RNA Interference Mediated Inhibition of Dengue Virus Multiplication and Entry in HepG2 Cells
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{"title"=>"RNA interference mediated inhibition of dengue virus multiplication and entry in HepG2 cells", "type"=>"journal", "authors"=>[{"first_name"=>"Mohammed Abdelfatah", "last_name"=>"Alhoot", "scopus_author_id"=>"54413809700"}, {"first_name"=>"Seok Mui", "last_name"=>"Wang", "scopus_author_id"=>"36457885800"}, {"first_name"=>"Shamala Devi", "last_name"=>"Sekaran", "scopus_author_id"=>"23100953400"}], "year"=>2012, "source"=>"PLoS ONE", "identifiers"=>{"pmid"=>"22457813", "sgr"=>"84863344879", "doi"=>"10.1371/journal.pone.0034060", "scopus"=>"2-s2.0-84863344879", "pui"=>"364494667", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "issn"=>"19326203"}, "id"=>"5d2dcc7a-c775-3511-93a3-a66f59690439", "abstract"=>"BACKGROUND: Dengue virus-host cell interaction initiates when the virus binds to the attachment receptors followed by endocytic internalization of the virus particle. Successful entry into the cell is necessary for infection initiation. Currently, there is no protective vaccine or antiviral treatment for dengue infection. Targeting the viral entry pathway has become an attractive therapeutic strategy to block infection. This study aimed to investigate the effect of silencing the GRP78 and clathrin-mediated endocytosis on dengue virus entry and multiplication into HepG2 cells.\\n\\nMETHODOLOGY/PRINCIPAL FINDINGS: HepG2 cells were transfected using specific siRNAs to silence the cellular surface receptor (GRP78) and clathrin-mediated endocytosis pathway. Gene expression analysis showed a marked down-regulation of the targeted genes (87.2%, 90.3%, and 87.8% for GRP78, CLTC, and DNM2 respectively) in transfected HepG2 cells when measured by RT-qPCR. Intracellular and extracellular viral RNA loads were quantified by RT-qPCR to investigate the effect of silencing the attachment receptor and clathrin-mediated endocytosis on dengue virus entry. Silenced cells showed a significant reduction of intracellular (92.4%) and extracellular viral RNA load (71.4%) compared to non-silenced cells. Flow cytometry analysis showed a marked reduction of infected cells (89.7%) in silenced HepG2 cells compared to non-silenced cells. Furthermore, the ability to generate infectious virions using the plaque assay was reduced 1.07 log in silenced HepG2 cells.\\n\\nCONCLUSIONS/SIGNIFICANCE: Silencing the attachment receptor and clathrin-mediated endocytosis using siRNA could inhibit dengue virus entry and multiplication into HepG2 cells. This leads to reduction of infected cells as well as the viral load, which might function as a unique and promising therapeutic agent for attenuating dengue infection and prevent the development of dengue fever to the severe life-threatening DHF or DSS. Furthermore, a decrease of viremia in humans can result in the reduction of infected vectors and thus, halt of the transmission cycle.", "link"=>"http://www.mendeley.com/research/rna-interference-mediated-inhibition-dengue-virus-multiplication-entry-hepg2-cells", "reader_count"=>59, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>2, "Student > Doctoral Student"=>5, "Researcher"=>14, "Student > Ph. D. Student"=>16, "Student > Postgraduate"=>4, "Student > Master"=>8, "Other"=>1, "Student > Bachelor"=>7, "Lecturer"=>1, "Lecturer > Senior Lecturer"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>2, "Student > Doctoral Student"=>5, "Researcher"=>14, "Student > Ph. D. Student"=>16, "Student > Postgraduate"=>4, "Student > Master"=>8, "Other"=>1, "Student > Bachelor"=>7, "Lecturer"=>1, "Lecturer > Senior Lecturer"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>4, "Biochemistry, Genetics and Molecular Biology"=>4, "Medicine and Dentistry"=>4, "Agricultural and Biological Sciences"=>38, "Neuroscience"=>1, "Pharmacology, Toxicology and Pharmaceutical Science"=>1, "Business, Management and Accounting"=>1, "Veterinary Science and Veterinary Medicine"=>1, "Social Sciences"=>1, "Computer Science"=>2, "Immunology and Microbiology"=>2}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>4}, "Neuroscience"=>{"Neuroscience"=>1}, "Social