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1 Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 2QQ, UK
2 Center for Human Virology, Division of Infectious Diseases, Thomas Jefferson University, Philadelphia, PA 19107-5587, USA
3 Christian Doppler Laboratory for Gene Therapeutic Vectors, Research Institute of Virology and Biomedicine, University for Veterinary Sciences, Vienna, Austria
Correspondence
Jane S. Greatorex
jg10018{at}mole.bio.cam.ac.uk
Andrew M. L. 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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) is recognized by the Gag polyprotein against the background of spliced and cellular mRNAs. consists of a series of three to four stem–loops (Harrison et al., 1998
). Stem–loop 1 contains the dimerization-initiation site (Laughrea & Jette, 1994; Skripkin et al., 1994) plus two internal loops termed loop A and loop B (Greatorex et al., 2002). These internal loops are highly conserved (Greatorex et al., 2002) and, importantly, so are the helices surrounding them, implying a role for the loops in the virus life cycle. The tertiary structure of loop A closely resembles that of the Rev-responsive element (RRE) (Fig. 1) and has been demonstrated to bind the Rev protein specifically (Gallego et al., 2003). A mutation of the loop changing adenine to guanine at positions 692 and 695 in the HIVHXB2 sequence (Fig. 1), termed 4G, reduces Rev binding significantly (Gallego et al., 2003). The aim of this work was to examine the effect of this mutation against the background of an infectious molecular clone, to help to elucidate the role of this novel Rev-binding site in the context of virus replication.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The 4G mutation was introduced into pKSBglII [described previously (Harrison et al., 1998)] and cloned back into pSVC21 by using SpeI and SfoI to create pSVC4G. Plasmid pT319R-RRE was used to generate cold competitor RNA and has been described previously (Dangerfield et al., 2005).
Plasmid KSIICS was used to generate the antisense probe for the ribonuclease-protection assays (RPAs) (Kaye & Lever, 1996).
RNA production.
Templates for RNA were generated by using forward primer 5'-TAATACCACTCACTATAGGAAACCAGAGGAGC-3' and reverse primer 5'-CTCTCTCCTTCTAGCTCCGC-3'. PCR and template purification were performed as described previously (Gallego et al., 2003). The template for the RRE RNA was generated by linearizing pT319R-RRE with EcoRI (Roche Molecular Biochemicals), cleaning the DNA using a QIAquick column (Qiagen) and ethanol precipitation. The template for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control probe came from the RPA II kit (Ambion).
In vitro transcription from the DNA templates was carried out by using a Promega Riboprobe kit, incorporating [
-32P]UTP (Amersham Biosciences) in the reaction. The concentration of labelled RNA was determined by trichloroacetic acid precipitation.
Dimerization assay.
In vitro-transcribed RNA (wild-type or 4G mutant) was resuspended in DEPC-treated water at 1 mg ml–1. RNA (1 µg) in dimer buffer [40 mM KCl, 0.1 mM MgCl, 10 mM Tris/HCl (pH 7.4)] was heated at 90, 70 or 50 °C for 2 min, snap-cooled on ice and incubated for 60 min at 37 °C. Native loading buffer was added (Ambion) and samples were analysed by electrophoresis on 2 % native agarose gels.
Electrophoretic mobility-shift assays.
Band-shift assays were performed as described previously (Gallego et al., 2003). Briefly, RNA (90 nM) was heated to 95 °C for 2 min in binding buffer [250 mM Tris/HCl (pH 7.4), 250 mM KCl, 250 mM MgCl2] with tRNA and DEPC. RRE RNA was added prior to heating at concentrations ranging from equimolar to 1000-fold excess. The RNA was snap-cooled, peptide (Bachem) was added and the mixture was incubated at room temperature for 30 min. Following incubation, non-denaturing loading buffer (Ambion) was added to the mixture and the samples were analysed by electrophoresis on 10 % TBE/polyacrylamide gels (Anachem).
Replication studies.
Equal numbers of virions, as judged by a reverse transcriptase (RT) assay, from transient DEAE/dextran transfections of COS-1 cells were used to infect 2x106 Jurkat cells in 2 ml RPMI 1640 (Invitrogen) containing 10 % fetal calf serum, 100 U penicillin ml–1 and 100 µg streptomycin ml–1. After 24 h, cells were pooled to give a total volume of 10 ml. Every 4–5 days, 10 µl samples of supernatant were removed and assayed for RT activity (Potts, 1990). Cells were split 1 : 4 weekly (7–8 days) or 1 : 2 every 4–5 days.
RNA extraction.
