Sequences within the R region of the long terminal repeat activate basal transcription from the HIV-1 promoter

Abstract

The importance of the R region in basal human immunodeficiency virus type 1 (HIV-1) transcription was addressed by comparing a panel of HIV-1 R region mutants using in vitro and in vivo assays. Using deletion, base substitution mutants, and compensatory mutants, the precise R region sequences essential for basal HIV-1 promoter activity in vitro were mapped to sequences between +17 to +21. Within this regulatory domain, nucleotides +19 and +21 appear to be critical. The effect of these mutations on steady state RNA levels in transfected cells has been analyzed by S1 nuclease protection assay using uniformly labeled probes. Two main conclusions may be drawn from these studies. First, HIV-1 basal transcription is abundant, with the majority of correctly initiated transcripts truncated between sequences +57 to +70. Second, analysis of the compensatory mutants indicates the secondary structure of the nascent R region RNA is not an obligate requirement for the production of the truncated transcripts. Mutations in R region primary sequence that selectively abolish the production of the truncated transcripts in vivo also exhibit reduced promoter activity in vitro. The appearance of high levels of truncated transcripts raise the interesting possibility that-similar to c-myc, c-myb, and c-fos--basal HIV-1 expression is regulated by transcription elongation.

Figure 1

HIV-1 LTR CAT R region deletion mutations. The HIV-1 LTR-CAT expression plasmid LM1 contains R sequences +1 to +82 inclusive of the TAR region (+19 to +44). The transcription start site (+1) is designated by an arrow. In LM2, R sequences (+1 to +57) are designated by a black bar and are labeled with the endpoint nucleotide. Subsequent deletions were constructed in LM2 by substitution of synthetic mutated oligonucleotide cassettes in the unique Kpn I/Hind III sites. The recognition sites for LBP-l/UBP-1 are underlined. Also indicated are the recognition sites for the restriction endonucleases Hind III and Kpn I, enhancer factor NF-kB (nuclear factor-kB), the SP1 binding sites, TATA box, and the chloramphenicol acetyltransferase (CAT) gene. B. HIV-1 R region substitution mutations. Substitution mutations in LM2 were constructed by substitution of synthetic oligonucleotides into the unique Kpn I/Hind III sites. Boundaries of mutated sequences and the altered residues are indicated. The predicted nascent RNA structure and free energy (kcal/mol) for each mutant (Zucker, 1987) are shown in the right panel.

Figure 2

Analysis of HIV-1 R region mutants in vitro. A. Strategy to generate homologous single-stranded ³²P-uniformly labeled complementary DNA probes for SI nuclease protection assays. Plasmid DNA was cleaved at Pvu II (–17) and hybridized to an antisense CAT primer (CAT sequences +49 to +80). High specific activity probes were generated in cycles of heat denaturation, annealing, and Taq I polymerase-mediated primer extension in the presence of [α-³²P]dCTP. Probes were purified using denaturing polyacrylamide gel electrophoresis and gel elution. Each R region mutant was used as a template to generate a homologous probe. The 179 nucleotide probe generated from template LM1 protected full length LM1 RNA as a 162 nucleotide product corresponding to 82 nucleotides of R sequence and 80 nucleotides of CAT sequence. The 154 nucleotide LM2 probe protected 137 nucleotides of full length LM2 RNA corresponding to 57 nucleotides of R sequence and 80 bases of CAT. B. R sequences were essential for in vitro transcription from the HIV-1 promoter. DNA templates (50 ng) were transcribed in vitro using HeLa whole cell extracts, treated with DNase, and analyzed by the SI protection assay using the homologous R region probe. Lanes and predicted transcription size are as follows. Lane 1: LM1, 162 nt; lane 2: LM2, 137 nt; lane 3: LM4, 85 nt; lane 4: LM5, 99 nt; lane 5: LM7, 116 nt; lane 6: LM8, 108 nt; lane M: ³²P-labeled pBR322 Msp I digested DNA markers. C. HIV-1 promoter activity in vitro was abolished in R region substitution mutants. Template DNA was truncated at the EcoR I site, and runoff transcripts (313 nt) were generated in vitro using HeLa whole cell extracts. Lane 1: LM3, 258 nt; lane 2: LM4, 261 nt; lane 3: LM5, 275 nt; lane 4: LM7, 292 nt; lane 5: LM8, 280 nt; lane 6: LM2, 313 nt; lane 7: TM22, 313 nt; lane 8: TM25, 313 nt; lane 9: TM37, 313 nt; lane 10: TM32, 313 nt; lane 11: TM28, 313 nt; lane 12: LM11, 313 nt; lane 13: TM24, 313 nt; lane 14: TM245′, 313 nt.

