probes for NS, G and M protein mRNAs were obtained from Dr C. Samuel, ..... Wickstrom, E. L., Bacon, T.A., Gonzalez, A., Freeman, D.L., Lyman, D.L. and ...
Volume 17 Number 22 1989
Nucleic Acids Research
Antiviral activity and possible mechanisms of action of oligonucleotides-poly(L-lysine) conjugates targeted to vesicular stomatitis virus mRNA and genonic RNA Genevieve Degols, Jean-Paul Leonetti, Corinne Gagnor, Marc Lemaitre* and Bernard Lebleu
Laboratoire de Biochimie des Proteines, UA CNRS 1191, Universite des Sciences et Techniques du Languedoc, Place E. Bataillon, 34060 Montpellier cedex, France Received June 30, 1989; Revised September 14, 1989; Accepted October 17, 1989
ABSTRACT Synthetic oligonucleotides (oligomers) complementary to vesicular stomatitis virus (VSV) N protein mRNA have specific antiviral properties at concentrations lower than 1 AM when they are covalently linked to poly(L-lysine) (PLL). Since it is generally postulated that antisense oligomers act at the translational level, oligomers with potential targets on VSV viral mRNA and/or genomic RNA have been tested here. In vitro translation experiments in reticulocyte lysates, in vitro transcription experiments with permeabilized viruses, measurement of viral RNA transcription and accumulation in VSV infected cells, and antiviral experiments demonstrate in our model that antisense oligomers probably also act at other levels. Difficulties in the choice of the most effective antisense oligomer targets are also discussed.
INTRODUCTION Oligodeoxyribonucleotides (which will be referred as oligomers throughout) of relatively short length (11 to 20-mer range are most frequently used) specifically hybridize to complementary DNA or RNA sequences. During the past five years their antiviral potential has been largely reported, in attempts to design novel and more specific antiviral drugs. These synthetic oligomers have been demonstrated to inhibit the multiplication of various viruses including Rous sarcoma virus (1), vesicular stomatitis virus (2,3), herpes simplex virus (4), influenza virus (5) and human immunodeficiency virus type 1 (6,7 and references therein). However this antisense approach has to cope with several problems, including the sensitivity of oligomers to nucleases and their poor penetration into cells. To overcome these restrictions different modifications of oligomers have been described (for a review see (8)). Several classes of modified oligomers have been chemically synthesized, including alkylphosphotriester, methylphosphonate, and phosphorothioate oligomers. Such analogues are more resistant to nucleases and have been shown to specifically inhibit viral protein synthesis as well as to prevent viral replication in cell cultures. Other approaches (reviewed in (8)), as for instance the introduction at the 5' or the 3' end of the oligomer of free radical-generating groups, alkylating, intercalating or photoactivable agents provide also interesting prospects. An alternative method for the delivery of antisense sequences into intact cells has been worked out in our laboratory which consists in the covalent conjugation of oligomers to poly(L-lysine) (PLL) (3). PLL is a well known drugs or macromolecules carrier (9), also used in in vivo studies to increase the efficiency of double stranded RNA interferon inducers in ternary complex with carboxymethylcellulose (10). Our first experiments with a 15-mer oligomer complementary to the 5' end of N protein mRNA of VSV, demonstrated that 9341
Nucleic Acids Research concentrations lower than 1 AM (that is one to two order of magnitude lower than most previously published data) efficiently inhibited the accumulation of VSV proteins and the production of infectious viruses in L929 cells (3) . In this paper, we extend our analysis to the use of oligomer-PLL conjugates directed against other targets of the VSV system. As several of them strongly inhibit virus output, we tried to localize, with in vitro translation experiments in reticulocyte lysates, in vitro transcription experiments with permeabilized viruses, measurement of viral RNA transcription and accumulation in VSV infected cells, the steps in the viral multiplication cycle affected by these oligomer-PLL conjugates.
