The strength of the HIV-1 3' splice sites affects Rev function

Abstract

Background: The HIV-1 Rev protein is a key component in the early to late switch in HIV-1 splicing from early intronless (e.g. tat, rev) to late intron-containing Rev-dependent (e.g. gag, vif, env) transcripts. Previous results suggested that cis-acting sequences and inefficient 5' and 3' splice sites are a prerequisite for Rev function. However, we and other groups have shown that two of the HIV-1 5' splice sites, D1 and D4, are efficiently used in vitro and in vivo. Here, we focus on the efficiency of the HIV-1 3' splice sites taking into consideration to what extent their intrinsic efficiencies are modulated by their downstream cis-acting exonic sequences. Furthermore, we delineate their role in RNA stabilization and Rev function.

Results: In the presence of an efficient upstream 5' splice site the integrity of the 3' splice site is not essential for Rev function whereas an efficient 3' splice site impairs Rev function. The detrimental effect of a strong 3' splice site on the amount of Rev-dependent intron-containing HIV-1 glycoprotein coding (env) mRNA is not compensatable by weakening the strength of the upstream 5' splice site. Swapping the HIV-1 3' splice sites in an RRE-containing minigene, we found a 3' splice site usage which was variably dependent on the presence of the usual downstream exonic sequence. The most evident activation of 3' splice site usage by its usual downstream exonic sequence was observed for 3' splice site A1 which was turned from an intrinsic very weak 3' splice site into the most active 3' splice site, even abolishing Rev activity. Performing pull-down experiments with nuclear extracts of HeLa cells we identified a novel ASF/SF2-dependent exonic splicing enhancer (ESE) within HIV-1 exon 2 consisting of a heptameric sequence motif occurring twice (M1 and M2) within this short non-coding leader exon. Single point mutation of M1 within an infectious molecular clone is detrimental for HIV-1 exon 2 recognition without affecting Rev-dependent vif expression.

Conclusion: Under the conditions of our assay, the rate limiting step of retroviral splicing, competing with Rev function, seems to be exclusively determined by the functional strength of the 3' splice site. The bipartite ASF/SF2-dependent ESE within HIV-1 exon 2 supports cross-talk between splice site pairs across exon 2 (exon definition) which is incompatible with processing of the intron-containing vif mRNA. We propose that Rev mediates a switch from exon to intron definition necessary for the expression of all intron-containing mRNAs.

Figure 1

Alternative splicing of HIV-1. (A) Organization of the HIV-1 genome. Filled boxes indicate open reading frames present in all isolates, light grey boxes indicate the Tev orf which is isolate specific. The long terminal repeats (LTR) are present at both ends of the proviral DNA. (B) Localization of splice sites, splicing regulatory elements and the Rev responsive element (RRE). 5' splice sites: D1a-5; 3' splice sites: A1-7. Splice sites A6/D5 are isolate specific and not functional in the isolate NL4/3 used in this study. Splice sites A1a/D1a defining exon 1a have been recently described [6]. The nomenclature of the 3'ss is according to Stoltzfus [17,18] and Purcell and Martin [2] (in brackets). Splicing regulatory elements: M1, M2 (this report); ESSV [16,64]; ESS2p [18]; ESE2/ESS2 [17,32,43,44,49]; GAR [23,28]; ESS/ESE [19,20]; ISS [22]; ESE3 [17,21,24,25,33,65]; ESS3a, b [17,21,24,33,66]. (C) Splicing pattern and proteins encoded by the different mRNA classes. The 1.8 and 4 kb mRNAs contain obligatory sequences (dark grey) as well as alternative sequences (light grey) due to alternative usage of the splice sites. The nuclear export of the 4 kb mRNAs and the genomic full-length 9 kb mRNA is dependent on Rev binding.

