Inhibition of Monkeypox virus replication by RNA interference

Monkeypox virus genome consists of 196,858-base pairs (bp) with 190 open reading frames of 60 amino acid residues or more [16]. Like other orthopox viruses, the MPV genome encodes for a number of enzymes and factors that are necessary for entry, self-replication, and maturation. The central region of the genome contains highly conserved genes that are essential for viral replication, and terminal regions contain less conserved genes that are important for virus-host interactions. In designing siRNA molecules, we selected 12 gene targets based on their temporal expression and functional significance, e.g., attachment, replication, and host immune modulation (Table 1). These targets varied in size from 132 to 3021 bp, and mapped to the region between 22056 and 114223 on MPV genome covering most of the conserved region.

Custom SMARTpool software (Dharmacon) was selected to design four siRNA sequences for each of the 12 selected MPV genes. Constructs of 19-21 nucleic acids were chemically synthesized and pooled at comparable concentrations to maximize efficacy. Screening was performed in rhesus macaque kidney epithelial cells (LLC-MK2). This cell line was selected based on experimental transfection efficiency, host relevance, and ability to support replication of used MPV isolate.

For a preliminary evaluation of antiviral properties of the 12 siRNA pools, cultured LLC-MK2 cells in 24 wells plate were transfected with each of the multiplexed siRNA pools at 100 or 200 nM overnight. Cells were then infected with 100 pfu/well of MPV-KK. Infected cells were incubated for 48 h post infection (PI) at which time the cells were harvested and viral titer was measured by using the plaque assay. Percentage of viral replication rates in siRNA-transfected cells relative to replication in mock-transfected cells are shown in Fig 1. Inhibition of viral replication varied significantly based on targeted gene and used siRNA concentrations. For example, while anti-L5 siRNA pool showed little or no effect on viral replication at 100 nM, pools targeting A6R gene (siA6) or E8L gene (siE8) exhibited 95% and 78% at 100 nM respectively (Fig. 1). Differences in inhibition potency of siRNA polls at high 200 nM concentration were less pronounced generally, and most pools showed more than 50% inhibition.

Because transfection with siRNAs can be cytotoxic, especially at high concentrations [17], and consequently will affect viral replication [18], we examined the morphology of transfected cells using phase-contrast light microscopy and followed emergence of any cytopathic signs such as changes in the refractive index or cytoplasmic shrinkage of the cells. Alternatively we used vital dyes, such as Trypan blue. No signs of cytotoxicity were observed in cells treated with all tested siRNA pools at 100 nM, and only three out of the 12 siRNA pools induced 15-35% decrease in cells viability after 72 h of transfection at concentration of 200 nM (data not shown).

Each of the two siRNAs that exhibited the most significant inhibition of viral replication, siA6 and siE8, consisted of a pool of four different siRNA sequences that targeted various regions of the A6R and E8L genes (Table 2). Hence, we anticipated that each construct of the two pools will have different antiviral properties. To identify the most potent sequence within the two pools, we transfected LLC-MK2 cells with each of the siRNA sequences individually at concentration of 40 nM for 18 h. Transfected cells were infected with MPV-KK, and viral replication was evaluated 48 h PI. All four sequences of siA6 pool severely inhibited viral replication, with siA6-a and siA6-b being the most potent, achieving more than 95% inhibition. Interestingly, treating cells with the siA6 pool didn't result in appreciably stronger viral inhibition than treatment with the most potent siA6-a alone. The antiviral properties of siRNA sequences targeting E8L varied significantly and while siE8-d inhibited viral replication by more than 90%, siE8-c exhibited only a little more than 20% inhibition (Fig. 2).

