siRNAs encapsulated in recombinant capsid protein derived from Dengue serotype 2 virus inhibits the four serotypes of the virus and proliferation of cancer cells.

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Contents lists available at ScienceDirect

Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec

siRNAs encapsulated in recombinant capsid protein derived from Dengue serotype 2 virus inhibits the four serotypes of the virus and proliferation of cancer cells

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A.S. Manoj Kumar a,∗ , G.E.C. Vidyadhar Reddy a , Yogesh Rajmane a , Soumya Nair a , Sangita Pai a , Greeshma Sreejesh a , Khalander Basha a , Shailaja Chile a , Kriti Ray a , Vivant Nelly b , Nilesh Khadpe b , Ravishankar Kasturi b , Venkata Ramana a a b

Therapeutic Proteins Molecular Biology Group, Dhirubhai Ambani Life Sciences Centre, Rabale, Navi Mumbai 400 701, Maharashtra, India Therapeutic Proteins Process Development Group, Dhirubhai Ambani Life Sciences Centre, Rabale, Navi Mumbai 400 701, Maharashtra, India

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Article history: Received 28 August 2014 Received in revised form 28 October 2014 Accepted 3 November 2014 Available online xxx

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Keywords: siRNA RNAi Dengue Capsid Aurorakinase Rho A

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1. Introduction

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siRNA delivery potential of the Dengue virus capsid protein in cultured cells was recently reported, but target knockdown potential in the context of specific diseases has not been explored. In this study we have evaluated the utility of the protein as an siRNA carrier for anti Dengue viral and anti cancer applications using cell culture systems. We show that target specific siRNAs delivered using the capsid protein inhibit infection by the four serotypes of Dengue virus and proliferation of two cancer cell lines. Our data confirm the potential of the capsid for anti Dengue viral and anti cancer RNAi applications. In addition, we have optimized a fermentation strategy to improve the yield of Escherichia coli expressed D2C protein since the reported yield of E. coli expressed flaviviral capsid proteins are low. © 2014 Elsevier B.V. All rights reserved.

The capsid of Dengue Virus (DENV) is a highly basic protein which encapsidates a ∼10 kb single stranded RNA genome. The 28 protein is 12 kDa in size, and has charged amino acids at the C 29 and N termini, and a stretch of internal hydrophobic amino acids 30 31Q3 (Ma et al., 2004; Jones et al., 2003; Lobigs, 1993). Escherichia coli expressed capsid protein of Dengue serotype 2 virus (here after 32 referred to as D2C) has been reported to form nucleocapsid like 33 34 particles (NLPs), ∼30 nM in diameter, in the presence of DNA oligos, 35 and these in vitro assembled particles have the ability to induce cell 36 mediated immunity against DENV infection in mice (Lopez et al., 37 2009; Gil et al., 2009). Being an internal protein in the viral archi38 tecture, the DENV capsid has been proposed as a viral protein with 39 a potential to be developed as a novel recombinant Dengue vaccine 40 that does not pose the risk of Antibody Dependent Enhancement 41 (ADE) (Lazo et al., 2007). 27

∗ Corresponding author. Tel.: +91 996 718 1502; fax: +91 226 767 8099. E-mail address: [email protected] (A.S.M. Kumar).

The ability of viral capsids to encapsulate and deliver siRNA into cells is known. Chimeric capsid protein of Hepatitis B Virus (HBV) was shown to assemble into 50 nM diameter particles in the presence of siRNA, and serve as nanocarriers for siRNA (Choi et al., 2013). Recently, D2C protein and a D2C based peptide, Pep M, were demonstrated to encapsulate and deliver fluorescently labelled siRNA into a variety of cell lines and inhibit TLR-3 in endothelial cells (Freire et al., 2013, 2014). To the best of our knowledge, the potential of either D2C protein, or its peptide derivatives, for functional siRNA delivery in the context of any specific disease model, has not been reported. In this study we have evaluated the potential of D2C protein as a carrier for siRNAs targeted against DENV infection and cancer cell proliferation, using cell culture systems. The DENV specific siRNAs that we have used in the study are targeted against conserved sites in the DENV genome. The anti cancer siRNAs that we have used have been reported earlier from our laboratory (Kumar et al., 2011; Addepalli et al., 2010; Chile et al., 2014). Furthermore, the reported yields of E. coli expressed flaviviral capsid proteins are extremely low (Jones et al., 2003). Therefore, in this study, a fermentation process was evaluated as a potential strategy to improve the yields of the protein.

http://dx.doi.org/10.1016/j.jbiotec.2014.11.003 0168-1656/© 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Kumar, A.S.M., et al., siRNAs encapsulated in recombinant capsid protein derived from Dengue serotype 2 virus inhibits the four serotypes of the virus and proliferation of cancer cells. J. Biotechnol. (2014), http://dx.doi.org/10.1016/j.jbiotec.2014.11.003

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2. Materials and methods

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2.1. Cell lines and viruses

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C6/36, BHK-21, HepG2, K-562 (ATCC) and Huh-7 cells (The Health Sciences Research Foundation, Osaka, Japan) were maintained according to the recommendation of the supplier. The source and early passage history of representatives of the four serotypes of DENV have been described earlier (Rajmane et al., 2013). Passaging of DENV serotypes 1, 3 and 4 in infant AG129 mouse brain (2 passages), followed by serial spleen to spleen passaging in adult AG129 mice (3 passages), and C6/36 cell line amplification (3 passages) resulted in all serotypes having a high plaque titre of 108 PFU/ml. All experiments involving DEN viruses were performed under Biosafety level-2 containment laboratories, with prior approval of an Institutional Biosafety Committee. Virus passaging in mice was carried out after obtaining approval from an Institutional Animal Ethics Committee.

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2.2. siRNA design and synthesis

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siRNAs used in the study (Supplementary Table 1) were designed according to the Tuschl rules (Elbashir et al., 2002) or according to the guidelines of the siRNA manufacturer (Qiagen, IDT or Biospring). The DENV specific siRNAs used in the study targeted conserved sites in the DENV genome reported in previous studies (Kyle et al., 2007; Villegas-Rosales et al., 2012; Swamy et al., 2006), but the target sequences of these siRNAs were not exactly the same as those reported in the above studies (Supplementary Table 1). To avoid interferon mediated non-specific gene silencing, the length of each strand of all siRNA duplexes was 21–27 nucleotides. siRNA targeting an unrelated virus (Encephalomyocarditis virus, EMCV) was used as a negative control in all anti DEN viral assays. siRNAs targeting Rho A and AURK B were validated earlier for specific gene knockdown via the RNAi pathway (Chile et al., 2014) and not by interferon stimulation (Addepalli et al., 2010; Kumar et al., 2011). In addition, a 27mer siRNA targeting EMCV was used as a negative control in all anti cancer assays. 2.3. D2C protein expression by shake flask culture and fermentation process

