Therapies to treat HCMV infections have been difficult to manage because of side effects associated with antiviral drugs. Effective vaccines to protect individuals from HCMV infection or protect them from infection-associated diseases do not exist. Silencing of gene expression by siRNAs is becoming a powerful tool for the development of new therapies. In this study, we demonstrated that an siRNA directed against a shared exon of UL122-UL123 reduces the expression of the targeted transcripts, which has the expected effects on IE, E, and L protein expression; DNA replication; viral propagation; and PML body integrity. Further, targeted depletion of IE1 and IE2 proteins or the viral DNA polymerase alters the formation of viral RCs and the recruitment of DDR proteins to the RCs at E and L time points. These events mark important hallmarks of productive replication, and their perturbation may explain the mechanism behind the efficacy of the siRNA treatments. Significantly, we also demonstrated therapeutic efficacy by treating cells with siRNAs after HCMV replication had commenced.
To our knowledge, no study has considered using one siRNA to simultaneously abolish the expression of multiple IE genes, even though reports have demonstrated that siRNA-mediated targeting of other open reading frames (ORFs) can affect HCMV replication (4
). A result of this present study is the demonstration that targeting a conserved and shared region of IE genes can inhibit HCMV replication, thereby illustrating the potential of using this region as a new drug target for RNAi-based therapeutics. Consistent with this possibility, our observations are similar to those made for an exon 3-deleted virus, IE delta 30–77 (78
). This mutant virus exhibits impaired growth at both high and low MOIs. Likewise, attempts to rescue virus from a ul122-deficient bacterial artificial chromosome (BAC) fail to induce cytopathic effects and the expression of several viral early genes is undetectable (46
The similarity of the results obtained here with siX3 to those made with an exon 3-deleted virus (78
) illustrates the potential of siRNA-based targeting of HCMV gene expression for performing quick and relatively inexpensive genetic studies of viral gene function. However, there are caveats to the siRNA approach, including the targeting of multiple viral genes due to overlapping transcription and incomplete suppression of gene expression. Complementary experiments such as the inclusion of multiple siRNAs that target the expression of the same gene and inclusion of appropriate controls can often mitigate these concerns.
The antiviral activity of siX3 and siUL54, which targets UL54-57
, translates into a >1- to 3-log reduction of virus replication in HEL fibroblasts, with some conditions producing no detectable infectious virus. This level of antiviral activity compares favorably with that of siRNAs against herpes simplex virus, where a 1.5-log reduction in virus titer in cell culture experiments was shown to be sufficient to protect mice from lethal intravaginal challenge when animals were pretreated with an anti-HSV siRNA (55
). Our results are tempered somewhat by the observations of HCMV replication recovering from siRNA treatment in HEL fibroblasts. Taken together with data obtained in U373MG cells, which are more efficiently transfected than HEL fibroblasts, it appears that efficient delivery of siRNAs is essential for efficacy. Still, these experiments demonstrate that a single prophylactic or therapeutic treatment of cells with anti-HCMV siRNA is sufficient to have a dramatic impact on viral replication and cytopathic effects.
The fact that siX3 effectively blocked both IE gene expression and nascent virus production in U373MG cells raises the potential for targeting exon 3 as an adjuvant therapy for malignant gliomas, which are often-lethal brain cancers. HCMV has been shown to be associated with malignant gliomas (18
; B. Bhattacharjee, N. Renzette, and T. F. Kowalik, submitted for publication), and the U373MG cell line is derived from an astrocytoma, which is a form of malignant glioma. While it is unclear whether viral replication occurs in these cancers, HCMV IE gene expression is a hallmark of this disease (18
). IE1 expression has also been shown to stimulate both cell proliferation and signature signaling pathways in glioma cells (19
). Thus, inhibiting IE gene expression may have therapeutic value as a component of an aggressive treatment regimen for this cancer.
One of the greatest challenges in the development of RNAi-based therapies has been delivery in vivo
. Efficacy of siRNAs has been related to the targeted cell type and delivery strategy. Different modifications of siRNA/siRNA-like molecules have been used to stabilize the molecules, such as using sulfate in place of phosphate in vivo
), or to improve uptake, such as tagging siRNAs with cholesterol (83
). However, in certain circumstances, we have been able to deliver functional unmodified siRNAs in vivo
). The effectiveness of siRNAs typically lasts for a few days to weeks. However, this effective window may be sufficient for antiviral applications with acute phenotypes. Others have recently developed a clever system that delivers an RNase P-based ribozyme to greatly reduce mouse CMV replication both in vitro
and in vivo
). An orally delivered carrier for delivery of siRNAs has also been shown to be effective in vitro
and in vivo
). It will be interesting to compare the effectiveness of delivery vehicles and RNA therapeutics (ribozyme versus siRNA) in treating CMV infections.
We attempted to improve the efficacy of the siRNAs through the use of multiple dosing of siRNAs, expecting that the siRNA effects would be more potent and sustained. However, the results of these experiments were no different than those of single-dose transfections. Why multiple dosing did not improve the effectiveness of the siRNAs is unclear. One possibility is that there is a subset of fibroblasts that are resistant to transfection. In contrast, we were successful in therapeutically reducing HCMV replication by introducing siRNAs postinfection. To our knowledge, this is the first study to demonstrate that cells previously infected with HCMV can be treated with siRNAs in this manner.
HCMV drug resistance, toxicity, and the limited repertoire of antiviral targets represent major challenges in managing HCMV infections in patients. The specificity and flexibility of siRNA approaches offer new ways for treating HCMV infection in place of conventional antiviral strategies, which are mainly based on chemical inhibitors of the HCMV polymerase (i.e., ganciclovir, foscarnet, or cidofovir). However, the potential for resistance to RNAi-based therapies exists, and given the genetic variability of viral genomes, it may be possible to select for point mutations that are resistant to infection, as has been observed in HIV-infected cells (20
). In principle, selection for escape mutants can be minimized by directing siRNAs against highly conserved viral genes or by combining siRNAs that target conserved and transcribed regions of the viral genome. The X3 siRNA was designed with both approaches in mind. Still, it might be worthwhile to consider targeting the major IE locus together with other ORFs to further reduce the potential for resistance to develop. Such considerations await further study.
In conclusion, our investigation shows that an siRNA designed against a shared exon of the major IE ORFs or siRNAs against a locus encompassing UL54-57, which encodes the viral DNA polymerase, among other proteins, are effective at inhibiting HCMV replication and reducing viral gene expression when used in prophylactic or therapeutic contexts. Moreover, both siRNAs prevent cellular cytotoxicity at the level of DNA damage signaling. Delivery of the siRNA against the major viral IE gene transcripts also results in retention of PML body integrity, which influences host gene expression and is an important component of the intrinsic immune defense against HCMV. The combined effects of reduced viral replication and cytotoxicity support a proposition for the continued development of siRNA therapies for the treatment of HCMV infections.