Basic Research Laboratory, Division of Basic Sciences, National Cancer Institute, Building 37, Room 5E10, National Institutes of Health, Bethesda, MD 20892, USA1
Author for correspondence: Suresh Arya. Fax +1 301 496 5839. e-mail aryas{at}dc37a.nci.nih.gov
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Abstract |
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Introduction |
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Encapsidation of transgene RNA by a retroviral vector is governed by the packaging signal located in the leader sequence. The packaging signal of HIV-1 and HIV-2 is multipartite with subelements located upstream (exonic) and downstream (intronic) of the splice donor site in the leader sequence (Lever et al., 1989 ; Aldovini & Young, 1990
; Luban & Goff, 1994
; Garzino-Demo et al., 1995
; McBride & Panganiban, 1996
; McCann & Lever, 1997
; Arya et al., 1998
; Poeschla et al., 1998
). Inclusion of the 5' end of the gag gene is thought to enhance RNA encapsidation (Luban & Goff, 1994
; Berkowitz et al., 1995
; Parolin et al., 1994
; McBride et al., 1997
; Miller, 1997
). However, gag of HIV-1, and presumably also of HIV-2, contains inhibitory/instability or cis-acting repressive (INS/CRS) sequences. These sequences downregulate expression post-transcriptionally, in part by causing nuclear retention of the transcripts accompanied by their splicing and/or degradation. This negative effect can be overcome by providing the Rev responsive element (RRE) in cis and rev in trans (Maldarelli et al., 1991
; Schwartz et al., 1992
; Malim & Cullen, 1993
; Schneider et al., 1997
).
We have previously presented our studies on the mapping of the HIV-2 packaging signal and the requirement for helper virus-free encapsidation (Garzino-Demo et al., 1996; Arya et al., 1998 ; Sadaie et al., 1998
). We report here that despite the provision of the required sequence elements, only a small fraction of the HIV-2 vector RNA was encapsidated by HIV-2. The vector RNA appeared to be spliced before it could be encapsidated. Modification of the splice donor resulted in increased packaging of the vector RNA. Vectors with or without the internal cytomegalovirus (CMV) early gene promoter were competent in transducing target cells. An unconcentrated vector titre of 105 transducing units (TU)/ml was readily obtained for vectors containing the neo or the green fluorescent protein (GFP) transgene. Transduction-competent vectors containing a number of other transgenes representing different disease entities and potential target cells have also been created.
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Methods |
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The wild-type HIV-2 (ROD) proviral clone (pROD) and its truncated version pROD(SD36) have been described previously (Arya et al., 1998 ). Clone pROD(SD36) is a deletion mutant of pROD where the subelements of the packaging signal located upstream (nt 306459) and downstream (nt 482538) of the splice donor have been deleted, but the splice donor site itself is preserved. It is replication defective, but produces all the proteins needed for packaging (Arya et al., 1998
). Clone pROD(SD36/EM) was created by deleting nt 63706640 of the env region from the clone pROD(SD36) and substituting them with a synthetic linker with multiple stop codons in all three open reading frames in the amino terminus region of the env gene. Clones pCM-ROD(SD36) and pCM-ROD(SD36/EM) were derivative clones of pROD(SD36) and pROD(SD36/EM) where the 5'-LTR had been substituted with the CMV promoter and the 3'-LTR with a heterologous pA signal. The VSV-G clone was kindly provided by Inder Verma of the Salk Institute (La Jolla, CA, USA) with additional advice from Didier Trono, now at the University of Geneva (Naldini et al., 1996
). Flossie Wong-Staal of the University of California, San Diego, kindly provided the HIV-2 vector clone termed pLAGC (Poeschla et al., 1998
).
DNA-mediated transfection.
Human epithelioid 293T cells were transfected by the calcium phosphate protocol (Arya & Gallo, 1988 ; Arya, 1993
). Typically, 1x106 cells from subconfluent monolayer culture were transfected with 10 µg of the vector DNA and 410 µg of the cotransfecting DNA. Cultures were incubated with calcium-DNA aggregates overnight, washed and reincubated with fresh medium. Cells and culture supernatant were harvested 3 days after transfection.
RNA analysis.
