1 Department of Microbiology, Immunology and Molecular Genetics, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095, USA
2 Departments of Microbiology, Immunology, and Molecular Genetics and Medicine, UCLA AIDS Institute, Los Angeles, CA 90095, USA
Correspondence
Debi P. Nayak
dnayak{at}ucla.edu
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ABSTRACT |
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INTRODUCTION |
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For therapeutic application against virus diseases, siRNA must be able to enter infected cells efficiently and inhibit an ongoing virus infection. As siRNAs are too short to induce an interferon (IFN) response in mammalian cells, siRNA-mediated interference bypasses the non-specific, IFN-mediated antivirus response (Gitlin et al., 2002; Kapadia et al., 2003
). After influenza virus infection, viral RNA (vRNA) (minus strand) is transcribed into two classes of plus strand RNAs: namely, the viral mRNA and viral complementary RNA (cRNA). Because of the complementarity of duplex siRNA strands, it could interfere directly with each class of virus-specific RNA in virus-infected cells, including vRNA, viral mRNA and viral cRNA. However, viral mRNAs are the specific target of siRNA-mediated interference (Ge et al., 2003
), as influenza vRNAs and cRNAs, bound to nucleoprotein (NP), may be protected from cleavage by RNAi machinery. Furthermore, whilst mRNAs are exported into the cytoplasm, cRNAs remain in the nucleus, thereby making mRNAs the preferred target of siRNA-mediated degradation.
siRNAs have been used recently to interfere with the replication of a number of viruses (reviewed by Ahlquist, 2002; Carmichael, 2002
; Lindenbach & Rice, 2002
; Saksela, 2003
). To investigate whether siRNA can be used to interfere with influenza virus replication, we used siRNAs from the M gene sequence of influenza virus, as its matrix (M1) protein plays a critical role in many aspects of virus replication, including virus assembly and budding (reviewed by Nayak & Hui, 2002
). The virus presents a potential target for siRNA-mediated interference and there are a number of advantages in using siRNA against influenza. Firstly, as influenza is restricted primarily to the lungs, a virus vector-based delivery system could be used effectively to reach the lungs via the intranasal route. Secondly, as influenza virus changes annually, siRNA from a conserved M1 sequence may be effective against influenza virus strains arising from either antigenic drift or antigenic shift. In this report, we have used siRNAs targeted against the M gene and show that M1 expression can be inhibited specifically in 293T cells by plasmids expressing siRNAs from a Pol III promoter. Furthermore, as lentivirus vectors with an siRNA cassette can efficiently infect and express siRNA in different cells, including stable cell lines (Abbas-Terki et al., 2002
), primary cells (Qin et al., 2003
; Stewart et al., 2003
) and fertilized eggs used in generating transgenic animals (Tiscornia et al., 2003
), we have used a lentivirus vector derived from human immunodeficiency virus (HIV)-1 to deliver M gene siRNA into MadinDarby canine kidney (MDCK) cells, which are commonly used for influenza virus propagation. Our data show that this lentivirus-mediated siRNA delivery method can be used to specifically silence the target M1, but not NP, expression and to inhibit influenza virus replication in MDCK cells.
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METHODS |
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Transfection of the siRNA expression cassette into 293T cells.
Transfection and protein pulse-labelling were performed as described previously (Hui et al., 2003a, b
). Briefly, 10 µg U6 promoter-driven siRNA cassette plasmid (pCR-M-89, pCR-M-331 or pCR-M-620) and 1 µg Pol IPol II construct of the M gene (Hoffmann et al., 2000
) were mixed with 16 µl TransIT LT-1 (Panvera), mixed with Opti-MEM-I, added to 293T cells (1x106) on a Becton Dickinson BioCoat poly-D-lysine/laminin six-well culture plate. The DNA transfection mixture was replaced with virus growth medium (VGM) 6 h later. For labelling, 293T cells at 13 h post-transfection (hpt) were starved and pulse-labelled with 100 µCi [35S]protein label (ICN Biomedicals). Cells were then lysed in radioimmunoprecipitation assay buffer, immunoprecipitated with anti-M1 antibodies (Biodesign International) and analysed by SDS-PAGE.