To characterize the antiviral properties of most potent siRNA constructs, namely siA6-a and siE8-d, LLC-MK2 cells were transfected with one fold serial dilution of each construct to cover a range of 40 to 1.25 nM in six concentrations. Overnight transfected cells were infected with MPV at 100 pfu/well and viral replication was examined at 48 hours PI (Fig 3). siA6-a showed average viral replication inhibition of 23% at the lowest tested concentration of 1.25 nM, and complete inhibition at concentrations of 20 nM and higher. In agreement with previous assays, siE8-d maintained weaker antiviral activity than siA6-a and concentrations of ≤ 2.5 nM were inefficacious. The estimated IC50 for siA6-a would be less than 5 nM under the experimental conditions described above.

We used 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to measure the effect of siA6-a and siE8-d on cells viability. Transfected mock-infected cells that had been handled identically to virus infected cells maintained more than 90% viability at all siA6-a or siE8-d concentrations (Fig. 3).

We used MPV expressing green fluorescence protein (MPV-GFP) [19] to reproduce the results obtained using wild-type MPV-KK, examine the relationship of viral replication rate and virucidal properties of siA6-a or siE8-d, and to assess the stability of these two siRNA constructs over extended culture. Cells were transfected for 18 h with either siA6-a or siE8-d at the same concentration range described above. Transfected cells were infected with 2000 pfu/well of MPV-GFP, and viral replication was followed for 7 days PI by measuring fluorescence increase of the culture once every 24 h. Controls of untransfected-infected, transfected-uninfected, and non-targeting scrambled siRNA transfected-infected cells were included for results normalization and to confirmation of siRNA specificity. Consistent with plaque assay results obtained from wild-type MPV, the viral replication rates in cells transfected with scrambled non-targeting siRNA and in mock-transfected cells (Fig. 4A, D upper panel) were identical. No increase in fluorescence was observed in cells transfected with siA6-a at concentrations of 40 and 20 nm, which resembled the results observed in cells treated with 100 μM Cidofovir (not shown). Furthermore, viral replication rate was kept at less than half of its values in control cells as late as 7 days PI in cells treated with 10 nM of siA6-a, and only concentrations ≤ 5 nM exhibited mild or no viral inhibition (Fig. 4b, D middle panel). These results highlight the unusual stability of siA6-a construct and confirm its antiviral function regardless of virus replication rate. In contrary to our finding while using wild-type MPV, cells treated with siE8-d were more permissive to MPV-GFP replication and sharp increase in fluorescence was detected in cells treated at concentration ≤ 20 nM across all PI time points (Fig. 4C), and significant viral replication was observed at concentration of 40 nM (Fig, 4C, D lower panel). This variation in antiviral properties of siE8-d and dependence on experimental conditions was likely due to the known gradual decline of siRNA stability during extended incubation duration. The use of MPV-GFP required relatively long incubation period to reach the exponential phase of fluorescence amplification needed for adequate assessment of viral replication. Examination of transfected cells by phase contrast microscopy showed no cytopathic signs, underscoring the specific antiviral properties of siA6-a.

Successful siRNA application in the treatment of diseases depends on a variety of parameters, including stability of the siRNA, effective delivery, and efficacy against a high viral burden [20, 21]. Stable and potent siRNA may have indications for post onset treatment as well as prophylaxis. We evaluated the antiviral properties of siA6-a through multiplicity of infection (MOI) range of 4 to 0.005. Our results showed that LLC-MK2 cells transfected with 20 nm of siA6-a remained refractory to viral replication after 8 days of MPV infection at an MOI of 0.01 or less (Fig. 5). Under identical conditions, siA6-a showed more than 50% inhibition of MPV-GFP replication at MOI of 0.1 in day 6 PI. Optimizing uptake and nuclease resistance properties of siA6-a, through chemical modifications that introduce lipophilic moieties and other chemical groups, would produce more potent derivatives that cover wider MOI range and last for longer durations [22].