Figure 2

Analysis of HIV-1 R region mutants in vitro. A. Strategy to generate homologous single-stranded ³²P-uniformly labeled complementary DNA probes for SI nuclease protection assays. Plasmid DNA was cleaved at Pvu II (–17) and hybridized to an antisense CAT primer (CAT sequences +49 to +80). High specific activity probes were generated in cycles of heat denaturation, annealing, and Taq I polymerase-mediated primer extension in the presence of [α-³²P]dCTP. Probes were purified using denaturing polyacrylamide gel electrophoresis and gel elution. Each R region mutant was used as a template to generate a homologous probe. The 179 nucleotide probe generated from template LM1 protected full length LM1 RNA as a 162 nucleotide product corresponding to 82 nucleotides of R sequence and 80 nucleotides of CAT sequence. The 154 nucleotide LM2 probe protected 137 nucleotides of full length LM2 RNA corresponding to 57 nucleotides of R sequence and 80 bases of CAT. B. R sequences were essential for in vitro transcription from the HIV-1 promoter. DNA templates (50 ng) were transcribed in vitro using HeLa whole cell extracts, treated with DNase, and analyzed by the SI protection assay using the homologous R region probe. Lanes and predicted transcription size are as follows. Lane 1: LM1, 162 nt; lane 2: LM2, 137 nt; lane 3: LM4, 85 nt; lane 4: LM5, 99 nt; lane 5: LM7, 116 nt; lane 6: LM8, 108 nt; lane M: ³²P-labeled pBR322 Msp I digested DNA markers. C. HIV-1 promoter activity in vitro was abolished in R region substitution mutants. Template DNA was truncated at the EcoR I site, and runoff transcripts (313 nt) were generated in vitro using HeLa whole cell extracts. Lane 1: LM3, 258 nt; lane 2: LM4, 261 nt; lane 3: LM5, 275 nt; lane 4: LM7, 292 nt; lane 5: LM8, 280 nt; lane 6: LM2, 313 nt; lane 7: TM22, 313 nt; lane 8: TM25, 313 nt; lane 9: TM37, 313 nt; lane 10: TM32, 313 nt; lane 11: TM28, 313 nt; lane 12: LM11, 313 nt; lane 13: TM24, 313 nt; lane 14: TM245′, 313 nt.

Figure 3

Steady-state HIV-1 RNA levels were significantly reduced in R region deletion mutants. SI nuclease protection analysis of RNA from cells transfected with the R region deletion mutants. The truncated transcripts are indicated by bars, full-length transcripts by arrows, and undigested probe by dots. A. RNA from transfected cells (20 μg) was hybridized to the homologous probes. Lane 1: LM2; lane 2: LM3; lane 3: LM4. B. LM2 probe was hybridized to each RNA sample, except for LM1, which was hybridized to LM1 probe. Lane 4: LM5; lane 5: LM7; lane 6: LM1; lane 7: LM1; lane M: ³²P-labeled pBR322 Msp I DNA markers.