Figure 2

3'ss A7 is nonessential for RNA stability and Rev responsiveness. (A) Schematic drawing of the HIV-1 genome and of the subgenomic env expression plasmid SV E/X tat^- rev^-. LTR: long terminal repeat, SV40: SV40early promoter, pA: SV40 polyadenylation sequence. Nucleotide sequences of the 5'ss D4 and its mutations D4^- and -1G3U as well as the 3'ss A7 and its mutations A7^- and A7⁺ are shown beneath. The splice sites (grey squares, including the minor 3'ss 7a and 7b), the reported or supposed branch point sequence (bold, asterix indicates the branch point nucleotide) and the mutated nucleotides (underlined) are marked. In the 3'ss mutants the reading frame was kept unchanged except for position 703 (Val→Ala) in A7⁺. (B) HeLa-T4⁺ cells were transiently transfected with the subgenomic env expression plasmids (SV E/X tat ^- rev ^-) containing either the wild type 5'ss D4 or the non functional D4^- mutation combined with either the wild type 3'ss A7 or the A7^- mutation in presence or absence of a Rev expression plasmid (SVcrev) as indicated above the lanes. The poly(A)⁺ RNA was analyzed by Northern blotting. s: spliced, us: unspliced transcript. Transfection efficiency was monitored by co-transfection of a human growth hormone (hGH) expressing plasmid (pXGH5).

Figure 3

Weakening of the 5'ss D4 does not compensate for the strength of 3'ss A7. HeLa-T4⁺ cells were transiently transfected with the subgenomic HIV-1 constructs (SV E/X tat ^- rev ^-) combining an efficient (A7⁺) or inefficient (A7) 3'ss with a 5'ss with high (D4) or lower (-1G3U, cf. Fig. 2A) complementarity to U1 snRNA. The p(A)⁺ RNA was analyzed by Northern blotting (cf. Fig. 2). (A) Northern blot with indication of the ratio of spliced (s) and unspliced (us) RNA in presence of Rev ([s/us], mean ± standard error) from three independent experiments. (B) Mean values of the relative amounts of spliced (s, black) and unspliced (us, grey) transcripts from three independent experiments, normalized to transcription efficiency (hGH). The spliced (s) and unspliceds (us) RNA populations were quantified from different exposure times of the blots to adjust for the different levels of signal intensities. The maximum values of both RNA populations were defined as 100%.

Figure 4

The strength of the 3'ss competes with Rev responsiveness. (A) Nucleotide sequence of one-intron constructs with mutations in the 3'ss A5. The reported branch point sequences for A5 (grey boxes, asterix indicates the branch point nucleotide), the 3'ss A4c, a, b, A5 (black boxes) and the PPT (hatched) are marked. Mutated nucleotides compared to the wild type are underlined. (B) Northern blot analyses of the p(A)⁺ RNA after transient transfection of HeLa-T4⁺ cells with one-intron constructs carrying SA5 mutations (cf. Fig.2). (C) Diagram of the hGH standardized relative amounts of spliced (left) and unspliced (right) transcripts from (B). The maximal amount was defined as 100%. The numbers below correspond to the lanes of the Northern blot.

Figure 5

Schematic drawing of the one-intron splicing reporter. Diagram of the one-intron construct used for comparison of the HIV-1 3'ss by a splice site swapping strategy. SV40: SV40early promoter, pA: SV40 polyadenylation sequence. RRE: Rev response element. Fragments including the different 3'ss (grey boxes) and branch sites (dashed line: assumed from consensus; underlined: reported BP, numbers are referring to the associated 3'ss, BP A2 [15], BP 4cab and A5 [14,30], BP A7 [14]) were inserted into the cassette. The ISS has been described by [22]. The 3'extended versions of the splice acceptor constructs additionally include the downstream exon sequences with cis-acting splicing regulating sequences (M1, M2 [this report]; ESSV [16,64]; ESS2p [18]; ESE2/ESS2 [17,32,43,44,49]; GAR [23,28]; ESE3 [17,21,24,25,33,65]; ESS3a, b [17,21,24,33,66]; splicing silencer (light grey boxes); splicing enhancer (dark grey boxes)).

