Abstract
Viral protein neutralizing antibodies have been developed but they are limited only to the targeted virus and are often susceptible to antigenic drift. Here, we present an alternative strategy for creating virus-resistant cells and animals by ectopic expression of a nucleic acid hydrolyzing catalytic 3D8 single chain variable fragment (scFv), which has both DNase and RNase activities. HeLa cells (SCH7072) expressing 3D8 scFv acquired significant resistance to DNA viruses. Virus challenging with Herpes simplex virus (HSV) in 3D8 scFv transgenic cells and fluorescence resonance energy transfer (FRET) assay based on direct DNA cleavage analysis revealed that the induced resistance in HeLa cells was acquired by the nucleic acid hydrolyzing catalytic activity of 3D8 scFv. In addition, pseudorabies virus (PRV) infection in WT C57BL/6 mice was lethal, whereas transgenic mice (STG90) that expressed high levels of 3D8 scFv mRNA in liver, muscle, and brain showed a 56% survival rate 5 days after PRV intramuscular infection. The antiviral effects against DNA viruses conferred by 3D8 scFv expression in HeLa cells as well as an in vivo mouse system can be attributed to the nuclease activity that inhibits viral genome DNA replication in the nucleus and/or viral mRNA translation in the cytoplasm. Our results demonstrate that the nucleic-acid hydrolyzing activity of 3D8 scFv confers viral resistance to DNA viruses in vitro in HeLa cells and in an in vivo mouse system.
Highlights
Viruses are pathogenic agents that cause potentially devastating diseases such as the flu, hepatitis, poliomyelitis, acquired immunodeficiency syndrome (AIDS), severe acute respiratory syndrome (SARS), avian influenza, and foot-and-mouse disease [1,2,3]
Most strategies for developing virus-resistant transgenic cells and animals are based on the concept of virusderived resistance, in which dysfunctional virus-derived products are expressed to interfere with the pathogenic process of the virus in transgenic cells or animals
We found that the transgenic cells and mice acquired complete resistance to two DNA viruses (HSV and pseudorabies virus (PRV)) without accumulating the virus, and showed delayed onset of disease symptoms
Summary
Viruses are pathogenic agents that cause potentially devastating diseases such as the flu, hepatitis, poliomyelitis, acquired immunodeficiency syndrome (AIDS), severe acute respiratory syndrome (SARS), avian influenza, and foot-and-mouse disease [1,2,3]. Qing Ge demonstrated that nucleocapsid siRNA or a component of the RNA transcriptase (PA) is a good antiviral drug to protect against influenza virus by inhibiting viral RNA transcription with siRNAs [5]. Acyclovir, which is the best antiviral agent against HSV-1, is a nucleotide analogue that shows an antiviral effect by inhibiting DNA replication [6]. Commercially-developed antiviral drugs such as viral DNA polymerases, viral reverse transcriptases, and neuraminidase inhibitors target one or two viruses [7,8,9,10,11]. A new strategy is needed to prepare for outbreaks caused by new viruses or new mutant viruses because of the high mutation rates of viral genomes and recombination events among closely-related viruses [12,13]
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