Abstract
Human cytomegalovirus (HCMV) causes severe sequelae in immunocompromised hosts. Current antiviral therapies have serious adverse effects, with treatment in many clinical settings problematic, making new therapeutic approaches necessary. We examined the in vitro efficacy of small interfering RNAs (siRNAs) targeting the HCMV gene transcripts UL54 (DNA polymerase), UL97 (protein kinase) and UL122/123 (immediate-early proteins) as inhibitors of viral protein expression and virus replication in cell cultures. Two siRNAs for each HCMV target (designated A and B) were assessed for inhibition efficacy using western blot and standard plaque assays. Continuous human embryonic kidney 293T cells were treated with HCMV or non-specific scrambled (siSc) siRNA followed by transfection with plasmids expressing the target transcripts. Human MRC-5 fibroblasts were HCMV-siRNA or siSc treated, infected with HCMV strain AD169 (1 pfu/cell) and HCMV immediate-early (IE1p72 and IE2p86), early (pp65), early-late (pUL97) and true late (MCP) protein and virus progeny production measured during a single round of replication. Concordant results showed siUL54B, siUL97A and siUL122B displayed the most potent inhibitory effects with a reduction of 92.7%, 99.6% and 93.7% in plasmid protein expression, 65.9%, 58.1% and 64.8% in total HCMV protein expression and 97.2%, 96.2% and 94.3% (p<0.0001) in viral progeny production respectively. Analysing the siRNA inhibitory effects during multiple rounds of HCMV replication at a multiplicity of infection of 0.001 pfu/cell, siUL54B, siUL97A and siUL122B treatment resulted in a reduction of 80.0%, 59.6% and 84.5% in total HCMV protein expression, 52.9%, 49.2% and 58.3% in number of cells infected and 98.5%, 91.4% and 99.1% (p<0.0001) in viral progeny production at 7 dpi respectively. These results suggest potential in vivo siRNA therapies targeting the HCMV gene transcripts UL54, UL97 and UL122/123 would be highly effective, however, the antiviral efficacy of siRNAs targeting UL97 may be more highly dependent on viral load and methods of administration.
Highlights
Human cytomegalovirus (HCMV) is a ubiquitous infection causing serious sequelae in immunocompromised patients and the developing fetus during pregnancy [1]
The efficacies of each small interfering RNAs (siRNAs) at silencing protein production were measured using western blot of the cell lysates of 293T cells treated with the HCMV siRNAs and transfected with plasmids expressing the specific HCMV proteins, at 72 hr post plasmid transfection (Figure 1A)
For UL54 siRNA silencing efficacies, siUL54A showed a modest reduction (58.7%) in pUL54 expression compared with cells transfected with scrambled siRNA, whereas siUL54B showed a substantial reduction (92.7%)
Summary
Human cytomegalovirus (HCMV) is a ubiquitous infection causing serious sequelae in immunocompromised patients and the developing fetus during pregnancy [1]. Any vaccine will take at least 20–30 years to reduce disease in pregnancy and immune compromise due to very high population seropositivity, and the importance of virus reactivation as the major cause of disease in transplant recipients and the fetus [3]. Few antiviral drugs are available for the treatment of HCMV infection - these include ganciclovir, valganciclovir, foscarnet, cidofovir, and fomivirsen. Treatment with these drugs is frequently associated with toxic side effects that make them unsuitable in some settings including during pregnancy. There is a current and pressing need for the development of novel drugs to treat HCMV infection, those with reduced toxicity. Novel developmental drugs such as letermovir (AiCuris, Wuppertal, Germany), which targets the viral DNA terminase complex [4,5,6], are presently undergoing clinical trials, some with promising results, final approval has not been achieved and the putative frequency of viral drug resistance has not been addressed in detail for most of these drugs
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