Cytoplasmic RNA was extracted from COS-1 cells as described previously (Harrison et al., 1998). In brief, cells were lysed in chilled lysis buffer [50 mM Tris/HCl (pH 8), 100 mM NaCl, 5 mM MgCl2, 0.5 % Nonidet P-40]. Debris and nuclei were removed by centrifugation for 1 min at 13 000 r.p.m. in a microcentrifuge (Heraeus Biofuge Pico). The supernatants were adjusted to 0.2 % SDS and 125 µg proteinase K (Ambion) ml–1, incubated at 37 °C for 15 min, extracted with phenol/chloroform and ethanol-precipitated. Nuclear RNA was purified post-lysis by using a Qiagen RNeasy kit. Extractions were performed following the manufacturer's instructions.
Supernatants from transfected cells were passed through a 0.45 µm filter and the filtrate was precipitated overnight with 0.5 vols 30 % PEG 8000 in 0.4 M NaCl at 4 °C. The precipitate was collected by centrifugation at 2000 r.p.m. in an MSE 43124-129 rotor at 4 °C and resuspended in 0.5 ml TNE [10mM Tris/HCl (pH7.5), 150 mM NaCl, 1 mM EDTA]. This material was layered onto an equal volume of TNE containing 20 % sucrose and centrifuged at 98 000 g for 2 h at 4 °C in a Beckman Optimax TLA-50 rotor. Virions were lysed in proteinase K buffer [50 mM Tris/HCl (pH 7.5), 100 mM NaCl, 10 mM EDTA, 1 % SDS, 100 µg proteinase K ml–1, 100 µg tRNA ml–1) for 30 min at 37 °C. Following this, the RNA was extracted with phenol/chloroform and ethanol-precipitated.
RPAs.
Nuclear and cytoplasmic RNA levels were normalized by RNA concentration, with 5 µg being used per reaction for the cytoplasmic preparations. Virion levels were normalized by RT count, as described above. The riboprobe was generated by using the KSIICS template described above, incorporating [-32P]UTP (Amersham Biosciences). Probes were gel-purified and 10 µl was added to 2 ml scintillant to determine the number of counts µl–1.
Reagents for the RPA were obtained commercially (RPA II kit; Ambion) and the assay was performed following the manufacturer's instructions. Each reaction used 2x105 counts of probe. Reactions were analysed on 6 % polyacrylamide gels and the relative quantities of viral RNA in each sample were counted by using a Packard Real-time Instant Imager or the NIH ImageJ program. The probe that was used protects fragments of the following sizes: DNA, 517 and 332 nt; genomic RNA, 375 and 238 nt; spliced RNA, 289 nt.
Protein production.
Equal numbers of cells were harvested at 72 h post-transfection (or later). For cytoplasmic-protein analysis, cells were washed twice with ice-cold PBS, trypsinized, resuspended in 1 ml PBS and spun gently. Cells were resuspended in 100 µl PBS and 200 µl loading buffer [6.25 mM Tris/HCl (pH 6.8), 2 % SDS, 10 % glycerol, 5 % 
-mercaptoethanol, 0.02 % bromophenol blue]. Protein from virions was prepared as follows: virions were harvested as described above. Following centrifugation at 2000 r.p.m. in an MSE 43124-129 rotor, the pellet was resuspended in 100 µl PBS and 200 µl loading buffer. Samples were subjected to sonication and the protein equivalent of 6.5x105 cells was loaded onto an SDS-polyacrylamide gel. Western blotting was performed as described previously (Harrison et al., 1998) using an anti-p55/p24 antibody (ADP313; NIBSC).
Northern blotting.
Virion RNA was prepared and levels were normalized as described above. RNA samples were electrophoresed on 1 % agarose gels (Ambion), transferred to nitrocellulose overnight at room temperature and the RNA was detected by using commercially available reagents (Northern Max-Gly and BrightStar BioDetect; Ambion).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, lanes 6 and 7).
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), to generate pSVC4G. The two plasmids were transfected into COS-1 cells and virus was harvested at 48–72 h post-transfection. Virion levels were normalized by RT assay and used to infect Jurkat T cells. Following infection, RT assays were performed at regular time points and cells were split either 1 : 4 every 7–8 days (Fig. 3a) or 1 : 2 every 5 days (Fig. 3b). Under both sets of conditions, the 4G-mutated virus showed a replication defect. Peak viral production was delayed and never reached wild-type levels, as judged by RT assays.