Wild-type monkeypox virus, strain Katako Kombe (MPV-KK) and MPV expressing green fluorescent protein (MPV-GFP) were used to evaluate siRNA efficacy [19]. The viruses were propagated in Vero E6 cells maintained in Eagle's minimum essential medium with non-essential amino acids (EMEM/NEAA) supplemented with 2 mM L-glutamine, 10% of heat-inactivated fetal bovine serum (FBS), 10 mg/L Gentamycin, 250 μg/L Fungizone, and buffered with 10 mM HEPES [46]. Viral titers were determined by the plaque assay as described in [29]. Briefly, monolayers of confluent Vero-E6 cells in six-well plates were overlaid with 100-200 μl aliquots of serial dilutions of examined viral sample in triplicate. The virus was allowed to absorb for 1 h at 37°C with gentle mix for 30 sec each 15 min. Two to three ml of complete culture medium was added to each well, and plates were incubated until the development of clear plaques (≈ 4-5 days). Stock solution of 1.3 g/L of crystal violet, 30% formalin, and 5% ethanol was diluted with PBS (1:2) and used to fix and stain the cells. Plates were incubated 15-20 min at room temperature to allow clear staining. Cells were rinsed gently with PBS and plaques were counted on a light box.

For experiments involving MPV-GFP, plates were infected with 2000 plaque-forming units (pfu) per well unless otherwise indicated. Fluorescence readings were taken every 24 h by using a Gemini EM fluorescence reader and Softmax 4.7 software (Molecular Devices, Union City, CA). The results were normalized to average fluorescence in control cells. Data were expressed as means ± SD. Statistical analysis was performed using Student's t-test when appropriate.

Chemically synthesized siRNAs were custom-designed to target selected MPV genes by Dharmacon (Chicago, IL). siRNA sequences were BLAST-searched against the human genome database to assess possible cross-reactivity. For each targeted gene, four siRNAs were synthesized and pooled. Dried siRNA pools were reconstituted in rehydration buffer (100 mM KCl, 30 mM HEPES pH 7.5, 1 mM MgCl₂) to a final concentration of 100 μM and stored at -80°C until used.

LLC-MK2 cells were used to evaluate the efficacy of the siRNAs. Confluent cells were briefly trypsinized, counted, and resuspended in culture medium at desired concentration. Cell suspension was used to seed 24-well culture plates (Costar, Lowell, MA), and plates were incubated for 24-48 h to allow multiplication of cells in antibiotic- and Fungizone-free medium before infection with virus. To transfect the cells, siRNA and the transfection reagent were complexed as recommended by the manufacturer (Dharmacon, Chicago, IL). Briefly, equal volume of 10× solution (relative to the final intended concentrations) of siRNA and transfection reagent were prepared and mixed together. The resultant 5× solutions mix was incubated for 30 min at room temperature to allow the formation of siRNA transfection complex. This was diluted to the intended final 1× concentration using OPTI-MEM-I (Invitrogen, Carlsbad, CA). Cells were washed twice gently with 0.5 ml of OPTI-MEM-I buffer and incubated with 1× transfection siRNA complex for 12 to 16 h prior to viral infection.

To infect siRNA transfected and non transfected control cells with MPV, incubation medium was replaced by the virus inoculum diluted to produce the desired MOIs using OPTI-MEM-I (GIBCO-Invitrogen, Carlsbad, CA). Cells were incubated with the virus seed for 30 min at room temperature to allow viral absorption, and gentle 15-sec shake every 10 min was done to ensure even viral distribution. Removed culture medium was added back, and cells were incubated in 93-95% relative humidity atmosphere at 37°C, 5% CO₂.

Cells viability was assessed using MTT Cell Proliferation assay (ATCC, Manassas, VA). Briefly, 10 μl of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to each well and the plates were incubated at 37°C, 5% CO₂ until clear purple crystals precipitant appeared in cells cytoplasm (4-8 h). Cells were lysed by adding 100 μl of detergent (proprietary, ATCC kit) and incubated overnight or until the purple crystals were dissolved. Color intensity was measured by spectrophotometer (Tecan, Pittsburgh, PA) at 570 nm, and cytotoxicity was calculated as a ratio of absorbance in treated versus untreated cells.