P. Knowlesi erythrocyte invasion assay


Blood was collected in 10% citrate phosphate dextrose (CPD) and stored at 4°C unwashed for up to 4 weeks, or washed in RPMI with malaria supplements and stored in malaria culture medium at 50% hematocrit for up to 2 weeks. The DARC+ human erythrocytes used in the erythrocyte binding assay and the P. knowlesi erythrocyte invasion assay had the phenotype Fy(a-b+) as determined by standard blood banking methods using anti-Fya and anti-Fyb antisera (Gamma Biologicals, Houston, TX). Erythrocytes were washed three times in DMEM (Gibco BRL) and resuspended to a hematocrit of 10% in complete DMEM for the erythrocyte binding assay. Erythrocytes used in the P. knowlesi erythrocyte invasion assay were washed three times and resuspended to a hematocrit of 10% using malaria complete RPMI.
Percoll purification of schizont-infected erythrocytes

Cultures of P. knowlesi at 5-10% infected erythrocytes were washed three times in RPMI with malaria supplements and 10% FBS and brought up to a hematocrit of 10%. A 50% Percoll solution was made by adding 0.45 vol 1× PBS, 0.05 vol 10× PBS and 0.5 vol Percoll (Sigma). Two ml of the washed culture was overlaid on 2 ml of the 50% Percoll solution in a 4 ml polystyrene tube and centrifuged for 20 min at 2100 RPM in a Sorvall centrifuge. The ring of cells at the interface was removed, pooled and washed three time in 1× PBS. The pellet was brought up in malaria culture medium to 2 × 107 cells/ml.

P. Knowlesi erythrocyte invasion assay

Human Duffy Fy(a-b+) erythrocytes were washed in complete malaria medium and 2 × 107 washed cells were added to increasing concentrations of sulfated polysaccharide in malaria culture medium at final volume of 900 μl for 1 h at room temperature. To each tube of sulfate polysaccharide-treated erythrocytes, 100 μl or 2 × 106 schizont-infected erythrocytes was added and placed in a well of a polystyrene 24-well plate (Becton-Dickinson). The cultures were maintained under a blood-gas atmosphere at 38°C for 8 h to allow the infected erythrocytes to rupture and release free merozoites capable of infecting new erythrocytes and developing to ring-stage trophozoites. The culture was centrifuged at 2100 RPM for 3 min and a thin smear was made from the pellet. The thin smear was fixed with methanol and stained with Leukostat Solution B (100 mg Eosin Y+ 300 μl 37% formaldehyde +400 mg sodium phosphate dibasic +500 mg potassium phosphate monobasic, q.s. to 100 ml with dH2O), rinsed, and stained with Leukostat Solution C (47 mg Methylene Blue +44 mpp Azure A +400 mg sodium phosphate dibasic +500 mg potassium phosphate monobasic, q.s to 100 ml with dH2O). The percentage of erythrocytes infected with ring-stage trophozoites per 2000 erythrocytes was determined at 1000×. Percentage inhibition of invastion was determined by dividing the percentage of ring-stage parasites at each polyanion concentration by the percentage of ring-stage parasites at 0 μg/ml of the polyanion, multiplying by 100 and subtracting this value from 100.

Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein. Part 2

One possible function of the HBM in chemokines, HIV and DBPs is to associate with cell surface proteoglycans. Alternatively, HBMs could participate in binding to negatively charged amino acid side chains on the chemokine receptors. RANTES is known to bind to sulfated polysaccharides as part of its processing and function, but tyrosine sulfation of CCR5 is also important for binding of chemokines and HIV, and sulfation of Tyr 41 on DARC is important for DBP binding. Here we designed a peptide from PvDBP subdomain 1 that contains the HBM, tested its ability to bind sulfated polysaccharides, and compared it to the binding of the PvDBP, PkDBP, P. knowlesi β and γ proteins, HIV V3 loop peptides and RANTES to see if they shared similar binding specificities.


