Adenovirus Delivered Short Hairpin RNA Targeting a Conserved Site in the 5′ Non-Translated Region Inhibits All Four Serotypes of Dengue Viruses

<div><h3>Background</h3><p>Dengue is a mosquito-borne viral disease caused by four closely related serotypes of Dengue viruses (DENVs). This disease whose symptoms range from mild fever to potentially fatal haemorrhagic fever and hypovolemic shock, threatens nearly half the global population. There is neither a preventive vaccine nor an effective antiviral therapy against dengue disease. The difference between severe and mild disease appears to be dependent on the viral load. Early diagnosis may enable timely therapeutic intervention to blunt disease severity by reducing the viral load. Harnessing the therapeutic potential of RNA interference (RNAi) to attenuate DENV replication may offer one approach to dengue therapy.</p> <h3>Methodology/Principal Findings</h3><p>We screened the non-translated regions (NTRs) of the RNA genomes of representative members of the four DENV serotypes for putative siRNA targets mapping to known transcription/translation regulatory elements. We identified a target site in the 5′ NTR that maps to the 5′ upstream AUG region, a highly conserved <em>cis</em>-acting element essential for viral replication. We used a replication-defective human adenovirus type 5 (AdV5) vector to deliver a short-hairpin RNA (shRNA) targeting this site into cells. We show that this shRNA matures to the cognate siRNA and is able to inhibit effectively antigen secretion, viral RNA replication and infectious virus production by all four DENV serotypes.</p> <h3>Conclusion/Significance</h3><p>The data demonstrate the feasibility of using AdV5-mediated delivery of shRNAs targeting conserved sites in the viral genome to achieve inhibition of all four DENV serotypes. This paves the way towards exploration of RNAi as a possible therapeutic strategy to curtail DENV infection.</p> </div

The effect of rAd-mediated shRNA expression on DENV RNA accumulation.

<p>Vero cells were pre-infected either with rAdsh-5b (red bars) or rAdsh-scr (green bars) followed 24 hours later by infection with DENV-1, DENV-2, DENV-3 and DENV-4. Total RNA was isolated on days 2 (panels A and B) and 7 (panels C and D) post-DENV infection and analyzed for DENV ’minus’ (panels A and C) and ‘plus’ (panels B and d D) sense viral genomic RNAs by strand-specific real time PCR analyses. DENV RNA was normalized to GAP RNA in each sample analyzed. The data depict DENV RNA levels in rAdsh-5b treated cells relative to those in the corresponding rAdsh-scr treated cells. Each experiment was carried out in triplicate wells and the entire experiment repeated twice.</p

Design and characterization of the rAd-sh viruses.

<p>(A) The linear genome of the rAd-sh virus constructed for this study. In constructing the rAd-sh virus, the E1 region (dashed line) is replaced by the sh DNA expression cassette (<i>sh DNA EC</i>), consisting of the U6 promoter (rightward arrow), the shDNA insert with the sense (s) and antisense (as) arms, of 21 base pairs each, followed by of the U6 terminator (empty box). The shaded box between the ‘s’ and ‘as’ arms is a 6-base pair loop sequence. The dotted lines flanking the expression cassette represent plasmid vector sequences. Other elements of the rAd-sh genome include a ∼2.7 Kb deletion in the E3 region (ΔE3), the left (L) and right (R) inverted terminal repeats, and the packaging signal (ψ). The nt sequences of the ‘s’ strands of the sh-5b and sh-scr constructs are shown below. (B) PCR analysis of wild type AdV5 (lanes 1 & 5), rAdsh-E (lanes 4 & 8), rAdsh-5b (lanes 2 & 6) and rAdsh-scr (lanes 3 & 7) using insert-specific (lanes 1–4) and AdV5 E1-specific (lanes 5–8) primers. DNA size markers (sizes in kb shown to the left) were analyzed in lanes ‘M’. The arrows to the right denote the positions of the predicted insert-specific (upper) and AdV5 E1 region-specific (lower) amplicons. (C) RNase protection assay to detect anti-sense strand of sh-5b siRNA. A radiolabeled sense probe was digested with RNases A and T1, either before (lanes 2 & 5) or after hybridization with total RNA isolated from rAdsh-5b-infected Vero cells, harvested on either day 3 (lane 3) or day 8 (lane 6) post-infection. Protected fragments (lower arrow) were analysed on 8 M urea gel and visualized using a phosphoimager. The un-hybridized probe (upper arrow) without any RNase treatment was analysed in parallel (lanes 1 & 4). It is to be noted that in lanes 3 and 6, the cells used for total RNA preparation were challenged with DENV-2 and DENV-4, respectively, at 24 hours post rAdsh-5b infection.</p