Plasmid pGEMT-Easy was procured from Promega. The cap101 sid gene of DENV2 was PCR amplified from cDNA generated from 102 total RNA isolated from DENV2 infected C6/36 cells, using spe103 cific forward and reverse primers (Supplementary Table 2), ligated 104 in pGEMT-Easy (Promega) and subcloned into pET24a (Novagen). 105 E. coli strains BL21-DE3, BL21-DE3 Codon Plus and BL21-DE3 PLysS 106 (Novagen) were transformed with pET24a carrying the capsid gene 107 insert, and the capsid (D2C) protein expression was initially eval108 uated by culturing the bacteria using standard LB medium, in a 109 shake flask at 37 ◦ C. Protein expression was induced by adding Isopropyl thiogalactopyranoside (IPTG) at a final concentration of 110 1 mM, when the culture reached an optical density (O.D600 ) of 0.6 111 and switching the culture to 30 ◦ C, based on published guidelines 112 Q4 (Botting and Kuhn, 2012). Protein expression was evaluated by SDS 113 PAGE. 114 E. coli BL21 DE3 PLysS harbouring the plasmid that encodes the 115 DENV2 capsid gene was then cultivated by fermentation using 116 3000 l YPD medium (yeast 10 g/l, soyatone 20 g/l, Dextrose 40 g/l, 117 Kanamycin 30 mg/l), in a 5 l fermenter (Sartorius Biostat) at 37 ◦ C 118 and pH 6.8. The dissolved oxygen probe was calibrated at 100% 119 saturation by purging air (1.0 vvm) before inoculation. A two 120 stage fermentation process, involving initial cell growth by batch 121 fermentation followed by fed-batch fermentation process, was fol122 lowed. Dissolved oxygen in the fermenter was maintained at 30% 123 100

saturation. The protein expression was induced by addition of IPTG at 1.0 mM final concentration when the optical density at 600 nm (O.D600 ) reached 3.0 ± 1.0. The temperature of the culture was then reduced to 31 ◦ C. The cultivation was continued till the 5th hour after the addition of IPTG. After the initial glucose was exhausted, glucose feeding was carried out and the pH of the production medium was raised above 6.8. The excess foam inside the fermenter was controlled by adding 10% v/v antifoam solution. Cultivation samples were taken at regular intervals and stored at −20 ◦ C for subsequent analysis of D2C protein expression by SDS PAGE as well as residual glucose levels by using a Glucose estimation kit (Randox GL364). After the 5th induction hour, the culture broth was harvested and centrifuged at 6500 rpm at 4 ◦ C, for 20 min. The supernatant was decanted and the the cell lysate was evaluated for expression of a protein of the expected size of 12 kDa. 2.4. Purification of D2C protein The method described to purify the alpha virus capsid (Tellinghuisen et al., 1999), with slight modifications, was adopted to purify D2C protein. The final pooled protein preparation was analyzed for purity by silver staining. Purity was further evaluated by reverse phase high pressure liquid chromatography (RP-HPLC) analysis. This was performed using a Interchim Interchrom C4 RP-HPLC Column (250 × 4.6 mm, 5 ␮m) in a HPLC system (Waters Alliance 2695 Separation module, 2489 UV/vis detector and Empower2 Software). The linear gradient used is summarized in Supplementary Table 3. 2.5. Characterization of D2C protein The RP-HPLC fractions were then subjected to Intact mass (MS) analysis on an AB-Sciex 3200 Q-TRAP ESI-mass spectrometer. The RP-HPLC collected fractions were infused with the MS solution (50% ACN, 50% water, 0.1% formic acid) and the acquisition was performed between 800 and 1700 m/z (Supplementary Table 4). 2.6. D2C based synthetic peptides Peptides corresponding to the different regions of D2C protein were custom synthesized at USV Ltd., Mumbai, India. A Clustal W alignment of D2C based peptides with the full length D2C protein and previously reported D2C based peptides Pep M and Pep R (Freire et al., 2013, 2014) is shown as Supporting information II. 2.7. Flow cytometry Flow cytometry (using Becton Dickinson FACS Calibur and Cellquest software) was adopted for quantification of viral antigen in cells with a rabbit polyclonal antibody that recognizes the four serotypes of DENV (US Biologicals) and FITC labelled secondary antibody, as well as determination of Cy3 labelled siRNA internalization into cells. Cy3 labelling kit was from Ambion. 2.8. siRNA transfection siRNA ransfection was carried out using HiPerfect reagent(Qiagen) and purified D2C protein (>90% pure as determined by RP-HPLC). HiPerfect mediated transfection was done according to the directions of the manufacturer. Transfection using D2C or D2C based peptides was carried out as follows. siRNA and D2C or D2C based peptide, diluted in the encapsulation buffer (100 mM Potassium Acetate, 25 mM HEPES, 1.7 mM Magnesium Acetate pH 7.4), were incubated at the desired molar ratio at 30 ◦ C for 30 min. siRNA encapsulation was evaluated by agarose gel mobility shift/quenching assay based on the method


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of Subramanya et al. (2010). For transfection, the encapsulated siRNA was applied to cells overnight, in the presence of low serum conditions. For MTS based antiviral siRNA screening assays to identify the lead serotype-specific siRNAs, an siRNA concentration of 20 nM was used. However, during optimizations of antiviral siRNA concentrations, we found that a high concentration of 200 nM in HiPerfect reagent gave the best virus inhibition results (data not shown), and hence this concentration was used in all experiments involving single DENV specific siRNAs and EMCV specific control siRNAs. DENV targeted siRNA cocktails contained 200 nM of each siRNA and equimolar concentration of the control siRNA was used in all antiviral experiments. 27mer siRNAs targeting AuRK B and Rho A, and the control siRNA targeting EMCV, were used at a pre-optimized concentration of 10 nM each as reported earlier (Addepalli et al., 2010; Kumar et al., 2011).

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2.9. Fluorescence microscopy

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Internalization of siRNA into cells was evaluated by Fluorescence microscopy using Cy3 labelled siRNA. We used two different fluorescence microscopes (Nikon Eclipse TE300 and Zeiss Observer.Z1) in these experiments, as specified under the legends.

