A hairpin structure in the R region of the human immunodeficiency virus type 1 RNA genome is instrumental in polyadenylation site selection

Abstract

Some retroviruses with an extended repeat (R) region encode the polyadenylation signal within the R region such that this signal is present at both the 5' and 3' ends of the viral transcript. This necessitates differential regulation to either repress recognition of the 5' polyadenylation signal or enhance usage of the 3' signal. The human immunodeficiency virus type 1 (HIV-1) genome encodes an inherently efficient polyadenylation signal within the 97-nucleotide R region. Polyadenylation at the 5' HIV-1 polyadenylation site is inhibited by downstream splicing signals, and usage of the 3' polyadenylation site is triggered by an upstream enhancer element. In this paper, we demonstrate that this on-off switch of the HIV-1 polyadenylation signal is controlled by a secondary RNA structure that occludes part of the AAUAAA hexamer motif, which we have termed the polyA hairpin. Opening the 5' hairpin by mutation triggered premature polyadenylation and caused reduced synthesis of viral RNA, indicating that the RNA structure plays a pivotal role in repression of the 5' polyadenylation site. Apparently, the same hairpin structure does not interfere with efficient usage of the 3' polyadenylation site, which may be due to the presence of the upstream enhancer element. However, when the 3' hairpin was further stabilized by mutation, we measured a complete loss of 3' polyadenylation. Thus, the thermodynamic stability of the polyA hairpin is delicately balanced to allow nearly complete repression of the 5' site yet efficient activation of the 3' site. This is the first report of regulated polyadenylation that is mediated by RNA secondary structure. A similar hairpin motif that occludes the polyadenylation signal can be proposed for other lentiviruses and members of the spumaretroviruses, suggesting that this represents a more general gene expression strategy of complex retroviruses.

FIG. 1

A tandem hairpin motif is present at both ends of HIV-1 genomic RNA. (A) A schematic of HIV-1 proviral DNA is shown at top with the LTRs subdivided into the U3, R, and U5 domains. The terminal R and U5 elements (R region is boxed) are also present in the primary transcript. This R-U5 region encodes the TAR and polyA hairpins, and details of the latter’s RNA structure are provided in Fig. 2. The polyA hairpin encompasses the polyadenylation signal (the AAUAAA hexamer motif, indicated by a triangle). This motif is apparently ignored in the 5′ context but is used efficiently at the 3′ end of the HIV-1 sequences. Cleavage and subsequent polyadenylation occur 19 nucleotides downstream of the hexamer. The resulting mature transcript represents the unspliced genomic HIV-1 RNA. (B) The pLAI-R37 construct used in this study contains a 3′LTR deletion that truncates the 3′R. The 3′ HIV-1 polyadenylation site is absent and replaced by the SV40 polyadenylation site (also marked as a triangle). The primary transcript and the products of 5′ and 3′ polyadenylation are shown. The full-length HIV-1 RNA was specifically detected by a gag-pol probe in dot blot assays (see Fig. 3). An RT-PCR protocol was used to specifically amplify the prematurely polyadenylated transcript form (see Fig. 6).

FIG. 2

The wild-type and mutant polyA hairpins. The wild-type polyA hairpin conformation was established by several lines of evidence: RNA structure probing, phylogenetic comparison, and the analysis of mutants (reviewed in reference 7). The hairpin was mutated in different ways (nucleotide numbers refer to positions in the 5′ R-U5 of HIV-1 RNA). The polyadenylation signal, the AAUAAA hexamer, is indicated in boldface type. The thermodynamic stability (free energy, ΔG) of the RNA structures was calculated by the Zuker algorithm (46) and is indicated below the stems (in kilocalories per mole). In mutant A, the hairpin was stabilized by a single nucleotide substitution (boxed) and deletion (▴) of two bulges. The hairpin was destabilized in mutant B by four nucleotide substitutions (boxed). The hairpins of the A revertant and the B revertant were obtained upon prolonged culturing of the corresponding mutant viruses (clones A120-3 and B127-4 in reference 18). We circled the reversion-based mutations that restore the wild-type hairpin stability. In mutants C and D, destabilizing mutations (boxed) were introduced in the right- and left-hand side of the stem, respectively. The mutated segments of C and D were combined in the double mutant CD, which repairs the lower part of the stem.

FIG. 3

Gene expression and virus production by HIV-1 genomes with a mutated 5′ polyA hairpin. C33A cells were transfected with the wild-type and mutant proviral constructs and cultured for 2 days. (A) The intracellular HIV-1 RNA content was measured with a gag-pol probe that specifically detects unspliced transcripts (illustrated in Fig. 1B). (B) The CA-p24 level was measured in the culture supernatant by ELISA. (C) The genomic RNA content of virions was determined by slot blot analysis and compared with the CA-p24 values. (D) The RNA packaging efficiency was calculated as the ratio of virion RNA to intracellular HIV-1 RNA. All parameters were arbitrarily set at 100% for the wild-type (wt) construct. The standard error of the mean was calculated for four to six independent transfections. For panels C and D, we first calculated the ratio per independent transfection and then calculated the mean value and standard error of the mean. Abbreviations: A, mutant A; Arev, A revertant; B, mutant B; Brev, B revertant; C, mutant C; D, mutant D; CD, double mutant CD.