Ca-spirulan, Na-spirulan, and Na-hornan (Na-HOR) were kindly provided by Toshimutsu Hayashi, Department of Virology, Toyama Medical and Pharmaceutical University, Sugitani, Toyama, Japan. Heparin, dextran sulfate, and pentosan polysulfide were obtained from Sigma-Aldrich (St. Louis, MO).

Peptide preparation

Peptides based on the wild type (wt) putative polyamine binding site of the PvDBP and a non-binding mutant, pvR22KARA (Figure 1) were obtained from Gene med Synthesis, Inc. (San Francisco, CA). The synthesis included N-terminal fluoresce in conjugation and HULK purification to greater than 80%. Peptides of the V3 loop were obtained from the NHI AIDS Reagent Program (NHI AIDS Reagent Program, Rockville, Md.)

Heparin-sepharose columns

The binding affinity of the PvDBP HBM and V3 loop peptides for heparin was determined by chromatography on a heparin-Sepharose column. Heparin-Sepharose CL-6B beads (Pharmacia Biotech) were swollen in 50 mM Tris-HCl pH 7.5 (column buffer), degassed for 1 h, and 1 ml of slurry was added to a 10 ml column. The column was equilibrated with 10 volumes of column buffer. Peptides were added at 1 mg/ml in 300 μl and allowed to enter the column. The column was washed with 3 ml of column buffer. The peptide was eluted with 3 ml volumes of increasing NaCl concentrations of 0.01, 0.15, 0.5, 1.0 and 2.0 M, and 0.5 ml fractions were collected. The column was regenerated between peptides by adding alternating 3 ml volumes of 0.1 M Tris-HCl, 0.5 M NaCl, pH 8.5 and 0.1 M NaOAc, 0.5 M NaCl, pH 5.0 for three cycles. The column was re-equilibrated with 10 vol. of column buffer before adding the next peptide. Fractions were measured for absorbance at 280 nm on a spectrophotometer.

P. Knowlesi in vitro culture

Whole blood from rhesus macaques was collected in 10% CPD and allowed to separate overnight at 4°C. The erythrocyte phase was washed in RPMI with L-glutamine and supplemented with 25 mM HEPES, 300 μM hypoxanthine, 10 μM thymidine, 1.0 mM sodium pyruvate, and 11 mM glucose. This RPMI with malaria supplements was then used to prepare malaria culture medium by adding to a final concentration of 0.24% sodium bicarbonate and 0.2% Albumax-I (Life Tech, Gibco BRL). Cultures were maintained at a hematocrit of 10% in malaria culture medium under an atmosphere of 5% O2, 5% CO2, balanced N2 (Air Liquide, Houston, TX) at 38°C.

Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein


The HIV surface glycoprotein gp120 (SU, gp120) and the Plasmodium vivax Duffy binding protein (PvDBP) bind to chemokine receptors during infection and have a site of amino acid sequence similarity in their binding domains that often includes a heparin binding motif (HBM). Infection by either pathogen has been found to be inhibited by polyanions.


Specific polyanions that inhibit HIV infection and bind to the V3 loop of X4 strains also inhibited DBP-mediated infection of erythrocytes and DBP binding to the Duffy Antigen Receptor for Chemokines (DARC). A peptide including the HBM of PvDBP had similar affinity for heparin as RANTES and V3 loop peptides, and could be specifically inhibited from heparin binding by the same polyanions that inhibit DBP binding to DARC. However, some V3 peptides can competitively inhibit RANTES binding to heparin, but not the PvDBP HBM peptide. Three other members of the DBP family have an HBM sequence that is necessary for erythrocyte binding, however only the protein which binds to DARC, the P. knowlesi alpha protein, is inhibited by heparin from binding to erythrocytes. Heparitinase digestion does not affect the binding of DBP to erythrocytes.


The HBMs of DBPs that bind to DARC have similar heparin binding affinities as some V3 loop peptides and chemokines, are responsible for specific sulfated polysaccharide inhibition of parasite binding and invasion of red blood cells, and are more likely to bind to negative charges on the receptor than cell surface glycosaminoglycans.


