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University of Cambridge Department of Medicine, Addenbrooke's Hospital, Cambridge CB2 2QQ, UK
Correspondence
Andrew Lever
amll1{at}mole.bio.cam.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) have been identified and characterized in a number of simple and complex retroviruses (Berkowitz et al., 1996
) and are found characteristically in the 5' leader region of the viral RNA and contain helix–loop motifs with terminal purine-rich loops. In human immunodeficiency virus type 1 (HIV-1) (Lever et al., 1989; Clavel & Orenstein, 1990; Aldovini & Young, 1990) and murine oncoretroviruses (Adam & Miller, 1988), regions are found predominantly downstream of the major splicing signal, the splice donor (SD) in the leader. However, in HIV-1, additional contributions to encapsidation are sometimes made by sequences upstream of the SD (Berkhout & van Wamel, 1996; Das et al., 1997) and for both HIV and murine leukaemia virus, these occur also in the Gag-encoding region (Parolin et al., 1994; Bender et al., 1987) as well as the dimer linkage site. The site of the major packaging signal ensures that it will be found only on the unspliced messages, thus confering further selectivity on the process. In Rous sarcoma virus (RSV) (Linial et al., 1978; Katz et al., 1986) and HIV-2 (Griffin et al., 2001), the major packaging signal is found upstream of the SD. RSV has only two RNA species: the genomic species (full-length genomic RNA) encodes Gag and Pol, whereas the second (spliced) species encodes the envelope glycoprotein. When this reaches a ribosome, the signal peptide sequence is translated and sequesters the complex to the rough endoplasmic reticulum away from the Gag protein, which is produced on free cytoplasmic ribosomes. This aids in the process of selectivity. For lentiviruses, regulatory and accessory proteins are encoded on multiply spliced mRNAs that are translated on free cytoplasmic ribosomes, where they could conceivably compete with the genomic RNA for packaging. For HIV-2, specificity is maintained by cotranslational packaging of the Gag-encoding (Kaye & Lever, 1999; Griffin et al., 2001) (unspliced) message and apparent limiting availability of Gag available to capture other RNAs (Griffin et al., 2001). Given the genetic relatedness of the primate lentiviruses, it was of interest to see where the encapsidation signal of simian immunodeficiency virus (SIV) would be found. Analysis of the SIV 5' leader region has shown evidence of cis-acting signals affecting packaging in the U5 stem and the DIS stem–loop (Guan et al., 2000, 2001b); the critical nature of the U5 stem for replication functions unrelated to packaging (Guan et al., 2001a) has been published. From vector (Schnell et al., 2000) and chimera (Rizvi & Panganiban, 1993) studies, there was also circumstantial evidence that the spliced mRNA was competent for packaging. However, comparison of the role of sequences upstream and downstream of the SD has not been assessed specifically in the context of the wild-type virus with the presence of competing viral RNA species. Our findings based on an analysis of packaging efficiency support the location of the major packaging determinant of SIV as being 5' of the SD, with relatively little contribution from 3' sequences. The position resembles that of HIV-2 more closely than HIV-1 and the position of the packaging signal relative to the cap site is remarkably similar in all three viruses. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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pC8delT-KS (a kind gift from M. Cranage, Jenner Institute, St George's Hospital, London, UK) is based on pC8, an infectious molecular clone of SIVmac (Rud et al., 1994). A 1973 bp BamHI–XhoI fragment was removed from pC8delT-KS and cloned into pBluescript KS (Stratagene) to create plasmid SIVKS+. Site-directed mutagenesis was then carried out on this plasmid.