For cellular RNA, transfected cells were lysed with Trizol reagent (Life Technologies) and RNA was recovered by isopropanol precipitation. RNA was further purified by extraction with phenolchloroform and re-precipitated with ethanol. It was then digested with DNase in excess and re-extracted and ethanol precipitated (Arya et al., 1998 ). Cytoplasmic RNA was prepared by lysing the cells with Triton X-100 in a hypotonic buffer and removing the nuclei by centrifugation. The cytoplasmic extract was mixed with Trizol reagent, extracted with chloroform and RNA was precipitated with ethanol. The RNA was further purified by DNase treatment, phenolchloroform extraction and ethanol precipitation. The recovery of RNA was quantified by absorbance at 260 nm. Viral RNA was prepared from partially purified virus particles. Culture supernatant was layered on a column of 20% (v/v) glycerol in a Beckman SW41 rotor tube and centrifuged at 32000 r.p.m. for 1·5 h (Arya et al., 1998
). The pellet was lysed with Trizol reagent and viral RNA extracted and DNase treated as described above. Final RNA was dissolved in 100 µl of 10 mM TrisHCl (pH 7·8)1 mM EDTA.
The abundance of vector RNA was estimated by slot-blot hybridization. Aliquots of cellular RNA (20 µg) or viral RNA (50 µl) were denatured and two dilutions (1:1 and 1:5) were slot-blotted and hybridized with the 32P-labelled neo probe. For Northern blot analysis, about 20 µg of cellular RNA was electrophoresed in denaturing formamideagarose gels, transferred to a nylon membrane by electroblotting and blot-hybridized with the neo probe. The filters were exposed to an X-ray film and subsequently to an imaging screen. The abundance of RNA was quantified by integrating the intensity of the bands with a PhosphorImager (Molecular Dynamics). Most results reported here represent multiple independent transfections done with cells at different passages.
p27 antigen capture assay.
The level of p27 viral antigen was determined with a commercially available ELISA kit (Coulter). Briefly, serial dilutions of the vector sample were lysed and applied to the mouse anti-SIV p27 antibody-coated wells of the microtitre plate. After washing, biotinylated anti-mouse antibody was added to the wells and binding quantified with the streptavidin-conjugated horseradish peroxidase reaction.
Transduction and vector titration.
The titre of the vector was determined by transducing 293 or 293T cells. Concentrated vector was obtained by pelleting the particles from the supernatant of the transfected culture by two cycles of ultracentrifugation at 50000 g for 1 h and resuspension in 1/2001/500 of the original volume. Serial dilutions of the supernatant with or without prior concentration were applied to the target cells to be transduced. Cultures of about 5x104 cells were incubated with the serial dilutions of the vector along with 8 µg/ml polybrene for 1620 h. To score for neo gene expression, cultures were incubated with G418 until there were no viable cells in the control cultures (1216 days) and the surviving colonies were counted. To score for GFP gene expression, transduced cultures were washed, cells detached from the surface with mild EDTA treatment, fixed with paraformaldehyde and analysed for GFP expression by flow cytometry.
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Results |
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The reference vector pSGT-5(RRE/RN) contained 300 nt of envelope sequence corresponding to RRE. With the objective of improving the vector RNA encapsidation, we tested the encapsidation of RNA transcribed by the second vector that contained 528 nt of RRE [pSGT-5(RRE2/RN)]. This vector was thus cotransfected with the helper virus clone pROD(SD36) or with pROD(SD36/EM) transcomplemented with the native envelope or VSV-G. When the particle and cellular RNAs were analysed by hybridization with the neo probe, intense signals were obtained for the cellular RNAs and only faint signals for the particle RNA (Fig. 2). These results showed that the vector RNA was abundantly expressed in the transfected cells but only a minor amount of it was encapsidated into particles.
Because the vectors contained a strong splice donor within the packaging signal, we wondered if the poor encapsidation was due to the excessive splicing of the vector RNA. We therefore mutated the splice donor away from the consensus sequences and created the vector pSGT-5(SDM/RRE/RN). The results of the transfection of this vector with different helper virus clones are included in Fig. 2. Hybridization of the cellular RNA with the neo probe showed that this vector synthesized abundant quantities of vector RNA in transfected cells and this abundance was roughly equivalent to the RNA synthesized by the reference vector pSGT-5(RRE/RN) or its RRE2 derivative. Apparently, the mutation of the splice donor did not affect the expression of the vector. However, there was a marked effect on encapsidation. The particle RNA gave clear hybridization signals showing that the vector RNA synthesized by the mutant vector was encapsidated. Comparison of the hybridization signals for the particle RNA suggested that the mutant vector [pSGT-5(SDM/RRE/RN)] encapsidated 7 to 10 times more RNA than the reference vector [pSGT-5(RRE/RN)] (Table 2
). This difference in encapsidation was observed regardless of whether the helper virus carried the envelope in cis [pROD(SD36)] or in trans [pROD(SD36/EM) plus pCM-ENV] or was pseudotyped with VSV-G [pROD(SD36/EM) plus pCM-VSV-G] (Fig. 2
, Table 2
). Both clones produced similar amounts of virus particles as judged by the supernatant p27 levels (Table 1
).