The human immunodeficiency virus type 1 (HIV-1), the human malaria, Plasmodium vivax, and the monkey malaria, P. knowlesi, have ligands that bind to chemokine receptors and mediate cell invasion. The surface glycoprotein gp120 (SU) of HIV-1 binds to CCR5 and CXCR4 as the major coreceptors for infecting CD4+ T-lymphocytes in vivo, and changes in the amino acid sequence of the V3 loop of gp120 can change viral tropism from CCR5 using (R5) to CXCR4 using (X4) to both (R5X4). The V3 loop region of gp120 also provides a neutralizing epitope, and can bind glycosaminoglycans and other polyanions which inhibit viral infection.

P. vivax uses a Duffy binding protein (PvDBP) to bind the Duffy antigen receptor for chemokines (DARC) and invade human reticulocytes. P. knowlesi has three proteins, the P. knowlesi α, β, and γ proteins which can mediate binding to rhesus erythrocytes, and the P. knowlesi α protein (PkDBP) can bind to human and rhesus DARC. PvDBP, PkDBP, P. knowlesi β, and γ proteins are members of a Duffy Binding Ligand (DBL) family of erythrocyte binding proteins with conserved regions of homology which bind to many receptors. Region II within the family, as defined by conserved cysteine residues, is responsible for erythrocyte binding, and region II of PkDBP has been shown to be inhibited by glycosaminoglycan binding.

In a separate report, we describe an amino acid sequence similarity between subdomain 1 in DBP region II and the V3 loop of HIV strain MN. Within subdomain 1 and this V3 loop are consensus BBXB heparin binding motifs (HBM), where B is a basic amino acid and X is any amino acid. This HBM is conserved in many DBL family members, and we previously found that alanine substitutions at this site in PvDBP and PkDBP abrogated DARC binding. RANTES is a natural ligand of both CCR5 and DARC and can inhibit both HIV and DBP binding to their respective receptors. SDF-1 is a natural ligand for CXCR4, and both RANTES and SDF-1 have HBM and are known to bind sulfated polysaccharides.

Inhibitory effects of IRF-3ER dimerization on HCV JFH-1 virus replication. Part 2

Host immunity, including innate immunity and adaptive immunity, is an important and complicated system dedicated to the task of defending the host from microbial infection and cancer development. Innate immunity provides an immediate (first line) reply to a microbial infection, specifically for viral infections, while also controlling the later antigen-specific adaptive response. A key aspect of the antiviral innate immune response is the synthesis and secretion of type I INFs (α and β), which exhibit antiviral, anti-proliferative, and immunomodulatory functions. Two key steps are required to elicit an effective antiviral innate immune response: a. detection of the invading virus by immune system receptors; b. initiation of protein signaling cascades that regulate the synthesis of IFNs. Viruses are highly infectious pathogens that depend on host cellular machinery for survival and replication. Most viral infections, like the common cold caused by Rhinoviruses, are efficiently resolved by the host innate and adaptive immune system. For other viral infections, such as chronic hepatitis B or C viral infection, the host innate and adaptive immunity response is unable to clear them effectively and they become persistent infections. Several families of PPRs have been demonstrated to inspect the cellular micro-environment for microbial infection to target the pathogen-associated molecular patterns (PAMPs), a conserved structural moiety essential for microbial survival. Toll-like receptors (TLRs 3, 4, 7, 8, and 9) in addition to RIG-I are major PPRs that recognize different types of virally-derived nucleic acids or intracellular dsRNA to initiate signaling cascades leading to production of type I IFNs (details in reviews). The mechanisms by which different viruses induce a unique IFN-mediated antiviral response appear to require selective activation of members of the IRF family of proteins (IRF-1 to IRF-9). Thus far, IRF-3 and IRF-7 have been shown to be major regulators of IFN gene expression.