MATERIALS AND METHODS Materials Media for cell culture were obtained from Eurobio (Paris) and sera from Boehringer. PLL (14,000 mean MW) was supplied by Sigma, reticulocyte lysate by Amersham and oligomers synthesis reagents by Milligen. Cell cultures and virus L929 cells were grown in minimal essential medium supplemented with 10% (v/v) fetal calf serum. VSV virus (Indiana strain) was grown in L929 cells and titrated by a dilution method. This method is easier to implement than plaque titration assays and gives similar results (11). Differences in VSV titer of more than 0.5 log should be considered as highly significant. Oligomers synthesis and covalent linkage to PLL Oligomers were synthesized on a riboadenosine derivatized support using a Biosearch Cyclone automatic DNA synthesizer and purified by reversed phase chromatography. Covalent linkage of oligomers to PLL through a N-morpholine ring was achieved by periodic acid oxidation and borocyanohydride reduction of the 3' end ribose, as previously described (12). Antiviral experiments Cells (2 x 105/well in 24 wells tissue culture plate) were generally incubated for 2 hours with oligomer-PLL conjugates at 1 ,uM concentration, and infected with VSV at a multiplicity of infection (m.o.i.) of 1. The cells were frozen at -20°C 18 hours after infection, and virus was titrated as described above. Translation of VSV mRNA in rabbit reticulocyte lysates Cell-free lysate translation experiments were performed as described before (13). Commercial template-free rabbit reticulocyte lysate was programmed with a mixture of mRNAs coding for VSV NS, N and M proteins. Oligomers were added at 200 nM, a concentration representing roughly a 1000 fold molar excess to mRNA concentration. 20 U/ml bacterial RNase H was added as indicated. Incubations with [35S] Met at 37°C were performed for 1 hour. Protein distributions were analyzed by SDS-PAGE gels (14), fluorography and autoradiography. Synthesis of VSV mRNA by purified virions VSV transcription by Triton-permeabilized virions purified on sucrose gradients was performed as described by Rose et al. (15). The reaction mixture contained 2 AM oligomer as mentioned ie,. an oligomer/virion ratio of 1000/1. Reactions were stopped after 2 hours. RNA samples were isolated as described (15) and analyzed on a 4% (w/v) polyacrylamide-7M urea gel (16). Their distribution in the gels was revealed by autoradiography. 9342
Nucleic Acids Research N
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Analysis of VSV related RNA Cytoplasmic RNAs were prepared according to Maniatis et al. (16) from VSV-infected cells at 5 hours post-infection. To analyze primary transcripts of VSV, 100 fig/ml cycloheximide was added 15 min before infection. For Northern blot analysis, RNA samples were denatured and analyzed by electrophoresis in a 1 % (w/v) agarose gel in the presence of 2.2 M formaldehyde. After electrophoresis, the RNAs were transferred by capillarity to a nylon filter (Hybond-N) with 20 x SSC (16) as the transfer buffer. The Northern blot was prehybridized 8 hours in 50% (w/v) formamide, 4 x SSC, 5 x Denhardt solution (16), 1% (w/v) sodium dodecyl sulfate (SDS), 0.5 mg/ml sonicated salmon sperm DNA and 25 mM phosphate buffer pH 6.8. Hybridization was carried out for 48 hours in the presence of [32p] labelled VSV cDNA probes. After hybridization filters were washed 4 times with 2 x SSC, 0.1 % (w/v) SDS at room temperature and then twice with 0.2 x SSC, 0.1 % (w/v) SDS at 55°C. Northern blots were autoradiographed with Kodak Xomat films in the presence of intensifying screens. The cDNA probe for N protein mRNA was obtained from Dr S. Dezel6e, Laboratoire de G6netique des Virus , CNRS, Gif sur Yvette; cDNA probes for NS, G and M protein mRNAs were obtained from Dr C. Samuel, U. Calif. Santa Barbara. Plasmids were amplified and isolated as described by Maniatis et al. (16). RESULTS
J.Antiviral activity of oligomer-PLL conjugates directed towards various targets on VSV mRNA and genomic RNA Most studies so far have defined the 5' untranslated sequences or sequences overlapping the initial codon of mRNA as the most effective in translation inhibition experiments (as 9343
Nucleic Acids Research
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