To create 
P1, positions 862–898 were deleted from the SIV leader sequence using the mutagenic oligonucleotide 5'-AGTGAGAAGAACTCCACCACGACGGACTGC-3'. For P2, positions 915–947 were deleted using the mutagenic oligonucleotide 5'-CCAACCACGACGGAGGCGTGAGGAGCG-3'. For P3, positions 995–1045 were deleted using the mutagenic oligonucleotide 5'-CGGTTGCAGGTAAGTGCAAGTGGGAGATGGGC-3'. For P4, positions 1011–1042 were deleted using the mutagenic oligonucleotide 5'-GCAACACAAAAAAAGAAATTAGAGTGGGAGATGGGC-3'.
pRSenvSL was derived from pC8. The firefly luciferase gene expressed from the simian virus 40 early promoter was blunt-end ligated into the PflMI (6780) and PmlI (7973) sites located in the env gene. The mutated leader regions were then inserted into pRSenvSL using the BamHI/XhoI sites to create the deletion mutants pRSenvSLP1, pRSenvSLP2, pRSenvSLP3 and pRSenvSLP4. All plasmids were sequenced to confirm the presence of mutated sequences.
Plasmids used as templates for the production of riboprobes were created as follows: SIVSKGS, used to detect genomic versus spliced RNA, was created by amplification of SIV sequences between positions 818 and 1068 using the primers 5'-ATGGGAATTCGTTTCGTTTCTCGCGCCCATCTCCCACTCT-3' and 5'-TAATGGATCCAGATTGGCGCCTGAACAGGG-3'. The PCR product was then cloned into the BamHI/EcoRI sites of pBluescript SK+ (Stratagene). SIVSKLTR, used to detect DNA versus RNA, was created by amplification of SIV sequences between positions 300 and 750 using the primers 5'-CTTTGAATTCACCGAGTACCGAGTTG-3' and 5'-TTTGGGATCCTACCCAGAAGAGTTTGG-3'. The PCR product was the cloned into the BamHI/EcoRI sites of pBluescript SK+.
The plasmid expressing the vesicular stomatitis virus G protein (VSV-G) driven from a cytomegalovirus promoter was a kind gift from L. Tiley (Department of Veterinary Medicine, University of Cambridge, Cambridge, UK).
Cell culture and transfection.
293T cells were maintained in DMEM (Gibco-BRL) supplemented with 10 % FCS, 100 µg streptomycin ml-1 and 10 U penicillin ml-1. Transient transfections were performed with 10 µg plasmid (or as described) using a modified calcium phosphate technique. At 48 h post-transfection, the cells and supernatants were harvested. Viral protein production from the wild-type and mutant constructs was assessed by immunoprecipitation of 35S-labelled proteins with SIV-specific antisera (a kind gift from M. Cranage) and polyacrylamide gel electrophoresis.
Isolation of virion and cytoplasmic RNA for RNase protection assays (RPAs).
Virion RNA was extracted by precipitation of the transfection supernatant with 0·5 vol. 30 % polyethylene glycol 8000 in 0·4 M NaCl for 16 h at 4 °C. The precipitate was then centrifuged at 2000 r.p.m. for 40 min at 4 °C at 1400 g. The resulting pellets were resuspended in 0·5 ml TNE (10 mM Tris/HCl, 150 mM NaCl, 1 mM EDTA). A 10 µl sample was used in a reverse transcriptase (RT) assay and the remainder was layered over an equal volume of TNE containing 20 % sucrose and ultracentrifuged at 4 °C for 2 h at 40 000 r.p.m. in a Beckman TL100 using a TL45 rotor. Virus particles were then lysed for 30 min in proteinase K buffer (50 mM Tris/HCl, 100 mM NaCl, 10 mM EDTA, 1 % SDS, 0·1 % w/v proteinase K, 0·1 % w/v tRNA). Viral RNA was extracted twice with phenol/chloroform and once with chloroform and then precipitated at -80 °C. Cytoplasmic RNA was extracted by resuspending the transfected cells in ice-cold lysis buffer (50 mM Tris/HCl pH 8·0, 100 mM MgCl2, 0·5 % v/v Nonidet P-40). The supernatant was cleared by centrifuging at 13 000 r.p.m. for 2 min at 4 °C. The supernatant was then mixed with 125 µg proteinase K ml-1 and incubated at 37 °C for 15 min. RNA was extracted twice with phenol/chloroform and once with chloroform and then precipitated in ethanol at -80 °C. Virion and cytoplasmic RNA was resuspended in 100 µl DNase buffer [10 mM Tris/HCl pH 8·0, 10 mM MgCl2, 1 mM DTT, 5 U RNase-free DNase (Promega), 4 U RNase inhibitor (Promega)] and incubated for 15 min at 37 °C. The reaction was stopped with 25 µl DNase stop mixture (50 mM EDTA, 1·5 M NaCl, 1 % SDS). Samples were then extracted once with phenol/chloroform and once with chloroform and then precipitated in ethanol.
