Short hairpin-loop-structured oligodeoxynucleotides reduce HSV-1 replication
© Falkenhagen et al; licensee BioMed Central Ltd. 2009
Received: 25 March 2009
Accepted: 27 April 2009
Published: 27 April 2009
The Herpes simplex virus (HSV) is known as an infectious agent and widespread in the human population. The symptoms of HSV infections can range from mild to life threatening, especially in immune-compromised individuals. HSV infections are commonly treated with the guanosine analogue Aciclovir, but reports of resistance are increasing. Efforts are made to establish single-stranded antisense oligodeoxynucleotides (as) and small interfering ribonucleic acids (siRNAs) for antiviral treatment. Recently, another class of short interfering nucleic acids, partially double-stranded hairpin loop-structured 54 mer oligodeoxynucleotides (ODNs), was shown to allow hydrolysis of HIV RNA by binding to the viral RNA. This leads to a substrate for the viral RNase H. To assess the potential of such ODNs for inhibition of HSV-1 replication, five partially double-stranded ODNs were designed based on the sequences of known siRNAs against HSV-1 with antiviral activity. Three of them are directed against early and two against leaky late genes. Primary human lung fibroblasts, MRC-5, and African green monkey kidney cells, Vero, were transfected with ODNs and subsequently infected. The effect on HSV-1 replication was determined by analyzing the virus titer in cell culture supernatants by quantitative PCR and plaque assays. An inhibitory effect was observed with all five selected ODNs, with two cases showing statistical significance in both cell types. The observed effect was sequence-specific and dose dependent. In one case the ODN was more efficient than a previously described siRNA directed against the same target site in the mRNA of UL5, a component of the helicase/primase complex. HSV-1 virions and ODNs can be applied simultaneously without transfection reagent, but at a 50-fold higher concentration to Vero cells with similar efficiencies. The results underline the potential of partially double-stranded hairpin loop-structured ODNs as antiviral agents.
The sequences of the antisense strands of the ODNs used here were chosen on the basis of known siRNA sequences, recently shown to significantly inhibit HSV replication [10, 11]. The structure, sequences, and target sites of the ODNs are shown in figure 1B. ODN 5, 29, and 30 target the mRNA of the E genes UL 5, UL 29, and UL 30 essential for DNA synthesis . UL 5 encodes a component of the helicase/primase complex, UL 29 a single strand binding protein and UL 30 the DNA polymerase. ODN 48 and 48 G target the mRNA of the LL gene UL 48, which encodes the virion associated VP16 needed in progeny virions for the transcriptional activation of IE genes in the next round of infection . As controls, we used different oligodeoxynucleotides without any sequence similarity to the HSV-1 genome (figure 1C) or phosphate buffered saline (PBS). The ODNs used were protected against nucleases by thioate modifications of the three terminal nucleotides at each end and in the T4 linker as described [6, 14].
We have chosen African green monkey kidney (Vero) cells and human embryonic lung fibroblast (MRC-5) to demonstrate the effect of the ODNs on HSV type 1 (HSV-1) replication in vitro. Both cell lines are permissive to HSV-1 strain McIntyre infection. Vero cells are defective in interferon production and HSV infections of monolayers produce a clear plaque phenotype. The MRC-5 cells were chosen to confirm the results in a primary human cell line.
To examine whether the effect is reproducible in other cell lines such as a primary human cell-line, we transfected MRC-5 cells with the ODNs at a final concentration of 50 nM. The cells were infected 5 h post transfection at a moi of 0.001 for 1 h and the viral titer was assayed 24 h after infection by qRT-PCR. HSV-1 replication was only impaired in cells treated with the HSV-specific ODNs (figure 3B). The controls ODN Cont and asS2 were negative. The effect was statistically significant for ODN 5, 30 and 48 G. The ODNs did not exhibit a cytotoxic effect in MRC-5 cells (data not shown). A direct comparison between ODN5 and an siRNA is shown for Vero cells in figure 3C.