Sciences"=>{"Social Sciences"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>38}, "Computer Science"=>{"Computer Science"=>2}, "Business, Management and Accounting"=>{"Business, Management and Accounting"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>4}, "Unspecified"=>{"Unspecified"=>4}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>1}, "Veterinary Science and Veterinary Medicine"=>{"Veterinary Science and Veterinary Medicine"=>1}}, "reader_count_by_country"=>{"United States"=>1, "United Kingdom"=>2, "Mexico"=>1, "Switzerland"=>1, "Indonesia"=>1, "French Polynesia"=>1}, "group_count"=>5}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/663783"], "description"=>"<p>HepG2 cells transfection experiment was optimized by treating the HepG2 cells with increasing amount of transfection reagent and concentrations of siRNA. (a) Result shows that the efficient silencing with minimal cytotoxicity was achieved using 1.0 µl of transfection reagent. (b) HepG2 cells transfected with increased concentration of GRP78 siRNAs and the result shows no evidence of toxicity regardless of the siRNA concentration as compared with scramble siRNA transfected HepG2 cells (One-way ANOVA with Dunnett's post-test, P>0.05). The optimal siRNA concentration is 50 nM. Similar results were observed in (c) CLTC siRNAs and (d) DNM2 siRNAs. (e) HepG2 cells were transfected and exposed to optimal siRNAs concentration (50 nM) that target GRP78, CLTC, and DNM2 in separated and combined transfection. Cytotoxicity was tested by measuring LDH level and results were confirmed by counting of viable cells using Trypan Blue exclusion assay. No evidence of cytotoxicity was observed for all pools of siRNA as well as in combined transfection (91.6%±2.0, 90.7%±1.9, 90.5%±0.4, and 89.9%±1.8 for GRP78, CLTC, DNM2, and combined transfection respectively). Trypan Blue exclusion assay showed also more than 90% viable cells. No significant difference was observed between separated and combined transfection by One-way ANOVA analysis (P>0.05). Results are expressed as mean ± SD from a representative experiment performed in triplicate.</p>", "links"=>[], "tags"=>["optimization", "sirna"], "article_id"=>334271, "categories"=>["Molecular Biology", "Chemistry", "Genetics", "Infectious Diseases"], "users"=>["Mohammed Abdelfatah Alhoot", "Seok Mui Wang", "Shamala Devi Sekaran"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0034060.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Cytotoxicity_and_optimization_of_siRNA_transfection_/334271", "title"=>"Cytotoxicity and optimization of siRNA transfection.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 07:23:59"}
  • {"files"=>["https://ndownloader.figshare.com/files/663897"], "description"=>"<p>Each of target genes was targeted with a specific pool of siRNA. An efficient gene silencing was achieved in both separated and combined transfection when compared with non-transfected control and normalized to reference genes (One-way ANOVA with Dunnett's post-test, P<0.0001, Results are expressed as mean ± SD from a representative experiment performed in triplicate). There are no significant differences between separated transfection (87.1%±0.9, 90.5%±0.5, and 87.4%±1.0) and combined transfection (87.2%±1.0, 90.3%±0.9, and 87.8%±1.6) on gene expression of GRP78, CLTC, and DNM2 respectively (Two-way ANOVA with Bonferroni post-test, P>0.05, Results are expressed as mean ± SD from a representative experiment performed in triplicate). Scrambled siRNA control had no inhibitory effect on any gene expression, and a similar expression to the non-transfected control was observed (One-way ANOVA with Dunnett's post-test, P>0.05).</p>", "links"=>[], "tags"=>["genetics and genomics", "chemistry", "Infectious diseases", "molecular biology"], "article_id"=>334381, "categories"=>["Molecular Biology", "Chemistry", "Genetics", "Infectious Diseases"], "users"=>["Mohammed Abdelfatah Alhoot", "Seok Mui Wang", "Shamala Devi Sekaran"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0034060.g002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Efficiency_of_silencing_target_genes_/334381", "title"=>"Efficiency of silencing target genes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 07:24:35"}