Figure 6

The strength of the 3'ss competes with Rev responsiveness. (A) Northern blot analysis (cf. Fig. 2) from HeLa-T4⁺ cells transfected with constructs containing the HIV-1 3'ss in absence of their authentic 3' exon sequences. The particular 3'ss and the co-transfection of a rev expressing plasmid (SVcrev) are given above the lanes. The 3'ss A7^- and A7⁺ were used as reference constructs for a nonfunctional and an efficient 3'ss. All lanes were derived from the same Northern blot. (B) Northern blot analysis from cells transfected with 3'ss in presence of their authentic 3' exon sequences (ex). All lanes were derived from the same blot. (C) Mean values of the relative amounts of spliced transcripts in absence and presence of the 3' exon sequences from three independent experiments, normalized to transcription efficiency (hGH, cf. Fig. 2). The amount of spliced transcripts derived from the construct containing the improved A7⁺ was defined as 100% (not shown).

Figure 7

Alignment of exon 2 of HIV-1 M group consensus sequences. Exon 2 sequences were obtained from the HIV Sequence Database [67] flanked by A1 and D2. The heptameric sequences (M1 and M2) found to enhance splicing are highlighted by grey boxes.

Figure 8

An ASF/SF2-dependent ESE within exon 2. (A) Sequence of exon 2 (top line) and analyzed mutations. The heptameric sequences (M1 and M2) of the bipartite ESE are boxed and the mutations are given below exon 2 sequence. A schematic drawing of the two-intron minigene with exon 2 as internal exon flanked by A1 and D2 is given below the sequences. (B) HeLa-T4⁺ cells were transfected with LTR ds ex2 (ex2) or its mutations (cf. (A)), cotransfected with SVctat and pXGH5 (hGH) and analyzed by RT-PCR as described in Materials and Methods. (C) Pull-down analysis of in vitro transcripts of either exon 2 or exon 2 carrying both mutations, M1 and M2 in HeLa cell nuclear extract. The immunoblot was detected with a polyclonal SF2/ASF antibody.

Figure 9

Mutation of M1 by a single, silent point mutation does not alter vif mRNA expression within an infectious molecular clone. HIV-1 splicing pattern of PM1 cells infected with NL4/3 or NL4/3 carrying a silent point mutation within M1 (ΔM1-43, cf. Fig. 8A). The upper part shows an RT-PCR amplification for the vif 1.2I mRNA (the letter I denotes incompletely spliced or intron-containing mRNA) using primer pair #1544/#2183. The middle part was obtained using primer pair #1544/#1542. The stars indicate that these bands were isolated from the gel and confirmed by sequencing. To compare the total amount of HIV-1 transcripts within both mRNA preparations a separate RT-PCR was performed using primer pair #2335/#480 amplifying exon 1 sequence. (B) RT-PCR analysis of RNA from HeLa-T4⁺ cells transfected with the minigene lacking D2 (cf. figure legend 8B).

Figure 10

Schematic overview of HIV-1 splice sites and their strength. (A) Schematic drawing of the HIV-1 pre-mRNA and the distribution of the splice sites. The relative strength of the splice sites, based on splice site swapping strategies in this and previous publications [27,29], is represented by the size of the letters. D5 and A6 (grey) are marked for better orientation but they are not used in HIV-1 NL4/3 and therefore their relative strength was not tested. D1A and A1A are recently published splice sites preferentially involved in RNA stabilization [6]. ESEs (hatched) and ESS/ISS (black) are marked. (B) Schematic drawing of the exon structure in the 1.8 kb and 4 kb HIV-1 RNA classes. The numbers represent the relative incidence of exon inclusion [%] calculated from previously published RT-PCR analyses [2]. Differences to 100% in the total amount of the possibilities of overlapping exon recognition are due to rounding errors. The darker the colour of the exons the more efficiently they are included in the alternatively spliced HIV-1 transcripts.