Reduction of HSV-1 DNA levels upon treatment with ODNs in Vero and MRC-5 cells.
Average fold reduction of the mean (median)
Transfection with subsequent infection
Antisense ODNs and small interfering RNAs are established antiviral agents that have been shown to reduce HSV replication [10, 11, 18]. Here we are demonstrating that a new class of oligodeoxynucleotides – partially double-stranded hairpin loop-structured ODNs with one strand completely complementary to the target mRNA – can reduce HSV-1 replication in vitro. The result is consistent with previous studies reporting that hairpin loop-structured ODNs have an antiviral effect on HIV-1 and Influenza virus [2–9, 19]. The modes of action of the ODNs used in this study may be due to steric hindrance of ribosomes and hybridization to the corresponding mRNA thereby creating a substrate for cellular RNases H, comparable to the modes of action of single-stranded ODNs . In a direct comparison with siRNAs and ODNs in Vero cells both oligonucleotides were differentially effective, e.g. the most effective ODN 5 in this study was 2.5-fold more effective than the corresponding siRNA-UL5.2  (figure 3C), whereas ODN 29 was 2-fold less effective than its analog (data not shown). This suggests that different target sites might have preferences for particular types of oligonucleotides. Furthermore, the second strand linked to the antisense strand can modulate the effect in a positive or negative manner by affecting stability, accessibility, localization or uptake of the ODN. Overall, this study underlines the potential of partially double-stranded hairpin loop-structured ODNs as antiviral agents.
We thank Dr. Walter Bossart for providing HSV-1, cells, primers and probes, and Shan Qiao for performing some of the infection experiments.
- Roizman B, Knipe DM, Whitley R: Herpes Simplex Viruses. In Field's Virology. Volume 2. Fifth edition. Edited by: Knipe DM, Howley PM, et al. Lippincott, Williams & Wilkins, Philadelphia, PA; 2007:2501-601.Google Scholar
- Heinrich J, Mathur S, Matskevich AA, Moelling K: Oligonucleotide-mediated retroviral RNase H activation leads to reduced HIV-1 titer in patient-derived plasma. AIDS 2009, 23: 213-221. 10.1097/QAD.0b013e32831c5480View ArticlePubMedGoogle Scholar
- Volkmann S, Jendis J, Frauendorf A, Moelling K: Inhibition of HIV-1 reverse transcription by triple-helix forming oligonucleotides with viral RNA. Nucleic Acids Res 1995, 23: 1204-1212. 10.1093/nar/23.7.1204PubMed CentralView ArticlePubMedGoogle Scholar
- Jendis J, Strack B, Volkmann S, Boni J, Molling K: Inhibition of replication of fresh HIV type 1 patient isolates by a polypurine tract-specific self-complementary oligodeoxynucleotide. AIDS Res Hum Retroviruses 1996, 12: 1161-1168. 10.1089/aid.1996.12.1161View ArticlePubMedGoogle Scholar
- Jendis J, Strack B, Moelling K: Inhibition of replication of drug-resistant HIV type 1 isolates by polypurine tract-specific oligodeoxynucleotide TFO A. AIDS Res Hum Retroviruses 1998, 14: 999-1005. 10.1089/aid.1998.14.999View ArticlePubMedGoogle Scholar
- Moelling K, Abels S, Jendis J, Matskevich A, Heinrich J: Silencing of HIV by hairpin-loop-structured DNA oligonucleotide. FEBS Lett 2006, 580: 3545-3550. 10.1016/j.febslet.2006.05.033View ArticlePubMedGoogle Scholar