  • {"files"=>["https://ndownloader.figshare.com/files/663974"], "description"=>"<p>Silenced and non-silenced HepG2 cells were infected by DENV-2 at MOI of 2. Result showed a marked reduction in percentage of infected cells by flow cytometry. This figure shows the percentage of DENV infected cells at different conditions. (a) Transfected non-infected HepG2 cells (0.0%) represent the negative control. (b) Transfected mock-infected HepG2 cells as a staining control (0.0%). (c) Scramble siRNA transfected infected HepG2 cells (45.7%) as a positive control. (d) GRP78 silenced infected HepG2 cells (8.5%). (e) CLTC silenced infected HepG2 cells (8.5%). (f) DNM2 silenced infected HepG2 cells (15.2%). (g) GRP78, CLTC, and DNM2 combined silenced infected HepG2 cells (4.7%). (h) Summarized the results of the flow cytometry experiments. Data is expressed as a percentage of infected cells compared with scramble siRNA transfected infected HepG2 cells which was defined as 100%. The percentages of the infected cells are 18.7%±0.7, 18.6%±2.6, 33.4%±7.9, and 10.3%±3.9 for GRP78, CLTC, DNM2, and combined silenced HepG2 cells respectively (One-way ANOVA with Dunnett's post-test, P<0.0001, Results are expressed as mean ± SD from a representative experiment performed in triplicate). Also, statistical analysis shows no significant difference between scramble siRNA transfected and non-transfected infected HepG2 cells (One-way ANOVA with Dunnett's post-test, P>0.05).</p>", "links"=>[], "tags"=>["infected", "cells"], "article_id"=>334461, "categories"=>["Molecular Biology", "Chemistry", "Genetics", "Infectious Diseases"], "users"=>["Mohammed Abdelfatah Alhoot", "Seok Mui Wang", "Shamala Devi Sekaran"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0034060.g003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Quantification_of_infected_cells_by_flow_cytometry_/334461", "title"=>"Quantification of infected cells by flow cytometry.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 07:25:01"}
  • {"files"=>["https://ndownloader.figshare.com/files/664122"], "description"=>"<p>Viral RNA levels were quantified by RT-qPCR and normalized to reference gene (RPL27). Data is expressed as relative-fold expression to scramble siRNA transfected HepG2 cells control, which defined as 1.0 fold. Intracellular viral RNA load reduced (0.72 fold ±0.11), (0.67 fold ±0.04), (0.61 fold ±0.04), and (0.92 fold ±0.004) in GRP78, CLTC, DNM2, and combined silenced HepG2 cells respectively (One-way ANOVA with Dunnett's post-test, P<0.0001, Results are expressed as mean ± SD from a representative experiment performed in triplicate). No significant difference between scramble siRNA transfected and non-transfected infected HepG2 cells (One-way ANOVA with Dunnett's post-test, P>0.05). (TNI, Transfected Non-Infected; NTI, Non-Transfected Infected; NTNI, Non-Transfected Non-Infected).</p>", "links"=>[], "tags"=>["intracellular", "dengue", "rna"], "article_id"=>334607, "categories"=>["Molecular Biology", "Chemistry", "Genetics", "Infectious Diseases"], "users"=>["Mohammed Abdelfatah Alhoot", "Seok Mui Wang", "Shamala Devi Sekaran"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0034060.g004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Quantification_of_intracellular_dengue_virus_RNA_load_/334607", "title"=>"Quantification of intracellular dengue virus RNA load.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 07:25:45"}