The effect of rAd mediated shRNA expression on DENV secretion.

<p>Vero cells were pre-infected either with rAdsh-5b (red curves) or rAdsh-scr (green curves) followed 24 hours later by infection with DENV-1 (A), DENV-2 (B), DENV-3 (C) and DENV-4 (D). Culture supernatants were drawn at daily intervals up to 7 days post DENV infection and analyzed for the presence infectious DENV using a standard plaque assay. Data shown are mean values (n = 6). The vertical bars represent SD.</p

siRNA targets in the DENV NTRs.

<p>A ClustalW2 multiple alignment of 5′ (A) and 3′ (B) NTR sequences of the prototypic representatives of DENV-1, -2, -3 and -4 (described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001735#s2" target="_blank">Methods</a>) showing the sites conserved in two or more serotypes targeted for RNAi in this study. NTR sequences that were utilized to design the sense strand of the sh constructs are shown in red fonts. The names of the sh constructs are shown in italics above the sequences in red fonts. The DENV-4 5′NTR seed sequence identical to the sh-5b target site is underlined.</p

The effect of rAd mediated shRNA expression on on-going DENV infection.

<p>(A) Vero cells in 12-well plates were sequentially infected with DENV-2 (∼25 PFU/well) and 24 hours later, with rAdsh-scr or rAdsh-5b, each at a m.o.i of 5 (top row) or 10 (bottom row). Cells were overlaid with methyl cellulose and plaques visualized as explained in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001735#pntd-0001735-g003" target="_blank">Figure 3</a> legend. Two wells, of four assayed for each sequential infection experiment, are shown. (B) Vero cells in 24-well plates were sequentially infected with DENV-2 (1000 PFUs/well), followed 24 hours later with rAdsh-5b (red curves) or rAdsh-scr (green curve), each at m.o.i. of 5 (solid curve) or 10 (dashed curve). Culture supernatants were drawn at 48 hour intervals up to 7 days post DENV infection and analyzed for the presence of NS1 antigen using BioRad's Platelia Dengue NS1ELISA kit. The data represent plots of NS1 ELISA absorbance as a function of time after DENV infection. Data shown are mean values (n = 4). The vertical bars represent SD. (C) Culture supernatants in (B) were analyzed for the presence infectious DENV using a standard plaque assay. Data shown are mean values (n = 4). The vertical bars represent SD.</p

The effect of NTR-specific shRNAs on DENV replication^a.

a<p>The production of NS1 antigen, measured using BioRad's Platelia assay kit, served as a marker of DENV replication; data shown are from one representative screening experiment.</p>b<p>This indicates the location of the 21 nts corresponding to the sense strand of the sh construct-encoded siRNA, on the DENV genome; numbers indicated correspond to DENV-2 NGC strain (Accession number AF038403).</p>c<p>SLA: stem-loop A; 5′ UAR: 5′ upstream AUG region; 3′ conserved sequence; 3′ UAR: 3′ upstream AUG region; 3′ SL: 3′ stem-loop; nr: not reported.</p>d<p>The percent inhibition was calculated with reference to DENV infectivity (in the presence of transfected sh-scr construct), which was taken as 100%. D-1, D-2, D-3 and D-4 denote DENV-1, DENV-2, DENV-3 and DENV-4, respectively.</p

Design and evaluation of the in-house TRF immunoassays.