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Viability was determined by MTS assay using MTS reagent (Promega) according to the guidelines of the manufacturer. 2.10.1. Plaque assay Quantification of infectious virus was done by plaque assay on BHK-21 monolayers, based on the guidelines of Morens et al. (1985). 2.10.2. Quantitative realtime RTPCR Realtime Reverse Transcription PCR (qRTPCR) for DEN viral RNA copy number calculation was carried out based on the method originally reported by Yong et al. (2007). Evaluation of siRNA mediated knockdown of Aurorakinase B and RhoA was carried out by qRTPCR, as described earler (Addepalli et al., 2010; Kumar et al., 2011), based on the qRTPCR relative quantitation method reported earlier (Kenneth and Thomas, 2001). 2.10.3. Colony formation assay Cancer cell proliferation was evaluated by Colony formation assay according to published guidelines (Srikantan et al., 2002). Briefly, PC3 and HepG2 cells, were either left untreated or transfected with siRNA 26, siRNA 52 and the control siRNA 14627 using HiPerfect and D2C protein as the transfection reagent. 24 h after transfection, cells were plated at a concentration of 200 cells per well in 6-well plates and incubated at 37 ◦ C in a 5% CO2 atmosphere. After 10 days of incubation, the resultant colonies were stained with 0.005% crystal violet and counted manually. Mean values of triplicate sets, with respect to non-transfected control, were obtained to determine the colony forming efficiency.

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2.10.4. Statistical analysis Quantitative data were analyzed using Student t test. Differences were considered significant at p value 2 log reduction in infectious virus output was observed (Fig. 4C). These results suggested that not only a single serotypespecific siRNA, but also an siRNA cocktail targeting the four DENV serotypes can be encapsulated in D2C protein and functionally delivered into cells and inhibit DENV infection, and D2C encapsulated antiviral siRNAs can remain functionally active after long term storage at 4 ◦ C. 3.5. D2C:siRNA 201.3.4 induces viral RNA degradation and inhibits viral antigen burden in myeloid cells Viral genomic RNA targeted RNAi is expected to result in the degradation of viral RNA. Since myeloid cells, such as macrophages and dendritic cells, are the major cell types that support DENV replication in vivo (Kyle et al., 2007; Prestwood et al., 2012; Jessie et al., 2004), we tested whether DENV2 genomic RNA degradation can be induced in the myeloid cell line, K-562, by D2C:siRNA 201.3.4


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Fig. 4. Evaluation of D2C protein as a carrier for siRNA targeted against DENV2. (A) Huh-7 cells were treated with DENV2 specific siRNA162 and equimolar concentration of EMCV specific control siRNA14621 encapsulated in HiPerfect (H:162, H:14621, respectively) or D2C protein (D2C:162 and D2C:14621, respectively). An Infected Control (IC) that was not treated with siRNA and Uninfected Control (UIC) were included. On the following day the cells were challenged with DENV2 at an m.o.i. of 10. Viral antigen positivity at 48 h post infection (h.p.i.), as a percentage of UIC, was determined by Flow cytometry (see Supplementary Fig. S3 for histogram overlays). Data shown represents average of 3 independent experiments. (B) C6/36 cells were treated with D2C:siRNA 201.3.4 or equimolar concentration of D2C:siRNA14621. After 24 h, the cells were challenged with DENV2 at an m.o.i. of 10. Progeny virus was harvested from the infected cells at the indicated time points, and titres were determined by plaque assay on BHK-21 cells. (C) D2C encapsulated siRNA 201.3.4 was stored at the indicated temperatures for 4 weeks and C6/36 cells were treated with the complexes. The following day the cells were challenged with DENV2 at an m.o.i. of 10. An uninfected control (UIC) which received no virus, and an Infected Control (IC) which received virus infection without D2C: siRNA pre-treatment, were included for comparison. At 24 h after infection, progeny virus was harvested by freeze–thaw method and viral titres determined by plaque assay on BHK-21 cells. ** P < 0.005.

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treatment prior to viral infection. As expected, >1 log reduction in viral RNA was observed in DENV2 infected K-562 cells pretreated with D2C:siRNA 201.3.4, but not in those pre-treated with equimolar concentration of D2C:siRNA 14621 (Table 1). The qPCR amplification curves for No Template Control (NTC) and the Standard samples (Supplementary Fig. S4A) and the analytes, viz., cDNA generated from total RNA from Uninfected Control (UIC), that from DENV2 Infected Control (IC) cells, that from DENV2 infected cells pre-treated with D2C:siRNA 14621 and that from DENV2 infected cells pre-treated with D2C:siRNA 201.3.4 (Supplementary Fig. S4B) are shown. Although NTC and UIC samples also showed amplification plots corresponding to high cycle numbers, their dissociation curves (Supplementary Fig. S4C and D) and Tm were different from those of the standards and the analytes, confirming that the validity of our qRTPCR assay. Based on this analysis, the average reduction in viral RNA copy numbers/␮g total RNA in

Table 1 Evaluation of viral RNA degradation mediated by D2C encapsulated siRNA 201.3.4 in K-562 cells. D2C mediated transfection No siRNA Negative control siRNA 14621 siRNA cocktail 201.3.4

Mean viral RNA copies/␮g total RNA (±SD) DENV2 infected

Mock infected

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ND ND ND

K562 cells were transfected with the indicated siRNA using D2C protein as the carrier. The cells were challenged with DENV2 at an m.o.i. of 10. Intracellular viral RNA burden at 72 h post infection (h.p.i.) was determined by qRTPCR. Data shown is average of each sample analyzed in triplicate sets. SD, standard deviation; ND, not detected.


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No siRNA siRNA 203 siRNA 162

Mean viral RNA copies/␮g total RNA (±SD) DENV2 infected

MOCK infected

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4.4 × 106 ± 1.5 × 105 2.6 × 105 ± 2.0 × 104 3.3 × 105 ± 1.2 × 104

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Huh-7 cells were transfected with the indicated DENV2 specific siRNAs or mock transfected with no siRNA, using D2C protein as the carrier. The cells were challenged with DENV2 at m.o.i. of 10 and 1.0. Intracellular viral RNA burden at 24 h post infection (h.p.i.) was determined by qRTPCR. Data shown is average of each sample analyzed in triplicate sets. SD, standard deviation; NA, not applicable; ND, not detected. 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385

