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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INTRODUCTION |
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METHODS |
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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 BamHIXhoI 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 862898 were deleted from the SIV leader sequence using the mutagenic oligonucleotide 5'-AGTGAGAAGAACTCCACCACGACGGACTGC-3'. For
P2, positions 915947 were deleted using the mutagenic oligonucleotide 5'-CCAACCACGACGGAGGCGTGAGGAGCG-3'. For
P3, positions 9951045 were deleted using the mutagenic oligonucleotide 5'-CGGTTGCAGGTAAGTGCAAGTGGGAGATGGGC-3'. For
P4, positions 10111042 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 pRS
envSL using the BamHI/XhoI sites to create the deletion mutants pRS
envSL
P1, pRS
envSL
P2, pRS
envSL
P3 and pRS
envSL
P4. 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 6072 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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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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DISCUSSION |
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Aldovini, A. & Young, R. A. (1990). Mutations of RNA and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus. J Virol 64, 19201926.[Medline]
Bender, M. A., Palmer, T. D., Gelinas, R. E. & Miller, A. D. (1987). Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region. J Virol 61, 16391646.[Medline]
Berkhout, B. & van Wamel, J. L. B. (1996). Role of the DIS hairpin in replication of human immunodeficiency virus type 1. J Virol 70, 67236732.[Abstract]
Berkowitz, R. D., Hammarskjold, M. L., Helga-Maria, C., Rekosh, D. & Goff, S. P. (1995). 5' regions of HIV-1 RNAs are not sufficient for encapsidation: implications for the HIV-1 packaging signal. Virology 212, 718723.[CrossRef][Medline]
Berkowitz, R., Fisher, J. & Goff, S. P. (1996). RNA packaging. Curr Top Microbiol Immunol 214, 177218.[Medline]
Buck, C. B., Shen, X., Egan, M. A., Pierson, T. C., Walker, C. M. & Siliciano, R. F. (2001). The human immunodeficiency virus type 1 gag gene encodes an internal ribosome entry site. J Virol 75, 181191.
Clavel, F. & Orenstein, J. M. (1990). A mutant of human immunodeficiency virus with reduced RNA packaging and abnormal particle morphology. J Virol 64, 52305234.[Medline]
Das, A. T., Klaver, B., Klasens, B. I. F., van Wamel, J. L. B. & Berkout, B. (1997). A conserved hairpin motif in the R-U5 region of the human immunodeficiency virus type 1 RNA genome is essential for replication. J Virol 71, 23462356.[Abstract]
De Guzman, R. N., Wu, Z. R., Stalling, C. C., Pappalardo, L., Borer, P. N. & Summers, M. F. (1998). Structure of the HIV-1 nucleocapsid protein bound to the SL3 psi-RNA recognition element. Science 279, 384388.
Garzino Demo, A., Gallo, R. C. & Arya, S. K. (1995). Human immunodeficiency virus type 2 (HIV-2): packaging signal and associated negative regulatory element. Hum Gene Ther 6, 177184.[Medline]
Griffin, S. D. C., Allen, J. F. & Lever, A. M. L. (2001). The major human immundeficiency virus type 2 (HIV-2) packaging signal is present on all HIV-2 RNA species: cotranslational RNA encapsidation and limitation of Gag protein confer specificity. J Virol 75, 1205812069.
Guan, Y., Whitney, J. B., Diallo, K. & Wainberg, M. A. (2000). Leader sequences downstream of the primer binding site are important for efficient replication of simian immunodeficiency virus. J Virol 74, 88548860.
Guan, Y., Diallo, K., Whitney, J. B., Liang, C. & Wainberg, M. A. (2001a). An intact U5-leader stem is important for efficient replication of simian immunodeficiency virus. J Virol 75, 1192411929.
Guan, Y., Whitney, J. B., Liang, C. & Wainberg, M. A. (2001b). Novel, live attenuated simian immunodeficiency virus constructs containing major deletions in leader RNA sequences. J Virol 75, 27762785.
Guesdon, F. M. J., Greatorex, J., Rhee, S. R., Fisher, R., Hunter, E. & Lever, A. M. L. (2001). Sequences in the 5' leader of MasonPfizer monkey virus which affect viral particle production and genomic RNA packaging: development of MPMV packaging cell lines. Virology 288, 8188.[CrossRef][Medline]
Katz, R. A., Terry, R. W. & Skalka, A. M. (1986). A conserved cis-acting sequence in the 5' leader of avian sarcoma virus RNA is required for packaging. J Virol 59, 163167.[Medline]
Kaye, J. F. & Lever, A. M. L. (1999). Human immunodeficiency virus types 1 and 2 differ in the predominant mechanism used for selection of genomic RNA for encapsidation. J Virol 73, 30233031.
Lever, A., Gottlinger, H., Haseltine, W. & Sodroski, J. (1989). Identification of a sequence required for efficient packaging of human immunodeficiency virus type 1 RNA into virions. J Virol 63, 40854087.[Medline]
Linial, M. L. & Miller, A. D. (1990). Retroviral RNA packaging: sequence requirements and implications. In Retroviruses Strategies of Replication, pp. 125152. Edited by R. Swanstrom & P. K. Vogt. New York: Springer-Verlag.
Linial, M., Medeiros, E. & Hayward, W. S. (1978). An avian oncovirus mutant (SE 21Q1b) deficient in genomic RNA: biological and biochemical characterization. Cell 15, 13711381.[Medline]
McCann, E. M. & Lever, A. M. L. (1997). Location of cis-acting signals important for RNA encapsidation in the leader sequence of human immunodeficiency virus type 2. J Virol 71, 41334137.[Abstract]
Ohlmann, T., Lopez-Lastra, M. & Darlix, J. L. (2000). An internal ribosome entry segment promotes translation of the simian immunodeficiency virus genomic RNA. J Biol Chem 275, 1189911906.
Parolin, C., Dorfman, T., Palu, G., Gottlinger, H. & Sodroski, J. (1994). Analysis in human immunodeficiency virus type 1 vectors of cis-acting sequences that affect gene transfer into human lymphocytes. J Virol 68, 38883895.[Abstract]
Rizvi, T. A. & Panganiban, A. T. (1993). Simian immunodeficiency virus RNA is efficiently encapsidated by human immunodeficiency virus type 1 particles. J Virol 67, 26812688.[Abstract]
Rud, E. W., Cranage, M., Yon, J., Quirk, J., Ogilvie, L., Cook, N., Webster, S., Dennis, M. & Clarke, B. E. (1994). Molecular and biological characterization of simian immunodeficiency virus macaque strain 32H proviral clones containing nef size variants. J Gen Virol 75, 529543.[Abstract]
Sadaie, M. R., Zamani, M., Whang, S., Sistron, N. & Arya, S. K. (1998). Towards developing HIV-2 lentivirus-based retroviral vectors for gene therapy: dual gene expression in the context of HIV-2 LTR and Tat. J Med Virol 54, 118128.[CrossRef][Medline]
Schnell, T., Foley, P., Wirth, M., Munch, J. & Uberla, K. (2000). Development of self-inactivating, minimal lentivirus vector based on simian immunodeficiency virus. Hum Gene Ther 11, 439447.[CrossRef][Medline]
Zeffman, A., Hassard, S., Varani, G. & Lever, A. M. L. (2000). The major HIV-1 packaging signal is an extended bulged stem loop whose structure is altered on interaction with the Gag polyprotein. J Mol Biol 297, 877893.[CrossRef][Medline]
Received 21 February 2003;
accepted 26 May 2003.