- Matskevich AA, Ziogas A, Heinrich J, Quast SA, Moelling K: Short partially double-stranded oligodeoxynucleotide induces reverse transcriptase/RNase H-mediated cleavage of HIV RNA and contributes to abrogation of infectivity of virions. AIDS Res Hum Retroviruses 2006, 22: 1220-1230. 10.1089/aid.2006.22.1220View ArticlePubMedGoogle Scholar
- Matzen K, Elzaouk L, Matskevich AA, Nitzsche A, Heinrich J, Moelling K: RNase H-mediated retrovirus destruction in vivo triggered by oligodeoxynucleotides. Nat Biotechnol 2007, 25: 669-674. 10.1038/nbt1311View ArticlePubMedGoogle Scholar
- Wittmer-Elzaouk L, Jung-Shiu J, Heinrich J, Moelling K: Retroviral self-inactivation in the mouse vagina induced by short DNA. Antiviral Res 2009, 82: 22-28. 10.1016/j.antiviral.2009.01.002View ArticlePubMedGoogle Scholar
- Palliser D, Chowdhury D, Wang QY, Lee SJ, Bronson RT, Knipe DM, Lieberman J: An siRNA-based microbicide protects mice from lethal herpes simplex virus 2 infection. Nature 2006, 439: 89-94. 10.1038/nature04263View ArticlePubMedGoogle Scholar
- Zhang YQ, Lai W, Li H, Li G: Inhibition of herpes simplex virus type 1 by small interfering RNA. Clin Exp Dermatol 2008, 33: 56-61.PubMedGoogle Scholar
- Challberg MD: A method for identifying the viral genes required for herpesvirus DNA replication. Proc Natl Acad Sci USA 1986, 83: 9094-9098. 10.1073/pnas.83.23.9094PubMed CentralView ArticlePubMedGoogle Scholar
- Herrera FJ, Triezenberg SJ: VP16-dependent association of chromatin-modifying coactivators and underrepresentation of histones at immediate-early gene promoters during herpes simplex virus infection. J Virol 2004, 78: 9689-9696. 10.1128/JVI.78.18.9689-9696.2004PubMed CentralView ArticlePubMedGoogle Scholar
- Tang JY, Temsamani J, Agrawal S: Self-stabilized antisense oligodeoxynucleotide phosphorothioates: properties and anti-HIV activity. Nucleic Acids Res 1993, 21: 2729-2735. 10.1093/nar/21.11.2729PubMed CentralView ArticlePubMedGoogle Scholar
- Pevenstein SR, Williams RK, McChesney D, Mont EK, Smialek JE, Straus SE: Quantitation of latent varicella-zoster virus and herpes simplex virus genomes in human trigeminal ganglia. J Virol 1999, 73: 10514-10518.PubMed CentralPubMedGoogle Scholar
- Crumpacker CS, Schaffer PA: New anti-HSV therapeutics target the helicase-primase complex. Nat Med 2002, 8: 327-328. 10.1038/nm0402-327View ArticlePubMedGoogle Scholar
- Biswas S, Jennens L, Field HJ: The helicase primase inhibitor, BAY 57–1293 shows potent therapeutic antiviral activity superior to famciclovir in BALB/c mice infected with herpes simplex virus type 1. Antiviral Res 2007, 75: 30-35. 10.1016/j.antiviral.2006.11.006View ArticlePubMedGoogle Scholar
- Kmetz ME, Ceruzzi M, Schwartz J: Vmw65 phosphorothioate oligonucleotides inhibit HSV KOS replication and Vmw65 protein synthesis. Antiviral Res 1991, 16: 173-184. 10.1016/0166-3542(91)90023-KView ArticlePubMedGoogle Scholar
- Kwok T, Helfer H, Alam MI, Heinrich J, Pavlovic J, Moelling K: Inhibition of influenza A virus replication by short double-stranded oligodeoxynucleotides. Arch Virol 2009, 154: 109-114. 10.1007/s00705-008-0262-zView ArticlePubMedGoogle Scholar
- Chan JH, Lim S, Wong WS: Antisense oligonucleotides: from design to therapeutic application. Clin Exp Pharmacol Physiol 2006, 33: 533-540. 10.1111/j.1440-1681.2006.04403.xView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.