Packaging efficiency was calculated as the ratio of virion to cytoplasmic unspliced RNA compared to wild-type which was given an arbitrary value of 1.
RPA.
[32P]UTP was incorporated into the linearized riboprobes SIVSKGS and SIVSKLTR by in vitro transcription with T7 RNA polymerase (Promega). The riboprobes were purified from a 5 % polyacrylamide/8 M urea gel before use. RPAs were carried out using a commercially available kit (Ambion). Cytoplasmic RNA (0·25 µg) and equalized (by RT) amounts of virion RNA were incubated with 2x105 c.p.m. of 32P-labelled probe and 3 µg carrier RNA from Torrula yeast (Ambion) in 20 µl hybridization buffer (Ambion) for 16 h at 42 °C. Unhybridized probe was then removed by the addition of 0·5 U RNase in 200 µl RNase digestion buffer (Ambion). The protected fragments were ethanol-precipitated, resuspended in RNA loading buffer and separated on a 5 % polyacrylamide/8 M urea gel. Gels were then subjected to autoradiography and the levels of RNA determined using an Instant Imager (Packard). Size determination of fragments was achieved by running 32P-labelled RNA molecular mass markers made using a Century marker template set (Ambion) in parallel.
SIV vector production.
The SIV luciferase virus vector was produced by cotransfection of 10 µg of the envelope-deleted SIV construct containing the luciferase gene in env together with the wild-type (pRSenvSL) or deletion mutant leader sequence, and 3 µg of the VSV-G envelope-expressing plasmid by the calcium phosphate method. At 60–72 h post-transfection, the supernatant was removed from the cells and pre-cleared by low-speed centrifugation for 10 min at 2000 r.p.m. in a bench-top centrifuge (MSE Falcon 6/300). Supernatants were then filtered through a 0·45 µm filter and concentrated by ultracentrifugation for 2 h at 25 000 r.p.m. in a Beckmann centrifuge using an SW28 rotor. The virus pellet was resuspended in 500 µl PBS and concentrated further by ultracentrifugation over a 500 µl sucrose cushion at 40 000 r.p.m. for 2 h at 4 °C in a Beckmann bench-top ultracentrifuge. The viral pellet was resuspended in 50 µl PBS and stored at -70 °C.
Virus quantification.
The concentrated vector was quantified by a commercially available RT assay (Cavidi Tech) using SIV RT standards. Several dilutions and replicates of each virus vector were assayed.
Virus transduction and luciferase assay.
SV2 cells were seeded in 6-well tissue culture plates at a density of 8·3x105 per well. Equivalent quantities of each virus vector (10 ng) were added to each well in the presence of polybrene (6 µg ml-1) in serum-free medium (DMEM) for 6 h. Medium was replaced with DMEM containing 10 % FCS and cells were incubated at 37 °C. At 48 h post-transduction, luciferase activity in the transduced cells was assayed using the Promega luciferase system, according to the manufacturer's instructions. Luciferase levels were measured using a manual luminometer.
Virion extraction and luciferase RNA RT-PCR.