Lentivirus vector particle production.
All virus stocks containing VSV G protein were produced by calcium phosphate-mediated, three-plasmid transfection of 293T cells, as described previously (An et al., 2003). Briefly, 293T cells (1·5x107 cells in a 175T flask) were transfected with 5 µg pHCMV-G, 12·5 µg pCMVR8.2DVPR and 12·5 µg LVV-M-331 or RRL-U6shLuc-cPPT-PGK-EGFP-SIN and cultured in DMEM supplemented with 10 % FBS and antibiotics. Virus supernatants were collected on days 3 and 4 post-transfection, filtered through a 0·22 µm pore-size filter, ultracentrifuged (50 000 g, 1·5 h, 4 °C) and resuspended in PBS. Virus stocks were titrated by infecting 293T cells (5x104 cells in a 12-well plate) with virus dilutions in DMEM supplemented with 10 % calf serum and 8 µg hexadimethrine bromide ml1 and analysed for enhanced green fluorescent protein (EGFP) expression by flow cytometry. Titres of vector virus stocks were routinely 108 infectious units (IU) ml1.
Lentivirus vector transduction and influenza virus superinfection.
MDCK cell monolayers (1x105 cells in 35 mm dishes, 10 % confluence) were washed with PBS+ (PBS plus 0·5 mM MgCl2 and 1 mM CaCl2) and incubated with various lentivirus vectors at an m.o.i. of 10 in 300 µl DMEM supplemented with 10 % FBS, antibiotics and 8 µg hexadimethrine bromide ml1 for 2 h at 37 °C. Unadsorbed viruses were removed by washing with DMEM and cell monolayers were incubated with 2 ml DMEM supplemented with 10 % FBS and antibiotics at 37 °C. After 3 days incubation, cells were analysed for EGFP expression by flow cytometry. Approximately 80 % of cells were EGFP-positive. Lentivirus-transduced MDCK cell monolayers were infected with influenza virus (m.o.i., 0·1) in 300 µl virus dilution buffer (Hui et al., 2003b) for 1 h at 37 °C. Unadsorbed viruses were removed by washing with VGM and the superinfected cell monolayers were then incubated at 33 °C. Supernatants were harvested at 6, 8 and 10 h post-infection (p.i.) and titrated by plaque assay.
Flow cytometric analysis.
EGFP expression was analysed with a fluorescence-activated cell sorter (FACS) Calibur flow cytometer (BD Bioscience Immunocytometry Systems). Data were processed with CellQuest software (BD Bioscience Immunocytometry Systems).
Plaque assay.
Plaque assays were done as described previously (Hui et al., 2003b). Briefly, diluted virus samples were layered on MDCK monolayers (37 °C, 1 h). Cell monolayers were then overlaid with agar overlay medium, incubated at 33 °C for 3 days and plaques were counted. Data are expressed as mean±SD. The significance of the difference between values was compared by using Student's t-test; P<0·001 was considered to be significant.
EGFP fluorescence and indirect immunofluorescence.
MDCK cells (4x105) that had been grown on tissue culture chamber slides (Nunc) were transduced with lentivirus (m.o.i., 10). After 3 days, cells were superinfected with A/WSN/33 (m.o.i., 0·2). At 12 h p.i., cells were washed with PBS+, fixed in 4 % paraformaldehyde for 30 min at room temperature and then permeabilized in 0·5 % Triton X-100 for 20 min at room temperature. Cells were washed with PBS and then incubated with goat anti-M1 antibodies (1 : 30) for 1 h at 37 °C. Cells were washed three times for 10 min each in PBS and incubated with Texas red-conjugated anti-goat IgG (Santa Cruz Biotechnology) (1 : 400) for 35 min at room temperature. Cells were then washed and mounted. Slides were viewed under an Axioskop 2 fluorescence microscope (Zeiss).