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, compare lanes 1 and 4 with lanes 2 and 5). Once Jurkat cells had been infected and virus was being produced (as shown by a rise in RT values), protein was harvested as before from equal numbers of cells and protein levels were compared. Total viral protein levels in the Jurkat cells were marginally reduced, consistent with a replication defect, but there was no marked qualitative difference in protein production or processing (Fig. 4b).
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, lanes 1–6). A GAPDH probe was also used in separate assays (data not shown) to confirm equivalent cytoplasmic RNA loading. A consistent difference in RNA packaging was seen between the wild-type and 4G-mutant virus. The latter packaged RNA at 65–70 % of wild-type levels (Fig. 5, compare lanes 11 and 12 with lanes 9 and 10). Experiments were performed independently three times.
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RNA with and without the 4G mutation was assessed on non-denaturing agarose gels. The RNA containing the 4G mutation had marginally less stability as a dimer than did the wild-type RNA. The latter was wholly dimeric, even after heating at 90 °C (Fig. 6). Unheated RNA containing the 4G mutation was 100 % dimeric (Fig. 6, lane 5), but heating revealed a lower dimer stability and the monomer was detected following heating at all temperatures tested (Fig. 6, lanes 6–8).
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). However, in agreement with the RPA data, the 4G-mutant virus packaged less RNA.
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At 72 h post-transfection, a difference in RNA trafficking could be detected (Table 1). The percentages shown in Table 1 represent the level of each nucleic acid detected (the probe also detects DNA, hence the total RNA does not reach 100 %). More full-length RNA was retained in the nucleus following transfection of the 4G-mutant construct compared with the wild type. The wild-type virus showed preferential export of genomic RNA, with twice as much genomic as spliced message in the cytoplasmic fraction. Actual levels of spliced RNA were higher in the cytoplasm of cells transfected with the mutant than in those transfected with wild type; however, due to the nature of the overexpression system used, these results must be interpreted with caution.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). It has also been suggested that Rev has other effects, such as enhancement of viral encapsidation and translation (D'Agostino et al., 1992; Kaye et al., 1995).
We have demonstrated previously that the packaging signal of HIV-1 contains within it a Rev-binding motif. Mutation of adenines at positions 692 and 695 within this motif was demonstrated to affect Rev binding (Gallego et al., 2003). It was not known, however, whether this RNA–protein interaction was essential for virus replication.
We first investigated the relative affinity of the loop A–Rev interaction compared with that of RRE and Rev. We demonstrated that an excess of cold competitor RRE RNA could outcompete loop A for Rev interaction. The RRE competed successfully even at equimolar concentrations, suggesting that it had a higher affinity for the protein.
The mutation described above was introduced into an infectious molecular clone, HIVHXB2, and its protein production, RNA composition and replication ability were compared with those of the wild-type virus.
Replication of the virus was severely compromised. The substitution of two adenine residues for two guanines produced a virus with significantly delayed replication, which never reached wild-type levels.
Viral protein production of the mutant appeared to be normal, as assessed by both transient transfection of cells and virus infection of cells. There was no significant difference between the mutant and wild-type viruses.
The mutant displayed a modest genomic RNA-packaging defect. This result was confirmed by Northern blotting (Fig. 7
). One possible cause of decreased packaging would be a change in RNA structure due to the mutations that were introduced into the stem–loop 1 RNA. RNAs were generated in vitro from wild-type and mutant templates and their stability and electrophoretic characteristics were compared under non-denaturing conditions. Whilst both RNAs formed dimers, there was a difference in dimer stability, the mutant being less stable than the wild type. However, genomic RNA in the virions from the mutant was dimeric, suggesting that this was unlikely to be responsible for either the packaging defect or the striking effect on replication unless such a minor dimerization defect has a significant impact on post-entry stages of replication.
We observed a reproducible reduction in the export of genomic RNA from the nucleus in the mutant virus. Whilst it was less marked than published effects on transport caused by mutation of the RRE (Cullen, 1998), it supported the possibility that Rev has more than one point of action on the RNA. The reduced export of genomic RNA was modest but reproducible compared with that seen in the wild-type virus. Spliced mRNA was exported very efficiently in the mutant, implying that there was no reduction in availability of Rev protein to facilitate export of the other RNA species. The level of spliced RNA seen in the 4G mutant may also be consistent with the mutation favouring splicing; however, the fact that viral protein production was equivalent in the mutant also suggests that sufficient unspliced RNA was arriving at cytoplasmic ribosomes to fulfil the translation function and to produce virion particles.