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D2C:siRNA 201.3.4 pre-treated cells, compared to that in DENV2 infected control cells that were not pre-treated as well as compared to that in D2C:siRNA 14621 pre-treated cells, was statistically significant. In a separate experiment we tested whether D2C:siRNA 201.3.4 treatment inhibits viral antigen burden in K-562 cells, since viral RNA degradation is expected to result in a corresponding reduction in viral antigen levels. Since infection by any of the four serotypes of DENV can cause Dengue disease, we carried out this evaluation against representatives of the four serotypes. Interestingly, in this experiment, we observed 100% inhibition of DEN2 viral antigen burden (Fig. 5B) much higher than the expected levels based on the qRTPCR data mentioned above. It is possible that the qRTPCR assay picked up all viral RNA including those of defective viral particles and truncated viral RNA species, thereby showing only a little over 1 log reduction in the DEN2 viral RNA load in the D2C:siRNA 201.3.4 treated cells. Viral antigen burden corresponding to serotypes 4 and 3 were also inhibited by 90% (Fig. 5D) and 84% (Fig. 5C), respectively, by D2C:siRNA 201.3.4 pre-treatment. D2C:siRNA 201.3.4 was found to be least effective against serotype 1, with 62.5% inhibition (Fig. 5A). Taken together, the above results suggested that the D2C:siRNA 201.3.4 can inhibit the four serotypes of DENV, and the underlying mechanism appears to be siRNA 201.3.4 mediated degradation of viral genomic RNA. 3.6. D2C encapsulated serotype specific siRNAs induce viral RNA degradation in Huh-7 cells The liver cell line, Huh-7, is known to support DENV replication. Therefore we tested whether two DENV2 specific siRNAs, 203 and 162, encapsulated in D2C protein, can induce DEN2 viral RNA degradation in this cell line upon infection with DENV2. qRTPCR analysis showed >1 log reduction in viral RNA burden in D2C:siRNA 203 and D2C:siRNA 162 pre-treated cells compared to mock transfected control cells, at two different MOIs, 10 and 1.0 (Table 2). The qPCR standard curve used in this experiment is shown (Supplementary Fig. S5). This result confirmed that DENV specific siRNA delivered into cells using D2C protein can induce degradation of viral genomic RNA, presumably through the RNAi pathway. 3.7. siRNA 162 encapsulated in D2C based synthetic peptides fail to protect BHK-21 cells from DENV2 challenge In another experiment we tested whether synthetic peptides designed based on the amino acid sequence of D2C protein may be of use for siRNA mediated inhibition of DENV. For this we custom synthesized five peptides, designated GS51, GS52, GS53, GS54 and GS55, spanning the D2C protein sequence (see Section 2 for details). All the five peptides encapsulated and delivered Cy3 labelled siRNA

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162 into BHK-21 cells, as determined by fluorescence microscopy (Supplementary Fig. S6A). But siRNA 162, encapsulated in the five peptides, failed to protect BHK-21 cells from DENV2 challenge, unlike siRNA 162 encapsulated in either D2C protein or HiPerfect which gave significant protection (Supplementary Fig. S6B). Presumably, the above mentioned peptides lack the ability to release the internalized siRNA in the cytoplasm, unlike D2C protein. 3.8. D2C delivered siRNAs knock down AURK B and Rho A, leading to inhibition of cancer cell proliferation Next we examined whether D2C protein can encapsulate and deliver our previously reported siRNA 52 targeting Rho A (Kumar et al., 2011) and siRNA 26 targeting AURK B (Addepalli et al., 2010; Chile et al., 2014) into HepG2 cells, and specifically knock down the respective targets. As determined by qRTPCR analysis, siRNAs 52 and 26 encapsulated in D2C protein, specifically knocked down Rho A (Fig. 6A) and AURK B (Fig. 6B), respectively, in HepG2 cells. In another experiment, treatment of with D2C encapsulated siRNA 52 and siRNA 26, but not equimolar concentration of siRNA 14627, significantly inhibited proliferation of PC3 cells (Fig. 6C) and HepG2 cells (Fig. 6D) as determined by colony formation assay. The qPCR amplification plots for Rho A (Supplementary Fig. S7) and those for AURK B (Supplementary Fig. S8) are shown. These results suggested that D2C protein can also be used as an siRNA carrier for anti cancer RNAi applications. 4. Discussion The potential of siRNA molecules as novel drugs is widely accepted, but their delivery into target cells is an impediment for clinical development of RNAi based drugs (Gavrilov and Saltzman, 2012; Burnett et al., 2011). Virus based delivery agents are generally considered to be highly efficient (Liu and Berkhout, 2011; Usme Ciro et al., 2013; Wei et al., 2009; Chou et al., 2010; Choi et al., 2013; Rao et al., 2013). The ability of viral capsids to assemble into nanoparticles in the presence of DNA oligonucleotides (Lopez et al., 2009; Tellinghuisen et al., 1999), yeast tRNA (Tellinghuisen et al., 1999), or siRNA (Choi et al., 2013) is known. Hepatitis B (Choi et al., 2011; 2013) and JC (Chou et al., 2010) Virus like particles (VLPs) have also been reported to deliver siRNA in vitro and in vivo, resulting in specific knockdown of the desired cellular target. pH shift assembly of adenoviral serotype 5 capsid protein nanosystems has been used for macromolecular delivery (Rao et al., 2013). Recently, E. coli expressed D2C protein and a D2C based peptide, designated Pep M, were shown to encapsulate and deliver siRNA into cultured cell lines, but target knockdown data was restricted to TLR-3 in endothelial cells (Freire et al., 2013, 2014). The utility of D2C protein or its peptide derivatives for siRNA delivery in the context of any particular disease indication is not known at present. Therefore, the significance of the present study lies in the fact that it presents first time in vitro proof of concept on the potential utility of this protein as an siRNA delivery agent in the context of two diseases, viz., DENV infection and cancer cell proliferation. 4.1. D2C protein as an siRNA carrier for anti Dengue viral application Our specific interest in exploring the potential utility of D2C protein for DENV targeted RNAi application was triggered by the reported potential of this protein to assemble into nucleo-capsid like particles (NLPs), of ∼30 nM in diameter, in the presence of DNA oligos (Lopez et al., 2009) and to induce immunity against DENV infection in a manner in which there may be no risk of Antibody Dependent Enhancement (ADE) (Gil et al., 2009; Lazo et al., 2007). Furthermore, the capsid being an internal protein in


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Fig. 5. Evaluation of D2C:201.3.4 mediated reduction of viral antigen levels in human myeloid cells infected by the four serotypes of DENV2. K-562 cells were transfected with siRNA201.3.4 targeting the four serotypes of DENV, encapsulated in D2C protein (D2C:201.3.4). Equimolar D2C:14621, IC (Infected control without siRNA pre-transfection) and UIC (uninfected control) were included in the assay. On the following day the cells were challenged with DENV serotypes 1, 2, 3, 4 at an m.o.i. of 10 and viral antigen positivity on the 3rd day of infection was determined by Flow cytometry. Closed histogram: UIC, Uninfected control cells; open histogram with black (print) or red (web) outline: Infected control with no siRNA treatment; open histogram with grey (print) or pink (web) outline: Infected cells with siRNA 14621 pre-treatment using D2C protein as the carrier; open histogram with grey (print) or green (web) dotted outline: Infected cells with siRNA 201.3.4 pre-treatment using D2C protein as the carrier.