FIG. 4

Analysis of 5′ and 3′ polyadenylated HIV-1 RNA by RNase protection. (A) Shown is the unspliced, genomic HIV-1 RNA that is polyadenylated at the 3′ end. The two R regions are blocked, and nucleotide numbers refer to positions on the HIV-1 RNA. The position of the major splice donor site (SD, position 290) is indicated. The riboprobe used in the RNase protection assay and the expected protected fragments are indicated (the R region is marked black in the riboprobe and the protected fragments). Premature 5′ polyadenylation will result in the protection of a 97-nucleotide probe fragment. Read-through transcripts protect fragments at both the 5′ and 3′ ends. At the 5′ side, two fragments of 381 or 290 nucleotides are protected by the unspliced and spliced HIV-1 RNAs, respectively. At the 3′ side, a 525-nucleotide fragment is protected by both the unspliced and spliced transcripts. (B) Individual ³²P-labeled RNA probes were hybridized to total RNA from cells transfected with the corresponding HIV-1 plasmids (indicated at the top of the panel). The samples were treated with RNase and subjected to denaturing polyacrylamide gel electrophoresis. Control samples from mock-transfected cells and mock-treated RNAs did not produce the signals that are indicated (not shown). Several RNA transcripts of distinct length were used as size markers (not shown). The somewhat aberrant migration of the 5′ spliced signal of the CD mutant was caused by folding of the gel during drying. The doublet bands observed for the 5′ spliced and unspliced signals of the wild-type (wt) construct were not observed in parallel probing experiments with RNase T1. Larger RNase digestion products were observed for the riboprobe of mutant A, which is most likely caused by stable secondary RNA structure in this antisense RNA. In fact, this probe even resisted complete digestion in the absence of HIV-1 RNA (not shown). The data were quantitated by PhosphorImager analysis and corrected for the differences in probe length (amount of incorporated labeled nucleotides), and these results are presented in Fig. 5. Abbreviations are as defined in the legend for Fig. 3.

FIG. 5

Premature polyadenylation is triggered by opening of the 5′ polyA hairpin. These results were derived from the RNase protection experiment shown in Fig. 4. Abbreviations are as defined in the legend for Fig. 3.

FIG. 6

Analysis of the prematurely polyadenylated HIV-1 transcript by RT-PCR. (A) The HIV-1 plasmids with a wild-type or mutated 5′ polyA hairpin (indicated on top of the panel) were transiently transfected into C33A cells. Total cellular RNA was extracted, and HIV-1 sequences were amplified by a RT-PCR protocol (illustrated in Fig. 1B). The products were separated on an agarose gel, blotted, and probed with a TAR region probe. No PCR products were obtained in an RT-minus control reaction, indicating that the input plasmid DNA sequences were not amplified. The 5′ polyadenylated HIV-1 RNA yields a short cDNA product. Please note that read-through transcripts will be polyadenylated at the SV40 polyadenylation site. This RNA is expected to produce a much longer PCR product that includes the cat gene. This product is not detected with the TAR probe because of incomplete complementarity. Quantitation of the PCR products was performed by PhosphorImager analysis, and the results are presented in panel B. (B) The 5′ polyadenylation efficiency of wild-type HIV-1 RNA was arbitrarily set at the value 1. Abbreviations are as in the legend for Fig. 3. (C) Viral plasmids were constructed with the wild-type (wt) or mutant A or B hairpin in both LTRs. C33A cells were transfected with the individual plasmids (indicated on top of the panel), and total cellular RNA was isolated and analyzed by RT-PCR as described for panel A.

FIG. 7

A multifactorial model for differential HIV-1 polyadenylation. Shown are the proviral DNA and the primary HIV-1 transcript with the typical tandem hairpin motif at both the 5′ and 3′ ends. The polyadenylation signal AAUAAA (triangle) is located in the polyA hairpin. Results presented in this study indicate that regulated HIV-1 polyadenylation is made possible by the polyA hairpin structure, which down-modulates this potentially efficient polyadenylation site (indicated by the ± sign). In the presence of additional repressive signals in the 5′ context (indicated is inhibition through the SD site, but promoter proximity may also play a negative role), polyadenylation is reduced to a low level (approximately 10%; see Discussion). In the presence of the USE enhancer motif in the 3′ context, the full potential of the HIV-1 polyadenylation sequence is realized. This scheme does not indicate which step of polyadenylation is regulated. However, because the polyadenylation signal is occluded by the hairpin structure, it is likely that the initial binding of CPSF to the hexamer motif is blocked.

FIG. 8

Lentiviruses and spumaviruses have their polyadenylation signals encoded within a hairpin structure. All these viruses have a relatively extended R region encoding the AAUAAA hexamer motif (shaded in structure), which necessitates differential polyadenylation. Similar stem-loop structures were predicted for other HIV and SIV viruses (9) and also for simian spumaviruses (not shown). The free energies of the stem-loop structures (including the terminal stacks) were calculated with the Zuker algorithm (46), and the ΔG values are presented in kilocalories per mole. EIAV, equine infectious anemia virus; HSRV, human spumaretrovirus; BIV, bovine immunodeficiency virus.