Figure 4

R region substitution mutations selectively abolished the truncated transcripts. SI nuclease protection analysis of steady-state RNA from R region substitution mutants. Cellular RNA (20 μg) and the homologous probe were hybridized, treated with SI nuclease, and analyzed on 8% sequencing gels. Truncated transcripts are labeled with bars, full-length transcripts with arrows, and undigested probe with a dot. Lane 1: TM22; lane 2: TM24; lane 3: TM25; lane 4: TM28; lane 5: LM11; lane 6: LM2; lane 7: TM32; lane 8: TM37; lane 9: LM2; lane 10: ³²P-labeled pBR322 Msp I DNA markers.

Figure 5

Mapping of the termini of the truncated transcripts. A. Correctly initiated HIV-1 transcripts terminated 57 to 70 nt downstream of the transcription start site and initiated at +1. The termini of the truncated transcripts were mapped in LM2 RNA using two uniformly-labeled probes (+1 probe and +19 probe). LM2 +1 probe extended from –17 to +137, as illustrated in Figure 2A, and protected the transcripts from +1 to +137 (lane 1, arrow) and truncated transcripts 57 to 70 nt (lane 1, bar). To generate the +19 probe, LM2 template DNA was truncated at Bgl II (+19). The +19 probe protected the 5′ end of R region-CAT transcript and truncated transcripts beginning 19 nt downstream of +1 (lane 2, bar). B. The positioning of the truncated transcripts was independent of R sequences downstream of +57. SI nuclease protection analysis of steady-state RNA from cells transfected with R region constructs containing extended 3′ termini. Each RNA was hybridized to the homologous probe and was compared to the homologous sequencing ladder, as shown to the left of each SI analysis. Lane 1: LM2 (R sequences +1 to +57); lane 2: LM15 (+1 to +69); lane 3: LM1 (+1 to +80); lane 4: CD12 (+1 to +126).

Figure 6

Truncated transcripts were present in cellular RNA prepared by NP40 extraction but not by GSCN/CsCI extraction. A. SI nuclease protection analysis of LM1 and LM2 RNA prepared using the NP40 procedure or the GSCN/CsCI gradient method. RNA was isolated from duplicate HeLa cell transfections of LM1 and LM2 DNA, and 20 μg was hybridized with equal molar amounts of LM1 and LM2 uniformly-labeled probes. Protected full-length transcripts are designated by arrows, and the truncated R region transcripts are designated by bars. Lane M: ³²P-labeled markers; lane 1: LM1 RNA isolated by the NP40 method, 162 nt R region-CAT transcript, and 57 nt-70 nt truncated transcripts; lane 2: LM1 RNA isolated by the GSCN/CsCI gradient method, 162 nt transcript; lane 3: LM2 RNA isolated by the NP40 method, 137 nt R region-CAT transcript, and 57–70 nt truncated transcripts; lane 4: LM2 RNA prepared by the GSCN/CsCI gradient method, 137 nt R region-CAT transcript. B. Comparison of the recovery of 70 nt ³²P-labeled R region transcript from NP40 and GSCN/CsCI RNA isolation procedures. Lane 1: untreated 70 nt R region transcript; lane 2: R region transcript recovery in NP40 RNA from HeLa cells supplemented with 1 × 10⁴ CPM R region transcript; lane 3: recovery in NP40 RNA from cells supplemented with 1 × 10⁶ CPM R region transcript; lane 4: recovery in GSCN/CsCI RNA from cells supplemented with 1 × 10⁴ CPM R region transcript; lane 5: recovery in GSCN/CsCI RNA from cells supplemented with 1 × 10⁶ CPM R region transcript. C. Comparison of the stability of 632 nt R region-CAT transcript in NP40 or GSCN/CsCI extracted and purified RNA. HeLa cells were supplemented with ³²P-labeled R region-CAT transcript (632 nt), and cellular RNA was extracted. Lanes 1 and 3: R region CAT transcript recovered from GSCN/CsCI extracted RNA; lanes 2 and 4: R region CAT transcript recovered from NP40 extracted RNA.