Despite the outstanding success of vaccination in eradicating smallpox, the process was underlined with significant adverse reactions including inadvertent inoculation, ocular vaccinia, generalized vaccinia, eczema vaccinatum, progressive vaccinia, postvaccinial encephalopathy and encephalomyelitis, and fetal vaccinia. These complications can occur among primary vaccinated individuals or in secondary patients who are accidentally inoculated upon their contact with vaccinated individual [23
]. Resorting to vaccine as a response measure to sudden poxvirus release is undermined further by increasing prevalence of immuno- compromised individuals and delayed immune protection inherent to vaccination. Broad use of vaccinia immune globulin (VIG) is hampered by its limited accessibility; hence, adhering to developing a chemical agent that is effective and safe for use in PI and chemoprophylaxis purposes presents a more favorable approach. While Cidofovir [25
] and ST-246 [8
] remain the drugs of choice for poxvirus infections, known side effects or propensity to develop resistant viral strains dictate cautious use [8
In this study, we showed that RNAi can be used as a potent approach to reduce MPV replication in a sequence-specific manner. We screened 48 siRNA constructs in 12 pools targeting 12 monkeypox genes and examined their effect on viral replication. We showed that the siRNAs affected MPV replication in various degrees. Two siRNA pools exhibited substantial antiviral properties and reduced viral replication to less than 10% of its propagation in control untreated cells. A single siRNA construct targeting A6R gene suppressed viral replication to near completion with IC50 less than 10 nanomoles.
The observed disparity in siRNA efficacy among screened pools is consistent with a number of previous reports [28
]. Although reasons for this variation are still not fully understood, it is accepted that delivery of siRNA in its optimal functional concentration to targeted cells, and other unpredictable sub-cellular events such as concentration of siRNA in endosomes or trapping in other vesicles are major causes of variation in siRNA efficacy studies [30
]. Furthermore, larger forms of RNA undergo a number of sequential processing steps before they interact with RNAi machinery and achieve the intended biological function. This include processing of miRNA and shRNA by Dicer, assembly of resultant guide strand into the RNA-induced silencing complex (RISC), recognition of target viral RNA sequences and cleavage of targeted mRNA. Because these steps involve interactions of various secondary molecular conformations defined by diverse primary sequences they are likely to exhibit unequal efficiencies, which would factor into the observed variation in siRNAs potency. Thus, identifying an effective siRNA empirically remains a common tool in therapeutic applications of RNAi [31
It is important to note that screened siRNA pools may have silenced the targeted genes, but without producing a phenotype or influencing MPV replication. This is essentially defined by the function of the targeted gene. Presence of viral or host compensating mechanisms for the function of knocked-down gene would obfuscate evaluation of siRNA efficacy further [32
], especially when reduction or inhibition of replication is the endpoint in assessing siRNA potency. Alternatively, multiple genes with varied copy number [33
] are found to contribute to the same phenotype or trait with different intensities. Using siRNA and reverse genetics to silence one or more of these genes and determine its function is intrinsically difficult. Similarly, genes associated with high translation turnover duration [34
] or highly efficient protein synthesis mechanisms can sustain viral replication at low copy number. Nevertheless, our results provided reverse genetic evidence for the vitality of A6R and E8L in MPV replication, and further work is needed to clarify the knockdown of the other targets at the gene or protein level.
Poxvirus is the only known double-stranded DNA viral family that propagates in host cytoplasm and encodes most of the enzymes and factors necessary for transcription and replication of its material [35
]. Once the virus enters into the host cytoplasm, it becomes uncoated to release its genetic information and component of early transcription system packaged within the core of the virion [36
]. Many targeted RNA sequences will interact with viral and/or cellular proteins which would hamper, if not prevent, the ability of RNA-induced silencing complex to recognize its viral targets [38
]. This may contribute to the variation we observed in the efficacy of single siRNA constructs within the same pool as in the case of siE8-d and siE8-c which target the same E8L gene.