It has been demonstrated that, in lentiviruses, translation to produce Gag/Pol and encapsidation can occur from the same unspliced RNA pool (Dorman & Lever, 2000). However, RNA trafficking may ultimately influence the destiny of an RNA. Swanson et al. (2004) described how the route that an mRNA takes affects the function and therefore the fate of the encoded protein. In addition, Poole et al. (2005) showed in HIV-1 that, for efficient packaging to occur, RNA and Gag had to interact at a certain point within the cell. Alterations to RNA transport could therefore affect when and where (or whether) these interactions occur and consequently result in a defect in virus encapsidation. Thus, a relatively modest global export defect may conceal a larger effect on trafficking of some of the RNA to a critical site where encapsidation may occur.
Interaction of the Rev protein with the loop A motif in the packaging signal of HIV-1 appears to be essential for optimal virus replication. In the context of the virus, a mutation in the RNA loop that caused impaired Rev binding in vitro led to a replication defect, characterized by a decrease in and loss of specificity of viral RNA encapsidation. The accompanying alteration in nuclear export of genomic RNA suggests that this defect resulted from altered RNA trafficking.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Cullen, B. R. (1998). Posttranscriptional regulation by the HIV-1 Rev protein. Semin Virol 8, 327–334.
D'Agostino, D. M., Felber, B. K., Harrison, J. E. & Pavlakis, G. N. (1992). The Rev protein of human immunodeficiency virus type 1 promotes polysomal association and translation of gag/pol and vpu/env mRNAs. Mol Cell Biol 12, 1375–1386.
Dangerfield, J. A., Hohenadl, C., Egerbacher, M., Kodajova, P., Salmons, B. & Günzburg, W. H. (2005). HIV-1 Rev can specifically interact with MMTV RNA and upregulate gene expression. Gene 358, 17–30.[CrossRef][Medline]
Dorman, N. & Lever, A. (2000). Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus. J Virol 74, 11413–11417.
Fisher, A. G., Collalti, E., Ratner, L., Gallo, R. C. & Wong-Staal, F. (1985). A molecular clone of HTLV-III with biological activity. Nature 316, 262–265.[CrossRef][Medline]
Gallego, J., Greatorex, J., Zhang, H. & 7 other authors (2003). Rev binds specifically to a purine loop in the SL1 region of the HIV-1 leader RNA. J Biol Chem 278, 40385–40391.
Greatorex, J., Gallego, J., Varani, G. & Lever, A. (2002). Structure and stability of wild-type and mutant RNA internal loops from the SL-1 domain of the HIV-1 packaging signal. J Mol Biol 322, 543–557.[CrossRef][Medline]
Harrison, G. P., Miele, G., Hunter, E. & Lever, A. M. L. (1998). Functional analysis of the core human immunodeficiency virus type 1 packaging signal in a permissive cell line. J Virol 72, 5886–5896.
Kaye, J. F. & Lever, A. M. L. (1996). trans-Acting proteins involved in RNA encapsidation and viral assembly in human immunodeficiency virus type 1. J Virol 70, 880–886.[Abstract]
Kaye, J. F., Richardson, J. H. & Lever, A. M. L. (1995). cis-Acting sequences involved in human immunodeficiency virus type 1 RNA packaging. J Virol 69, 6588–6592.[Abstract]
Laughrea, M. & Jette, L. (1994). A 19-nucleotide sequence upstream of the 5' major splice donor is part of the dimerization domain of human immunodeficiency virus 1 genomic RNA. Biochemistry 33, 13464–13474.[CrossRef][Medline]
Poole, E., Strappe, P., Mok, H.-P., Hicks, R. & Lever, A. M. L. (2005). HIV-1 Gag–RNA interaction occurs at a perinuclear/centrosomal site; analysis by confocal microscopy and FRET. Traffic 6, 741–755.[CrossRef][Medline]
Potts, B. (1990). ‘Mini’ reverse transcriptase (RT) assay. In Techniques in HIV Research, pp. 103–106. Edited by A. Aldovini & B. D. Walker. New York: Stockton Press.
Skripkin, E., Paillart, J.-C., Marquet, R., Ehresmann, B. & Ehresmann, C. (1994). Identification of the primary site of the human immunodeficiency virus type 1 RNA dimerization in vitro. Proc Natl Acad Sci U S A 91, 4945–4949.
Swanson, C. M., Puffer, B. A., Ahmad, K. M., Doms, R. W. & Malim, M. H. (2004). Retroviral mRNA nuclear export elements regulate protein function and virion assembly. EMBO J 23, 2632–2640.[CrossRef][Medline]
Received 4 November 2005;
accepted 30 May 2006.
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