  • {"files"=>["https://ndownloader.figshare.com/files/664213"], "description"=>"<p>RT-qPCR was used to quantify the DENV RNA in the culture supernatant of silenced and non-silenced HepG2 cells. Marked reduction in viral RNA was achieved (65.1%±1.7), (65.4%±9.8), (60.9%±10.6), and (71.4%±5.7) for GRP78, CLTC, DNM2, and combined silenced HepG2 cells respectively. This result is statistically significant (One-way ANOVA with Dunnett's post-test, P<0.0001, Results are expressed as mean ± SD from a representative experiment performed in triplicate). There is no significant difference between scramble siRNA transfected and non-transfected infected HepG2 cells (One-way ANOVA with Dunnett's post-test, P>0.05). (TNI, Transfected Non-Infected; NTI, Non-Transfected Infected; NTNI, Non-Transfected Non-Infected).</p>", "links"=>[], "tags"=>["extracellular", "dengue", "rna"], "article_id"=>334704, "categories"=>["Molecular Biology", "Chemistry", "Genetics", "Infectious Diseases"], "users"=>["Mohammed Abdelfatah Alhoot", "Seok Mui Wang", "Shamala Devi Sekaran"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0034060.g005"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Quantification_of_extracellular_dengue_virus_RNA_load_/334704", "title"=>"Quantification of extracellular dengue virus RNA load.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 07:26:15"}
  • {"files"=>["https://ndownloader.figshare.com/files/664296"], "description"=>"<p>The ability to produce infectious virions was investigated by plaque assay. Figure shows marked reduction of the plaque forming units (0.56 log), (0.41 log), (0.34 log), and (1.07 log) for GRP78, CLTC, DNM2, and combined silenced HepG2 cells respectively compared to scramble siRNA transfected cells. All plaques were counted at day 7 of incubation. Plaques are expressed as plaque forming unit per mL (pfu/mL). This result is statistically significant (One-way ANOVA with Dunnett's post-test, P<0.0001, Results are expressed as mean ± SD from a representative experiment performed in triplicate) and there is no significant difference between scramble siRNA transfected and non-transfected infected HepG2 cells (One-way ANOVA with Dunnett's post-test, P>0.05). (TNI, Transfected Non-Infected; NTI, Non-Transfected Infected; NTNI, Non-Transfected Non-Infected).</p>", "links"=>[], "tags"=>["plaque", "forming", "infectious", "virions"], "article_id"=>334782, "categories"=>["Molecular Biology", "Chemistry", "Genetics", "Infectious Diseases"], "users"=>["Mohammed Abdelfatah Alhoot", "Seok Mui Wang", "Shamala Devi Sekaran"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0034060.g006"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Quantification_of_plaque_forming_infectious_virions_in_culture_supernatant_/334782", "title"=>"Quantification of plaque forming infectious virions in culture supernatant.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 07:26:39"}
  • {"files"=>["https://ndownloader.figshare.com/files/664383"], "description"=>"<p>Primer sequences of the target genes, positive control gene, optimal reference genes and DENV NS5.</p>", "links"=>[], "tags"=>["genetics and genomics", "chemistry", "Infectious diseases", "molecular biology"], "article_id"=>334875, "categories"=>["Molecular Biology", "Chemistry", "Genetics", "Infectious Diseases"], "users"=>["Mohammed Abdelfatah Alhoot", "Seok Mui Wang", "Shamala Devi Sekaran"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0034060.t002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Primer_sequences_/334875", "title"=>"Primer sequences.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-02-20 07:27:06"}
  • {"files"=>["https://ndownloader.figshare.com/files/664421"], "description"=>"<p>Three siRNAs for each target gene were designed. The experiment includes also one Positive control siRNA for GAPDH gene and one scramble siRNA.</p>a<p>The position refers to the siRNA sequence position on the target gene.</p>b<p>Scrambled siRNA is a control used to markup any changes to the gene expression profile that may result from the siRNA delivery method.</p>", "links"=>[], "tags"=>["sirna", "oligonucleotide"], "article_id"=>334909, "categories"=>["Molecular Biology", "Chemistry", "Genetics", "Infectious Diseases"], "users"=>["Mohammed Abdelfatah Alhoot", "Seok Mui Wang", "Shamala Devi Sekaran"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0034060.t001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Sequences_of_siRNA_oligonucleotide_template_/334909", "title"=>"Sequences of siRNA oligonucleotide template.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-02-20 07:27:17"}

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Relative Metric

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