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the flaviviral architecture, which gets exposed only upon internalization into the host cells (Lindenbach and Rice, 2001), a Dengue patient’s serum may be expected to contain no or low levels of capsid specific antibodies. Therefore, we reasoned that it may be possible to simultaneously utilize recombinant D2C protein for siRNA based antiviral therapy as well as immunization against future infections. However, the first step towards this end is in vitro demonstration of proof of concept that siRNA encapsulated in the capsid protein of DEN virus can be used to inhibit DENV infection. Dengue disease can be caused by any of the four serotypes of DENV (Guzman et al., 2010; Kyle and Harris, 2008; Molyneux and Maitland, 2005), and severity of the disease is directly correlated to viremia in the infected host (Vaughn et al., 2000). While vaccination is the best strategy to combat human pathogens, the lack of an approved vaccine and the risks involved in conventional anti flavivirals such as Ribavirin necessitates the development of novel therapeutics against this emerging virus (Botting and Kuhn, 2012). The therapeutic potential of siRNA against viruses (Haasnoot and Berkhout, 2009; DeVincenzo et al., 2010), including DENV (Zhang et al., 2004; Ng et al., 2007; Wu et al., 2010; Subramanya et al., 2010; Caplen et al., 2002; Stein et al., 2011), is known. Although this virus has been recently reported to escape from antiviral siRNA (Kakumani et al., 2013), Stein et al. (2011) have shown reasonable amelioration of lethal DENV2 infection in the AG129

mouse model (Johnson and Roehrig, 1999; Shresta et al., 2006; Williams et al., 2009; Yauch and Shresta, 2008; Tan et al., 2010) by administering chemically modified siRNA encapsulated in Invivofectamine reagent (Invitrogen) by retro-orbital intravenous (i.v.) injection. Our D2C:siRNA encapsulation data was comparable to that reported earlier for a cell penetrating peptide (Subramanya et al., 2010) and the chimeric capsid protein of Hepatitis B virus (Choi et al., 2011). The intracellular distribution of Cy3 labelled siRNA delivered using our D2C protein preparation, in C6/36 cells and BHK-21 cells, appeared to be comparable to the observations of Freire et al. (2014) using the same protein derived from a different DENV2 strain (Freire et al., 2013), and those of Choi et al. (2011) using the Hepatitis B virus capsid protein, as the siRNA nanocarrier. We have successfully demonstrated that DENV2 targeted siRNA, encapsulated in D2C, can inhibit DENV2 infection with comparable efficiency as that of a widely used commercial siRNA transfection reagent, HiPerfect, in the human liver cell line, Huh-7. This is significant because elevated transaminase levels in the serum of Dengue patients suggest the possible impact of DENV infection on liver function. Hepatocytes are one of the host cell types that support DENV infection (Lin et al., 2000; Brandler et al., 2005) and ∼105 PFUs of virus/g liver tissue has been reported in DENV2 infected AG129 mice (Shresta et al., 2006). Furthermore, developing DENV2 targeted antiviral therapeutics is important because serotype 2 has


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Fig. 6. Evaluation of D2C:201.3.4 mediated knockdown of Rho A and Aurora Kinase B and inhibition of cell proliferation in cancer cells. (A) and (B) HepG2 cells were separately transfected with 10 nM each of siRNA 52 targeted to Rho A and siRNA 26 targeted to Aurorakinase B (AURK B), using HiPerfect reagent (comparator) and D2C protein as the transfection agents. Equimolar concentration of D2C encapsulated siRNA 14627 was used as a control for comparison. At 72 h after transfection, percent knock down of Rho A (A) and AURK B mRNA (B), compared to ␤ Actin internal control mRNA, was determined by quantitative realtime RTPCR (qRTPCR), relative quantitation, as described under Section 2. C&D: HepG2 cells were separately transfected with 10 nM each of siRNA 52 and siRNA 26, or equimolar concentration of siRNA 14627, using D2C protein as transfection reagent. Cell proliferation was determined by Clonogenic assay as described under Section 2. Colony formation, as a percentage of non-transfected control cells (%CFU) for each siRNA is shown. *** P < 0.0001 compared to Untreated control. H, HiPerfect.

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been shown to be more virulent than the other three serotypes in mice (Schul et al., 2007; Rajmane et al., 2013). Myeloid cells constitute the major host cell type that supports DENV replication in vivo (Kyle et al., 2007; Prestwood et al., 2012; Jessie et al., 2004). We have shown that a single siRNA cocktail, encapsulated in D2C, can inhibit the four serotypes of DENV in the myeloleukemic cell line, K-562. We have also demonstrated that D2C:siRNA 201.3.4 mediated inhibition of DENV in this cell line is due to viral RNA degradation, which is the expected mechanism of action of antiviral siRNA in the cell. Interestingly, D2C:siRNA complexes were found to remain functionally stable for a long time, and when stored under appropriate conditions, can inhibit infectious virus output by 2 logs compared to a control siRNA encapsulated in D2C protein. These data confirm that D2C protein has the potential to be developed as a carrier for antiviral siRNAs targeting multiple serotypes of DENV in major host cells.

An interesting observation of this study is that five D2C based cell penetrating synthetic peptides, GS51 to GS55, encapsulate siRNA in a comparable fashion as the full length D2C protein and deliver DENV2 specific siRNA into BHK21 cells, but only siRNA delivered using full length D2C protect the cells from DENV2 challenge. The amino acid sequence of peptide GS52 was almost identical to peptide PepM (Freire et al., 2013, 2014), except for the additional two capsid residues at the N terminus and a stretch of six positively charged residues at the C terminus (Supplemetary information-II). Presumably, the extra 8 amino acids in GS52 may be a disadvantage for effective siRNA release after intracellular delivery. 4.2. D2C as an siRNA carrier for anti cancer application A previous study from our laboratory has reported siRNA 52 targeting Rho A GTPase in the context of angiogenesis. Using HiPerfect (Qiagen) as the transfection reagent, we have shown that siRNA 52