We also analysed the abundance of the vector RNA in the cytoplasmic fraction of the transfected cells (Fig. 3). As with the previous analysis, hybridization of the cytoplasmic RNA from cells transfected with different vectors gave signals of equivalent intensities with the neo probe. The intensity of the hybridization signal of the particle RNA for the mutant vector pSGT-5(SDM/RRE/RN) was about 8- to 10-fold the intensity for the reference vector pSGT-5(RRE/RN). The data displayed in Fig. 3
were obtained with the packaging clone pCM-ROD(SD36). Similar results were obtained with the packaging clones pCM-ROD(SD36/EM) plus pCM-VSV-G.
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Discussion |
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We then considered the possibility that the vector RNA was being excessively spliced, affecting its packaging. Splicing of viral RNA, a normal process in virus replication, involves the major splice donor in the leader and a number of splice acceptors downstream. It apparently is a controlled process in virus replication where sufficient viral genomic RNA is left unspliced to be packaged for the production of the progeny virus. In our vectors, the splice donor, located within the packaging signal, could utilize a cryptic splice acceptor resulting in reduction of the amount of vector RNA available for packaging. The results with the splice donor mutant vector support this possibility. The titre of the vector with the splice donor mutation was uniformly higher than the titre of the unmutated vector. This was the case for both Neo and GFP vectors. However, other explanations are possible. For HIV-1 and equine infectious anaemia virus, the splice donor itself has been reported to have a negative effect on viral gene expression, which is overcome by RevRRE (Tan et al., 1996 ; Borg et al., 1997
). The RevRRE was functional in our vectors. Thus, this mechanism may not be important here. In addition, the introduction of a splice-donor as a part of a synthetic intron had only a minimal effect on vector titre. For HIV-1 vectors, others have reported the curious observation that a deletion of the splice donor markedly reduces vector RNA expression but has no effect on vector titre (Cui et al., 1999
). One interpretation of this result is that with the HIV-1 vector, RNA is always in excess such that even when its abundance in the packaging cell is reduced because of splice donor mutation, there is no effect on vector RNA encapsidation or titre.
Successful packaging of some HIV-2 vectors with an intact splice donor has been reported previously (McCann & Lever, 1997 ; Poeschla et al., 1998
). We do not know if a modification of the splice donor in those vectors also will not enhance encapsidation and vector titre. When we directly compared the vector with an intact splice donor provided by F. Wong-Staal and colleagues with our vector with the mutated splice donor, the titre of the vector with the splice donor mutation was 15-to 30-fold higher. Thus, the splice donor mutation was advantageous. Furthermore, in situations where vector mobilization is desired, such as gene therapy of HIV infection, splice donor-mutant vector will have an added advantage. It will curtail vector RNA splicing and promote encapsidation and vector production. This will occur only in HIV-infected cells and not in uninfected cells as only infected cells can provide the packaging machinery in trans.
We think it is important to recognize the significant role of the splice donor in vector design. With a splice donor in place in the vector, a cryptic splice acceptor brought in with a transgene, or sequences surrounding it, will undermine the integrity of the vector and reduce the titre by a log or more. While the importance of a log difference may be debatable, it would seem desirable to take advantage of this observation in designing vectors, especially considering that low titre is one of the major shortcomings of the presently designed lentiviral vectors. With the splice donor mutated, we have generated HIV-2 vectors with a number of transgenes representing several disease models, including aromatic amino acid decarboxylase (AADC) gene for Parkinsons disease, Bax gene for tumour apoptosis, -galactosidase A gene (AGA) for Fabry disease and the chemokine (RANTES) gene for HIV infection. These vectors successfully transduced appropriate target cells, thus setting the stage for in vivo studies.
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Acknowledgments |
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References |
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Received 25 August 2000;
accepted 12 October 2000.