<p>(A) Schematic representation of the in-house TRF immunoassay. The design of both the TRF immunoassays was same, except for different incubation times and tracer amounts. The numbers represent the following: (1) microtiter well surface, (2) streptavidin, (3) r-Bio-p15-17-47, (4) serum anti-Tp IgG antibody, (5) serum anti-Tp IgM antibody, and (6) r-p15-17-47 coated on Eu³⁺ chelate-doped nanoparticles. (B) Scatter plot with the S/Co values of all serum samples (n = 311) analyzed in this study, using the in-house TRF immunoassays with 1 h (<i>x</i> axis) and 10 min (<i>y</i> axis) incubation times. Different symbols represent positive (+) or negative (-) serum samples, either from commercial panels based on the results provided by the panel supplier, or from six different categories (#1 – #6) of the in-house samples based on their reactivities with the reference assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084050#pone-0084050-t001" target="_blank">Table 1</a>. Symbols for different kind of samples are as the following: PSS202 (+), filled blue triangles; PSS202 (-), empty blue triangles; QSS701 (+), filled cyan triangles; QSS701 (-), empty cyan triangle; samples of category #1, filled red circles; samples of category #2, filled green circles; samples of category #3, filled blue circles; samples of category #4, filled magenta circles; sample of category #5, filled olive square; samples of category #6 (except one sample), empty red circles; and exceptional sample of category #6 which gave positive results with in-house TRF immunoassays, black star. Dashed vertical and horizontal lines represent the cutoffs (at S/Co = 1) for the two TRF immunoassays.</p

Evaluation of the TRF immunoassays using Syphilis Qualification Panel (QSS701).

<p>^a Members of the panel are manufactured from human serum or plasma, as provided by the panel supplier. Five members are formulated with various reactivities of Syphilis. Non-reactive member was formulated from Syphilis non-reactive pools.</p><p>^b Results were provided by the panel supplier. R, reactive; NR, non-reactive.</p><p>^c Values indicate S/Co ratios obtained in this study, using the in-house TRF immunoassays with 10 min and 1 h incubation times. Samples with S/Co values <1.0 are designated as negative (−) and those with values ≥1.0 are designated as positive (+).</p

Results from the non-conclusive primary and their respective follow-up samples mostly from category #3 and #4 in Table 1.

<p>^a Results obtained from the primary samples using the three reference assays, and both the TRF immunoassays (with 10 min and 1 h incubation times), as indicated. All the primary samples are from category #3 and #4, as listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084050#pone-0084050-t001" target="_blank">Table 1</a>. ‘Enzg.’ indicates Enzygnost syphilis EIA. ‘+’, ‘−’ and ‘+/−’ indicate positive, negative and indeterminate results, respectively, as obtained with the mentioned assays.</p><p>^b Results obtained from the thirteen follow-up samples of their respective primary samples using Enzygnost syphilis, TPHA, VDRL and I-L (Inno-Lia Syphilis score line immunoassay) as reference assays, and both the TRF immunoassays (with 10 min and 1 h incubation times), as indicated. Ten out of 13 samples are from category #3 and #4, as listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084050#pone-0084050-t001" target="_blank">Table 1</a>. Each primary sample had either one or none follow-up sample. ‘NA’ indicates that a follow-up sample was not available to us.</p><p>sample belongs to category #2 as divided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084050#pone-0084050-t001" target="_blank">Table 1</a>.</p><p>Sample not tested with TRF immunoassays in this study.</p><p>*The antibody titer obtained in TPHA assay is shown in parentheses. ‘ND’, indicates that the test was not done.</p><p>^c Syphilis status indicates whether the person has, or has had syphilis previously, and is based upon the results of previous or follow-up samples and on the available clinical data. ‘N’ indicates a negative and ‘P’ indicates a positive status for syphilis. ‘?’ indicates that true syphilis status is unknown.</p><p>sample from a new-born baby, with borderline level of maternal antibodies (data not shown).</p