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. 2015 Sep;28(7):378-84.
doi: 10.1089/vim.2015.0034. Epub 2015 Jul 8.

A Molecular Approach Designed to Limit the Replication of Mature DENV2 in Host Cells

Ummar Raheel  1 , Muhsin Jamal  1 , Najam Us Sahar Sadaf Zaidi  1
Affiliations

A Molecular Approach Designed to Limit the Replication of Mature DENV2 in Host Cells

Ummar Raheel et al. Viral Immunol. 2015 Sep.

Abstract

Dengue virus (DENV) is an arthropod-borne virus, which belongs to the Flaviviridae family, and completes its life cycle in two hosts: humans and mosquitoes. For DENV maturation, the surface pre-membrane (prM) protein is cleaved to form a mature membrane protein (M) by furin, which is a cellular enzyme subsequently releasing the mature virus from the host dendritic cell. The objective of the current study was to inhibit mature DENV isotype 2 (DENV2) by RNA-interference in a Vero-81 cell line. Mature DENV2 was propagated in and isolated from U937 cells expressing dendritic cell-specific intracellular adhesion molecule-3-grabbing non-integrin. Maturation of DENV2 was confirmed by Western blot analysis, where virus stock lacking prM was considered mature. Inhibition studies were carried out by transfection of Vero-81 cells with six synthetic siRNAs along with a control siRNA. Reduction in cellular DENV2 was observed also by focus-reduction assay, immunofluorescence assay (IFA), and real-time quantitative polymerase chain reaction (RT-qPCR). Cells transfected with DENV2SsiRNA2, which was targeting the structural region M of mature DENV2, was able to reduce DENV2 titer by up to 85% in focus reduction assays. A significant reduction in mature DENV2 RNA load was observed by RT-qPCR, confirming the previous findings. IFA also revealed reduced levels of cellular DENV2. These results demonstrated that mature DENV2 can be effectively inhibited by synthetic siRNA targeting the structural region of the genome. Mature DENV2 can be successfully inhibited by siRNAs, and specifically high knock-down efficiency is observed by siRNAs against M region of mature DENV2. This study shows that M represents a potential target for RNAi based inhibitory approaches.

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Figures

<b>FIG. 1.</b>
FIG. 1.
Western blot analysis of mature and immature dengue virus isotype 2 (DENV2). Top image: Western blot was performed by using an anti-envelope (E) protein 8A1 antibody and anti-pre-membrane (prM) mixture of monoclonal antibodies (mAbs). The first three lanes (1–3) show DENV 2 grown on C6/36 cell line having both E and prM bands, while the remaining three lanes (4–6) exhibit DENV2 grown on U937 dendritic cell-specific intracellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) cells lacking a prM band. Bottom image: DENV2 E-protein as a loading control. The experiment was repeated twice showing similar results.
<b>FIG. 2.</b>
FIG. 2.
Focus assay of siRNA-transfected Vero-81 cells. The focus assay was conducted on Vero-81 cells transfected with the six selected anti-dengue synthetic siRNAs (displayed in Table 1). Focus assays revealed that siRNAs from the 5′UTR (DENV2UTR5′siRNA1 and DENV2UTR5′siRNA2) had no effect on virus titers, and steady virus replication was observed as shown by bars 1 and 2. DENV2SsiRNA2, which targets the structural region of DENV2 genome, was able to achieve maximum reduction in DENV2 titer, while a slight decrease was also observed with DENV2SsiRNA1 (bars 2–3). DENV2UTR3′siRNA2, designed from the 3′UTR managed to limit DENV2 replication to some extent. Experiments were repeated with similar results, thus reducing the chances of error.
<b>FIG. 3.</b>
FIG. 3.
Immunofluorescence assay (IFA) images of siRNA-treated Vero-81 cells. IFA was done 48 h post-infection with anti-E antibody (2H2-488) for monitoring cellular levels of DENV2 E-protein. (A) and (B) represent cells transfected with DV2UTR5′siRNA1 and DV2UTR5′siRNA2. DENV2SsiRNA1 and DENV2SsiRNA2 transfected cells are shown in (C) and (D). DENV2SsiRNA2 in (D) shows a significant drop in cellular DENV2 E-protein as seen by reduced fluorescence. siRNAs designed from 3′UTR images (E) and (F) had a limited inhibitory effect shown by DENV2UTR3′siRNA2. Finally, (G) and (H) are positive controls with PCsiRNA and no DENV2, respectively. Color images available online at www.liebertpub.com/vim
<b>FIG. 4.</b>
FIG. 4.
Real-time quantitative polymerase chain reaction (RT-qPCR) for checking DENV2 RNA levels. Quantitative analysis of DENV2 RNA levels was performed by RT-qPCR. RNA from the cells was extracted 48 h post-infection for each individual sample. From left to right, bars 1 and 2 show no significant decrease in DENV2 RNA levels. Bars 3 and 4 show a steady decrease in DENV2 RNA, with DENV2SsiRNA2 showing least DENV2 RNA. Bar 5 is uniform without any prominent change in DENV2 RNA, while bar 6 shows almost a 60% reduction in DENV2 RNA. The last bar represents PCsiRNA (which is a plant origin siRNA and used as a negative control).
See this image and copyright information in PMC

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