Virion RNA from concentrated SIV virus vector was extracted using the QIAamp RNA Extraction system (Qiagen), according to the manufacturer's instructions. Of each concentrated virus vector, 10 ng (as determined by RT assay) was used for virion RNA extraction. Of the extracted virion RNA, 30 µl was treated with 1·5 U RQ DNase 1 (Promega) for 15 min at 37 °C followed by inactivation for 15 min at 70 °C. Preliminary experiments confirmed degradation of transfected plasmid DNA. Reverse transcription of luciferase RNA was performed using the ImProm-II Reverse Transcriptase system (Promega) with the antisense luciferase primer Luc R (5'-AATCTCACGCAGGCAGTTCT-3'). cDNA was then diluted 1/10 and serially double diluted to 1/2560. PCR amplification of the diluted luciferase cDNA was performed for 32 cycles using the sense primer Luc F (5'-CCAGGGATTTCAGTCGATGT-3') and the antisense Luc R primer in a 50 µl reaction volume containing 50 pmol of each primer, 10 mM dNTPs and 0·5 U Taq polymerase. PCR products were electrophoresed on a 2 % agarose gel.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, were assessed for their effect on viral protein production following transfection of the individual constructs into 293T cells and virion purification. P1–3 all produced similar quantities of viral Gag protein, as demonstrated by an immunoprecipitation assay with SIV antisera (Fig. 2). There was slightly more uncleaved Gag polyprotein precursor noted in P1, but this was not an invariable finding. P4 produced virtually no detectable protein on repeated assays. On sequencing, there appeared to be no other mutation in the leader or the start of Gag other than the mutation introduced. P4 produced comparable quantities of mRNA compared to wild-type and the other mutants. Analysis of the predicted secondary structure of the leader RNAs in the four mutant viruses and the wild-type virus shows that wild-type and P1–3 RNAs would all maintain a stem–loop structure proximal to the AUG stem–loop. In P4 alone, the region 5' to the AUG loop is relatively unstructured (Fig. 3).
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. The results of a representative RPA are shown in Fig. 5. The cellular bands of unspliced SIV RNA (Fig. 5, 
) confirm the abundant intracellular production of RNA from all the constructs, including P4. Spliced messages (Fig. 5, 
) were seen in the cellular samples but were greatly diminished in relative concentration compared to the unspliced message in the wild-type virions and the P3 mutant virions (the apparent size discrepancy for the unspliced message in virions of P3 is accounted for by the asymmetric running of this gel). In contrast, this selectivity was lost for P1 and also for P2, in which the spliced to unspliced ratio of RNA in the virion was similar to that in the cell. P2 packaged RNA consistently at a lower level than the wild-type virus or P3. The virus sample for P4 was essentially the whole transfected supernatant, as the very low level of protein produced made it impossible to equate values for particles by RT, as was done for the wild-type and for P1–3. Relative to the wild-type, P1 had a packaging efficiency of around 0·2, P2 was <0·1 and P3 was 0·5. Thus, P2 had the most profound packaging-defective phenotype, although mutations upstream and downstream of this region had some effect.
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P4 mutant was demonstrating an aberrant phenotype inconsistent with a packaging defect and thereafter only P1–3 were assessed in comparison to the wild-type sequence.
A second method of measuring virion RNA packaging is RT-PCR, which is quantitative and complementary to the RPA. Fig. 6 demonstrates the similarity in levels of packaged RNA between P3 and the wild-type virus, whereas the two deletions upstream of the SD show a more severe packaging-defective phenotype.