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RESULTS |
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To determine whether these M gene-specific siRNAs can suppress the expression of M1 protein, 293T cells were cotransfected with plasmids expressing the M1 protein and siRNA. 293T cells were used because of their high transfection efficiency. Transfected 293T cells were pulse-labelled at 13 hpt and cell lysates were immunoprecipitated with anti-M1 antibodies. The results (Fig. 2) show that M1 protein expression decreased by 52, 70 or 97 % when cells were cotransfected with pCR-M-620 (lane 5), pCR-M-89 (lane 3) or pCR-M-331 (lane 4) plasmids, respectively. These results demonstrate that siRNAs from different regions of the M gene had a varying effect in suppressing M1 protein synthesis and that the siRNA targeting the 331351 nt region of the virus M gene (pCR-M-331) was most effective in inhibiting translation of the M1 protein.
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DISCUSSION |
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In two recent reports (Ge et al., 2003; McCown et al., 2003
), the role of siRNA in influenza virus replication has been investigated. In both reports, siRNAs against the M gene targeted to M1 mRNA had only a partial effect against influenza virus replication. Ge et al. (2003)
observed that siRNAs against M1 caused, at most, 50 % reduction of M1 mRNA. However, NP and PA gene targets were most effective at virus inhibition, implying the role of segment-specific siRNA in virus interference. On the other hand, McCown et al. (2003)
reported that, within the M gene, two siRNAs against M1 caused only partial inhibition, whereas one against M2 interfered most effectively against M2 protein expression and virus replication. Our results with M gene siRNAs differ significantly from these published reports. We found that one M gene-specific siRNA (M-331) was a potent inhibitor of both M1 protein synthesis and virus replication, whereas two other M gene siRNAs caused only partial inhibition of M1 protein expression. In addition to the different delivery systems and host cells used in these reports, our results point towards the importance of the specific sequence within the M gene that is targeted by the siRNA in gene silencing and virus interference. Poor inhibition of M1 protein expression by 5' M1 siRNA and 3' M1 siRNA (McCown et al., 2003
) was probably due to targeting of siRNAs to the M1M2 splice sites. Similarly, partial inhibition by M-39 (nt 3957) and M-731 (nt 731749) [reported here (Fig. 2
)] could be due to the proximity of these sequences to the 5' M1M2 splice donor site (nt 51 and 52) and 3' splice acceptor site (nt 739 and 740). Complex regulatory proteins that are involved in splicing may bind to these regions and, thereby, may interfere with the siRNA interaction with mRNA (Elbashir et al., 2002
). Moreover, some sequences on the viral mRNA might be buried within secondary structures or highly folded regions of the target mRNAs and may not be accessible for interaction with siRNA (Elbashir et al., 2002
). In addition, the level of production and stability of siRNAs may also affect their interfering ability.
We have also shown here that the siRNA can be delivered effectively by a lentivirus vector system and used to suppress protein expression and virus replication. In fact, siRNA has been delivered and expressed successfully by retroviruses (Devroe & Silver, 2002; Yang et al., 2003
), lentiviruses (Abbas-Terki et al., 2002
; An et al., 2003
; Matta et al., 2003
; Qin et al., 2003
; Stewart et al., 2003
; Tiscornia et al., 2003
) and adenoviruses (Xia et al., 2002
; Shen et al., 2003
) to suppress specific host protein synthesis and interfere with virus replication. The levels of inhibition of protein expression and virus replication observed in our report were similar to those reported in other studies using lentivirus vectors (An et al., 2003
; Qin et al., 2003
). Furthermore, we show that the suppression of M1 protein synthesis is specific for M1 siRNA, as NP protein expression was not inhibited by M1 siRNA and M1 protein expression was not suppressed by luciferase siRNA.
In conclusion, we have shown that sequence-specific M1 siRNA and lentivirus-mediated siRNA expression could be used to interfere with viral protein synthesis and to inhibit virus replication and, therefore, has a potential application in therapy against influenza. Results from this and other studies suggest that lentivirus vector expression of siRNA could be a useful tool in addressing the complex interaction of viral and cellular regulatory proteins that are involved in the virus life cycle. This system can also be used to determine the role of specific host proteins in virus budding. However, as the level of virus inhibition was only moderate (80 %), further optimization of conditions for choosing the targeting siRNA sequence and delivering the siRNA in virus-infected cells is needed.
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ACKNOWLEDGEMENTS |
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Received 16 December 2003;
accepted 22 March 2004.