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mediated Rho A knockdown results in the inhibition of actin stress fiber formation in endothelial (Huvec) cells (Kumar et al., 2011). Rho A is also known to be important in the proliferation of cervical cancer cells (Liu et al., 2014). In two other studies reported from our laboratory, siRNA 26 targeting Aurorakinase B (AURK-B) in the prostate cancer cell line, PC3 (Addepalli et al., 2010), and the hepatocellular carcinoma cell line, HepG2 (Chile et al., 2014), was reported. Using HiPerfect as the transfection reagent, these studies have shown that siRNA 26 mediated knockdown of AURKB results in the inhibition of cancer cell proliferation. Therefore, in the second aspect of this study we have examined whether siRNAs 52 and 26, encapsulated in D2C protein, can knock down the respective targets in HepG2 cells. We also examined whether D2C:siRNA 52 and D2C:siRNA 26 treatment results in the inhibition of cancer cell proliferation using PC3 and HepG2 cells. We show that D2C encapsulated siRNA 52 and siRNA 26, delivered using D2C protein, specifically knock down their respective targets and inhibit cancer cell proliferation. These data showed preliminary proof of concept that D2C protein has the potential to be exploited for anticancer RNAi applications as well. 4.3. Yield of E. coli expressed D2C protein To obtain sufficient quantities of D2C protein for the experiments described in this study, we have adopted a fermentation strategy as described under Section 2. Previous studies that examined the yield of recombinant capsid proteins of two Flaviviruses in E. coli Codon Plus reported it to be 1–2 mg for Yellow Fever Virus and 3–5 mg/l for DENV2 Botting and Kuhn (2012). At shake flask level, using LB medium, we also observed that the D2C protein yield ranged between 1 and 5 mg/l, and the reason for this was the loss of cell viability associated with D2C expression (data not shown). In contrast, culturing of the bacteria under fermentation conditions considerably improved the yield of viable cells, and thereby the final yield of the purified D2C protein was enhanced to 15–20 mg/ml, although we do observe considerable loss of D2C protein in flow through fractions during downstream processing. We are aware that even this increased yield is not sufficient for using the protein for large scale in vivo studies, and further optimizations on the downstream process will be required to eliminate D2C protein loss in flow through fractions.

5. Conclusions In conclusion, the results of this study are not only consistent with the reported potential of D2C protein as a functional siRNA carrier, but also presents first time proof of concept that this capsid can be used as a delivery agent for siRNA against infection by the four serotypes of DENV and proliferation of different types of cancer cells. Thus the study presents an advancement over existing knowledge in the field of in vitro assembled viral capsid mediated siRNA delivery in the context of specific disease indications. Further studies using electron microscopy will be necessary to understand the structural features of the NLPs assembled by encapsulating siRNA in D2C protein. Furthermore, studies using animal models of DENV infection and cancer will be required to translate the above in vitro proof of concept data in vivo.

Acknowledgements

The study was funded by Reliance Life Sciences Pvt Ltd. TechniQ6 604 cal assistance by Shailendra Gaur, Shabih Shakeel, Vikas Trivedi and 605 Abhijeet Satwekar (former colleagues in our laboratory) are ack606 owledged. We thank Dr. Harinarayana Rao and Sameer Shaikh of 603 Q5

the Laboratory of Animal Research Services, Reliance Life Sciences Pvt. Ltd., for their help with mouse passaging of DEN viruses. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.jbiotec.2014.11.003. References Addepalli, M.K., Ray, K.B., Kumar, B., Ramnath, R.L., Chile, S., Rao, H., 2010. RNAimediated knockdown of AURKB and EGFR shows enhanced therapeutic efficacy in prostate tumor regression. Gene Ther. 17, 352–359. Botting, C., Kuhn, R.J., 2012. Novel approaches to flavivirus drug discovery. Expert Opin. Drug Discovery 7, 417–428. Brandler, S., Brown, N., Ermak, T.H., Mitchell, F., Parsons, M., Zhang, Z., Lang, J., Monath, T.P., Guirakhoo, F., 2005. Replication of chimeric yellow fever virusdengue serotype 1–4 virus vaccine strains in dendritic and hepatic cells. Am. J. Trop. Med. Hyg. 72, 74–81. Burnett, J.C., Rossi, J.J., Tiemann, K., 2011. Current progress of siRNA/shRNA therapeutics in clinical trials. Biotechnol. J. 6, 1130–1146. Caplen, N.J., Zheng, Z., Falgout, B., Morgan, R.A., 2002. Inhibition of viral gene expression and replication in mosquito cells by dsRNA-triggered RNA interference. Mol. Ther. 6, 243–251. Chile, S.A., Ray, K.B., Sameer, S., Rajagopal, V., Rao, H.S., Ramana, V., Kumar, A.S., 2014. Evaluation of target mRNA cleavage by Aurorakinase B specific siRNA in Prostate and Hepatic cancer cells and its therapeutic potential in mouse models of Liver cancer. Ind. J. Exp. Biol. (In press). Choi, K.M., Choi, S.H., Jeon, H., Kim, I.S., Ahn, H.J., 2011. Chimeric capsid protein as a nanocarrier for siRNA delivery: stability and cellular uptake of encapsulated siRNA. ACS Nano 22, 8690–8699. Choi, K.M., Kim, K., Kwon, I.C., Kim, I.S., Ahn, H.J., 2013. Systemic delivery of siRNA by chimeric capsid protein: tumor targeting and RNAi activity in vivo. Mol. Pharm. 10, 18–25. Chou, M., Hsieh, Y.F., Wang, M., Chang, J.T., Chang, D., Zouali, M., Tsay, G.J., 2010. In vitro and in vivo targeted delivery of IL-10 interfering RNA by JC virus-like particles. J. Biomed. Sci. 17, 51. DeVincenzo, J., Lambkin-Williams, R., Wilkinson, T., Cehelsky, J., et al., 2010. A randomized, double-blind, placebo-controlled study of an RNAi-based therapy directed against respiratory syncytial virus. PNAS USA 107, 8800–8805. Elbashir, S.M., Harborth, J., Weber, K., Tuschl, T., 2002. Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 26, 199–213. Freire, J.M., Veiga, A.S., Conceicao, T.M., Kowalczyk, W., Borges, R.M., Andreu, D., Santos, N.C., Da Poian, A., Castanho, M., 2013. Intracellular nucleic acid delivery by the supercharged dengue virus capsid protein. PLoS One 8, e81450. Freire, J.M., Veiga, A.S., Rego de Figueiredo, I., de la Torre, B.G., Santos, N.C., Andreu, D., Da Poian, A.T., Castanho, M.A., 2014. Nucleic acid delivery by cell penetrating peptides derived from dengue virus capsid protein: design and mechanism of action. FEBS J. 281, 191–215. Gavrilov, K., Saltzman, W.M., 2012. Therapeutic siRNA: principles, challenges, and strategies. Yale J. Biol. Med. 85, 187–200. Lindenbach, B.D., Rice, C.M., 2001. Flaviviridae. In: Knipe, D.M., Howley, P.M., Griffin, D.E., Lamb, R.A., Martin, M.A., Roizman, B., Straus, S.E. (Eds.), Fields Virology. Lippincott Williams & Wilkins, Philadelphia, PA, pp. 991–1041. Gil, L., Lopez, C., Lazo, L., Valdes, I., Marcos, E., Alonso, R., Gambe, A., Martin, J., Romero, Y., Guzman, M.G., Guillen, G., Hermida, L., 2009. Recombinant nucleocapsid-like particles fromdengue-2 virus induce protective CD41 and CD81 cells against viral encephalitis in mice. Int. Immunol. 21, 1175–1183. Guzman, M.G., Halstead, S.B., Artsob, H., Buchy, P., Farrar, J., Gubler, D.J., Hunsperger, E., Kroeger, A., Margolis, H.S., Martinez, E., Nathan, M.B., Pelegrino, J.L., Simmons, C., Yoksan, S., Peeling, R.W., 2010. Dengue: a continuing global threat. Nat. Rev. Microbiol. 8, S7–S16. Haasnoot, J., Berkhout, B., 2009. Nucleic acids-based therapeutics in the battle against pathogenic viruses. Handb. Exp. Pharmacol. 189, 243–263. Jessie, K., Fong, M.Y., Devi, S., Lam, S.K., Wong, K.T., 2004. Localization of Dengue virus in naturally infected human tissues by immunohistochemistry and in situ hybridization. JID 189, 1411–1418. Jones, C.T., Ma, L., Burgner, J.W., Groesch, T.D., Post, G.B., Kuhn, R.J., 2003. Flavivirus capsid is a dimeric alpha helical protein. J. Virol. 77, 7143–7149. Johnson, A., Roehrig, J.T., 1999. New mouse model for Dengue virus vaccine testing. J. Virol. 73, 783–786. Kakumani, P.K., Ponia, S.S., Rajgokul, K.S., Sood, V., Chinnappan, M., Banerjea, A.C., Medigeshi, G.R., Malhotra, P., Mukherjee, S.K., Bhatnagar, R.K., 2013. Role of RNA interference in Dengue virus replication and identification of NS4B as an RNAi suppressor. J. Virol. 87, 8870–8883. Kariko, K., Bhuyan, P., Capodici, J., Ni, H., Lubinski, J., Friedman, H., Weissman, D., 2004. Exogenous siRNA mediates sequence-independent gene suppression by signaling through toll-like receptor 3. Cells Tissues Organs 177, 132–138. Kenneth, J.L., Thomas, D.S., 2001. Analysis of relative gene expression data using real-time quantitative PCR and 2−Ct method. Methods 25, 402. Kumar, B., Chile, S.A., Ray, K.B., Reddy, G.E.C.V., Addepalli, M.K., Kumar, A.S., Ramana, V., Rajagopal, V., 2011. VEGF-C differentially regulates VEGF-A expression in