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P1–3 were introduced into an SIV-based vector system encoding the luciferase gene under the control of the viral LTR. Gene transfer was measured by detection of luciferase expression in the target cells and used as a surrogate marker for encapsidation. Again, luciferase expression was transferred most easily by the wild-type vector and that containing the P3 deletion, whereas P1 and P2 both caused defects in this single-round replication marker assay (Fig. 7). In the context of normal virion protein production, the three assays confirm that the packaging signal is predominantly 5' of the SD. By RPA, the P2 deletion was consistently the most profoundly defective for packaging, although it was virtually identical to P1 in the RT-PCR and vector transfer assays. P2 probably encompasses the major packaging signal with an additional significant contribution from the P1 region.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). However, it is likely that some of the mutants described have had secondary effects on the three-dimensional structure of the adjacent packaging signal, thus complicating interpretation. Despite this, in HIV-1 and HIV-2, a region has been identified that has the most effect on packaging, as determined by a fall in packaging efficiency when this sequence is deleted. For HIV-1, there is a consensus that the region between the major SD and the Gag initiation codon is site (Lever et al., 1989; Clavel & Orenstein, 1990; Aldovini & Young, 1990). This region has been studied by two- and three-dimensional analysis and a conserved helix–loop structure which changes shape as the Gag protein binds to it has been identified (De Guzman et al., 1998; Zeffman et al., 2000). In HIV-2, the site is more controversial. Some groups have found packaging signals in positions analogous to those in HIV-1 (Garzino Demo et al., 1995; Sadaie et al., 1998). We have found consistently only a minor effect on packaging attributable to this region (McCann & Lever, 1997; Griffin et al., 2001; Kaye & Lever, 1999). Most groups agree that there is some contribution to packaging from regions upstream of the SD and we have, on repeated experiments, found this region to be the dominant packaging signal. A single deletion upstream of SD led to a reduction in packaging to around 5 % of wild-type. We have shown that HIV-2 can use alternative strategies to overcome the apparent disadvantage of having a packaging signal in a region common to all its viral mRNAs (Kaye & Lever, 1999; Griffin et al., 2001). For SIV, deletion analysis of the leader directly comparing the contribution of regions 5' and 3' to the major splice site has not been published previously, nor has direct RNA quantification of encapsidation by RNase protection for such mutants. Here we show that, analogous to HIV-2, the encapsidation sequences localize to regions upstream of the SD, as suggested previously (Guan et al., 2000, 2001b), making it more akin to HIV-2, to which it is also more closely related than it is to HIV-1. The effects of deletions 5' to the P2 deletion and the very modest effect of a deletion 3' of the SD are a consistent finding in lentiviruses, where mutations flanking the major impinge on its function. P2 showed consistently the most profound packaging defect to an almost undetectable level, which, in our experience, is unusual in lentiviruses but indicates loss of the key encapsidation signal. Why different viruses should apparently site their regions in a functionally different region and incur selectivity problems is not clear; however, it is striking that the HIV-1 leader is much shorter than that of HIV-2 and SIV. If one superimposes the three sequences, then the site of the packaging signal relative to the 5' methyl cap is actually very similar, with the signal being approximately 300–400 bases 3' of the cap (Fig. 8). We hypothesize that this distance is critical to the encapsidation function and may relate to the competition between translation and encapsidation, which this RNA species must undergo. This positioning would imply that the site of relative to the SD is of secondary importance to the requirements of cap– distance and that the viruses adapt their RNA capture mechanism to cope with the relative constraints imposed by the site. Interestingly, the sites of the packaging signals of Mason–Pfizer monkey virus (Guesdon et al., 2001), spleen necrosis virus and Moloney murine leukaemia virus (Linial & Miller, 1990) are also at a relatively similar distance (300–400 bases) from their respective cap sites. Experiments to explore this are currently ongoing.
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P4 deletion phenotype was unexpected. From sequencing analysis, we are confident that this construct does not have other deletions in relevant regions, which would explain the failure of protein production. P4 clearly produces adequate mRNA, as can be seen in the RPA assay; however, this appears not to be translated into protein. There is evidence in the literature to suggest that some lentiviruses use internal ribosomal entry to initiate translation (Ohlmann et al., 2000; Buck et al., 2001). It is conceivable that this is the case for SIV and that the deletion has somehow altered the structural context of the IRES or that of the Gag initiation codon and, in doing so, disrupted the process. Further experiments are under way to explore this.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 21 February 2003;
accepted 26 May 2003.
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