shown were normalized to β-actin and β-galactosidase mRNA levels and represent the average of three independent experiments. RNA was also subjected to RPA analysis using the following riboprobes: DPHV (G), A1D2 (H), A2D3 (I), and 601c (J). Individual panels are representative of a single experiment. (K) Viral splice site utilization is represented relative to NL4-3 for each splice site. The values shown represent the average of three independents experiments and were normalized to β-actin and β-galactosidase mRNA levels.