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ocular and cancer cells: promotes angiogenesis via RhoA mediated pathway. Angiogenesis 14, 371–380. Kyle, J.L., Beatty, P.R., Harris, E., 2007. Dengue virus infects macrophages and dendritic cells in a mouse model of infection. J. Infect. Dis. 195, 1808–1817. Kyle, J.L., Harris, E., 2008. Global spread and persistence of Dengue. Annu. Rev. Microbiol. 62, 71–92. Lazo, L., Hermida, L., Zulueta, A., Sanchez, J., Lopez, C., Silva, R., Guillen, G., Guzman, M.G., 2007. A recombinant capsid protein from Dengue-2 induces protection in mice against homologous virus. Vaccine 25, 1064–1070. Lin, Y.L., Liu, C.C., Lei, H.Y., Yeh, T.M., Lin, Y.S., Chen, R.M., Liu, H.S., 2000. Infection of five human liver cell lines by dengue-2 virus. J. Med. Virol. 60, 425–431. Liu, Y.P., Berkhout, B., 2011. MiRNA cassettes in viral vectors: problems and solutions. Biochim. Biophys. Acta 1809, 732–745. Liu, X., Chen, D., Liu, G., 2014. Overexpression of Rho A promotes the proliferation and migration of cervical cancer cells. Biosci. Biotechnol. Biochem. 7, 1–7. Lobigs, M., 1993. Flavivirus pre-membrane protein cleavage and spike heterodimer secretion require the function of the viral proteinase NS3. PNAS USA 101, 3414–3419. Lopez, C., Gil, L., Lazo, L., Menendez, I., Marcos, E., Sanchez, J., Valdes, I., Falcon, V., Rosa, M.C., Marquez, G., Guillen, G., Hermida, L., 2009. In vitro assembly of nucleocapsid like particles from purified recombinant capsid protein of Dengue2 virus. Arch. Virol. 154, 695–698. Ma, L., Jones, C.T., Groesch, T.D., Kuhn, R.J., Post, C.B., 2004. Solution structure of Dengue virus capsid protein reveals another fold. PNAS USA 101, 3414–3419. Molyneux, E.M., Maitland, K., 2005. Intravenous fluids—getting the balance right. N. Engl. J. Med. 353, 941–944. Morens, D.M., Halstead, S.B., Repik, P.M., Putvatana, R., Raybourne, N., 1985. Simplified plaque reduction neutralization assay for Dengue viruses by semi micro methods in BHK-21 cells: comparison of the BHK suspension test with standard Plaque reduction neutralization. J. Clin. Microbiol. 22, 250–254. Ng, C.Y., Gu, F., Phong, W.Y., Chen, Y.L., Lim, S.P., Davidson, A., Vasudevan, S.G., 2007. Construction and characterization of a stable subgenomic Dengue virus type 2 replicon system for antiviral compound and siRNA testing. Antiviral. Res. 76, 222–231. Okamoto, M., Hirai, H., Taniguchi, K., Shimura, K., Inaba, T., Shimazaki, C., Taniwaki, M., Imanishi, J., 2009. Toll-like Receptors are expressed by myeloid leukaemia cell lines, but fail to trigger differentiation in response to the respective TLR ligands. Br. J. Haematol. 147, 585–587. Prestwood, T.R., May, M.M., Plummer, E.M., Morar, M.M., Yauch, L.E., Shresta, S., 2012. Trafficking and replication patterns reveal splenic macrophages as major targets of dengue virus in mice. J. Virol. 86, 12138–12147. Rajmane, Y., Shaikh, S., Basha, K., Reddy, G.E.C.V., Nair, S., Kamath, S., Sreejesh, G., Rao, H., Ramana, V., Kumar, A.S., 2013. Infant mouse brain passaged Dengue serotype 2 virus induces non-neurological disease with inflammatory spleen collapse in AG129 mice after splenic adaptation. Virus Res. 173, 386–397. Rao, V.R., Upadhyay, A.K., Kompella, U.B., 2013. pH shift assembly of adenoviral serotype 5 capsid protein nanosystems for enhanced delivery of nanoparticles, proteins and nucleic acids. J. Controlled Release 172, 341–350. Schul, W., Liu, W., Xu, H., Flamand, M., Vasudevan, S.G., 2007. A Dengue fever viremia model in mice shows reduction in viral replication and suppression of inflammatory response after treatment with antiviral drugs. JID 195, 665–674.