The function of siA6-a and E8-d targets in MPV remain unidentified; however, their orthologs in vaccinia, A5R, and D8L, function as a precursor of RNA polymerase [39
] and cell attachment protein [40
]. Mutant vaccinia virus with dysfunctional A5 or D8 genes show no or severely perturbed viral replication. Soluble vaccinia D8 protein, which binds chondroitin sulfate (CS), interferes with adsorption of wild-type vaccinia and decreases viral propagation rate. The significant decrease of MPV replication in siE8-d treated cells, and the disruptive effect of soluble D8 on vaccinia adsorption with consequent lower rates of replication imply a sort of similarity in both gene functions and suggests possible role for CS in MPV cell entry. The presence of alternative MPV cell-attachment and adsorption mechanisms, such as binding of A27L protein with cell heparan sulfate HS described in vaccinia [41
], may account for the incomplete replication inhibition of MPV replication despite knock-down of E8 gene.
Persistence of RNAi occurs for short period of time mainly due to the relatively short half life of siRNA and lack of an amplification mechanism in mammalian cells. The estimated 66 hours of RNAi persistence is relatively short due to siRNA hydrolysis and dilution over the course of cell division [42
]. Recently D5R gene in vaccinia virus strain Western Reserve was introduced as a valid siRNA target in vitro
]. Targeting this gene in vaccinia WR, CPV, and MPV led to 70% inhibition of viral replication at nanomolar levels of siRNA using different cell lines. The same work report an impressive prolonged prophylactic antiviral effect that lasted for 72 h at concentration of 100 nm. Surprisingly, our siA6-a maintained solid viral replication inhibition for more than 7 days PI in cell culture at concentration of 20 nm. This unusual stability may be due to a unique molecule tertiary structure and/or highly sensitive target. Further work is under way to access the pharmacokinetics of A6-a construct and address this point.
The antiviral effects of 20 nm of siA6-a and 100 nm of Cidofovir were comparable and seem to inhibit the replication of all virus forms. These two drugs target the virus directly by silencing gene expression or interfering with vDNA replication without perturbing host cell biology. However, easy synthesis and adjustment of siRNA sequence represent an extra advantage over other chemically synthesized drugs. This, in addition to recent advancements in sequencing capabilities and bioinformatics, enabled unprecedented flexibility to readapt siRNA molecules to function on any emerging resistant viral strains, enhance siRNA specificity, and reduce potential side effects. These tasks are made easier when the targeted microorganism and its host are genetically different. In our case, siA6-a didn't induce any signs of cytotoxicity and seemed not interfere with host cell biology even when used at concentrations up to ten times its IC50.
An alternative approach for developing antiviral drugs targets specific host functions necessary for viral replication. A good example of this class of targets is the epidermal growth factor receptor family of tyrosine kinases [44
], which disrupt viral maturation and replication cycle when antagonized. A member of this family (ErbB-1) was inhibited by CI-1033, a drug that has been developed originally for its anticancer properties, and led to significant reduction in poxvirus activity [45
]. However, CI-1033 seems to be more specific in targeting IMV and not EEV forms. This was evident from the described reduction in the size and not overall plaque count, and from the synergistic antiviral effect observed in cells co-treated with neutralizing virus antibodies and CI-1033. It remains unclear how inhibiting ErbB would affect host homeostasis. The complete inhibition of MPV replication by targeting A6R gene suggests that, unlike CI-1033, siA6-a acts on an indispensable viral function at a stage upstream to viral differentiation into distinct forms, and abolishes virus replication regardless of its form.
In conclusion, using RNAi pathway we identified two MPV genes that serve as potential drug targets during infection in cell culture. A6R and E8L genes of MPV were antagonized effectively using siRNA molecules, and constructs siA6-a and siE8-d disrupted MPV replication severely. siA6-a construct exhibited considerable stability and promising antiviral potency with IC50 less than 10 nm. Chemical modification study aiming to enhance A6-a stability and development of suitable siRNA delivery system are needed before assessing siA6-a in animal models.