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Shresta, S., Sharar, K.L., Prighozhin, D.M., Beatty, P.R., Harris, E., 2006. Murine model for Dengue virus induced lethal disease with increased vascular permeability. J. Virol. 80, 10208–10217. Srikantan, V., Valladres, M., Rhim, J.S., Moul, J.W., Srivastava, S., 2002. HEPSIN inhibits cell growth/invasion in prostate cancer cells. Cancer Res. 62, 6812. Stein, D.A., Perry, S.T., Buck, M.D., Oehmen, C.S., Fischer, M.A., Poore, E., Smith, J.L., Lancaster, A.M., Hirsch, A.J., Slifka, M.K., Nelson, J.A., Shresta, S., Fruh, K., 2011. Inhibition of dengue virus infections in cell cultures and in AG129 mice by an siRNA targeting a highly conserved sequence. J. Virol. 85, 10154–10166. Subramanya, S., Kim, S.S., Abraham, S., Yao, J., Kumar, M., Kumar, P., Haridas, V., Lee, S.K., Shultz, L.D., Greiner, D., Swamy, N., Shankar, P., 2010. Targeted delivery of siRNA to human dendritic cells to suppress Dengue viral infection and associated pro-inflammatory cytokine production. J. Virol. 84, 2490–2501. Swamy, M.N., Shankar, P., Kumar, P., Lee, S.K. Method to treat flavivirus infection with siRNA. US Pat. Appl. 20090047338A1, Oct, 2006. Tan, G.K., Ng, J.K., Trastei, S.L., Schul, W., Wip, G., Alonso, S., 2010. A non mouse adapted Dengue virus strain as a new model of severe Dengue infection in AG129 mice. PLoS Negl. Trop. Dis. 4, e673. Tellinghuisen, T.L., Hamburger, A.E., Fisher, B.R., Ostendorp, R., Kuhn, R.J., 1999. In vitro assembly of Alphavirus cores by using nucleocapsid protein expressed in Escherichia coli. J. Virol. 73, 5309–5319. Usme Ciro, J.A., Campillo-Pedroza, N., Almazán, F., Gallego-Gomez, J.C., 2013. Cytoplasmic RNA viruses as potential vehicles for the delivery of therapeutic small RNAs. Virol. J. 10, 185. Vaughn, D.W., Green, S., Kalayanarooj, S., Innis, B.L., Nimmannitya, S., Suntayakorn, S., Endy, T., Raengsakulrach, B., Rothman, A.L., Ennis, F.A., Nisalak, A., 2000. Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. JID 181, 2–9. Villegas-Rosales, P.M., Méndez-Tenorio, A., Ortega-Soto, E., Barron, B.L., 2012. Bioinformatics prediction of siRNAs as potential antiviral agents against Dengue viruses. Bioinformation 8, 519–522. Wei, B., Wei, Y., Zhang, K., Wang, J., Xu, R., Zhan, S., Lin, G., Wang, W., Liu, M., Wang, L., Zhang, R., Li, J., 2009. Development of an antisense RNA delivery system using conjugates of the MS2 bacteriophage capsids and HIV-1 TAT cell-penetrating peptide. Biomed. Pharmacother. 63, 313–318. Williams, K.L., Zompi, S., Beatty, P.R., Harris, E., 2009. A mouse model for studying dengue virus pathogenesis and immune response. Immunology and pathogenesis of viral hemorrhagic fevers. Ann. N.Y. Acad. Sci. 1171, E12–E23. Wu, X., Hong, H., Yue, J., Wu, Y., Li, X., Jiang, L., Li, L., Li, Q., Gao, G., Yang, X., 2010. Inhibitory effect of small interfering RNA on Dengue virus replication in mosquito cells. Virol. J. 7, 270. Yauch, L.E., Shresta, S., 2008. Mouse models of dengue virus infection and disease. Antiviral Res. 80, 87–93. Zhang, W., Singam, R., Hellerman, G., Kong, X., Juan, H.S., Lockey, R.F., Wu, S.J., Porter, K., Mohapatra, S.S., 2004. Attenuation of Dengue virus infection by AAV mediated siRNA delivery. Genet. Vaccines Ther. 2, 8. Yong, Y.K., Thayan, R., Chong, H.T., Tan, C.T., Sekara, S.D., 2007. Rapid detection and serotyping of Dengue virus by multiplex RT-PCR and real-time SYBR green RTPCR. Singapore Med. J. 48, 662.


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Generation and characterization of potential dengue vaccine candidates based on domain III of the envelope protein and the capsid protein of the four serotypes of dengue virus.

A small molecule inhibitor of dengue virus type 2 protease inhibits the replication of all four dengue virus serotypes in cell culture.

Properties and Functions of the Dengue Virus Capsid Protein.

Honokiol, a Lignan Biphenol Derived from the Magnolia Tree, Inhibits Dengue Virus Type 2 Infection.

Dengue virus serotype-2 impairs proliferation of healthy donors' T lymphocytes.

Isolation and characterization of dengue virus serotype 2 from the large dengue outbreak in Guangdong, China in 2014.

Understanding Dengue Virus Capsid Protein Interaction with Key Biological Targets.

Measurement of antibody-dependent infection enhancement of four dengue virus serotypes by monoclonal and polyclonal antibodies.

Direct and indirect interactions in the recognition between a cross-neutralizing antibody and the four serotypes of dengue virus.

Widespread signatures of local mRNA folding structure selection in four Dengue virus serotypes.

GNF-2 Inhibits Dengue Virus by Targeting Abl Kinases and the Viral E Protein.

Continued circulation of a single genotype of dengue virus serotype 2 in the Philippines.

Immunization of mice with recombinant vaccinia virus expressing authentic dengue virus nonstructural protein NS1 protects against lethal dengue virus encephalitis.

Structural insight and flexible features of NS5 proteins from all four serotypes of Dengue virus in solution.

Detection of dengue virus serotypes 1, 2 and 3 in selected regions of Kenya: 2011-2014.

Cissampelos pareira Linn: Natural Source of Potent Antiviral Activity against All Four Dengue Virus Serotypes.

Production of a Recombinant Dengue Virus 2 NS5 Protein and Potential Use as a Vaccine Antigen.

Full-length infectious clone of a low passage dengue virus serotype 2 from Brazil.

Isolation of dengue virus from the upper respiratory tract of four patients with dengue fever.

Phylogenetic analyses of the complete nucleotide sequence of the capsid protein (VP3) of Australian epizootic haemorrhagic disease of deer virus (serotype 2) and cognate genes from other orbiviruses.

36 cells and Aedes aegypti mosquitoes.

Dengue 2 virus protects mice from challenge by both dengue virus and JEV virulent virus.

Dengue Virus Serotype-2 Interferes with the Formation of Neutrophil Extracellular Traps.

Characterization of N-Glycan Structures on the Surface of Mature Dengue 2 Virus Derived from Insect Cells.