The type I IFNs, represented by multiple subtypes of IFN-α in addition to one subtype IFN-β, are key cytokines in this process, mounting an immediate antiviral response as well as adaptive immunity. IFN-mediated anti-viral effects are carried out using different mechanisms that are dependent on the type of viral infection, but these anti-viral effects are all dependent on IRF-3 activation. Activation of IRF-3 proteins appears to recruit the Tank Binding Kinase 1 (TBK1) and inhibitor of IκB-related kinase epsilon (IKKε) through their interaction with the RIG-I RNA helicase, resulting in phosphorylation of IRF-3, its dimerization, nuclear translocation, and transcriptional activation through binding to IFN-stimulated response elements (ISREs). Activated IRF-3 interacts with nuclear factor-κB (NF-κB) and transcriptional factor-2/c-Jun to form a transcriptionally active enhanceosome complex on IFNA1 and IFNB gene promoters.

In our studies, we utilized an IRF-3/mouse ER fusion protein expressing plasmid in order to achieve IRF-3ER activation in a cytokine/receptor-independent fashion. Our results demonstrated that IRF-3ER homodimers triggered the downstream pathways to produce IFN-α and IFN-β (Figure 2A and 2B). The anti-HCV effects, induced by 4-HT in Huh7.5-IRF3ER cells, were achieved by decreasing HCV RNA replication and HCV IRES-mediated translation. This is consistent with our previous studies which achieved activation of STAT1/and STAT3/mouse ER fusion proteins. Activation of the IRF-3ER fusion protein by 4-HT treatment provides strong evidence that this is necessary and sufficient to increase IFN-α and IFN-β expression in Huh7.5-IRF3ER cells. Our data showing that IRF-3ER activation triggers the downstream pathway, activating the JAK/STAT pathway and regulating ISG expression. Detection of p-STAT1 (S727) and p-STAT3 (Y705) in Huh7.5-IRF3ER cells provides a strong evidence for activation of Jak/STATs pathway by IFNs. Although the mechanism of IFN action against HCV replication has not been well defined, recent studies suggest that IFNs have a great impact on HCV replication by interrupting HCV IRES-mediated translation. Clinical data confirmed these findings in a study of HCV IRES-mediated translation in chronic HCV patients receiving IFN treatment, in which the efficiency of HCV IRES-mediated translation was reduced in IFN-treated HCV patients. In our study, the inhibitory effects of HCV RNA replication and HCV IRES-mediated translation were confirmed in Huh7.5-IRF3ER cells after treatment with 4-HT.

Inhibitory effects of IRF-3ER dimerization on HCV JFH-1 virus replication

Huh7.5-IRF3ER cells were further examined for its inhibitory effects on HCV JFH-1 viral replication after 4-HT treatment. Huh7.5-IRF3ER cells were inoculated with 0.5 MOI of JFH-1 virus stock and cultured for 14 days to achieve full HCV JFH-1 infected Huh7.5-IRF3ER cell state. The infected Huh7.5-IRF3ER cells were treated with 4-HT (1 μM) at the indicated times and harvested at the last-sample collection point for analysis of HCV RNA by real-time PCR. The infected Huh7.5-IRF3ER cells were used as control without 4-HT treatment for 72 hours. HCV JFH-1 replication decreased to 50% of control after 24 hours of 4-HT treatment. This data indicates that IRF-3ER dimerization after 4-HT treatment has inhibitory effects on HCV JFH-1 replication and was correlated with the production of IFN-α and IFN-β. To further separate HCV JFH-1 viral RNA replication and viral translation, the plasmid pRL-HL, containing Cap-dependent Renilla luciferase translation and HCV IRES-mediated Firefly luciferase translation start sites, was used in this study. After transfection of pRL-HL, Huh7.5-IRF3ER cell lysates were harvested at various times after 4-HT treatment for analysis of luciferase activity. In Figure 4B, both Cap-dependent and HCV IRES-mediated translation was reduced in Huh7.5-IRF3ER cells after 4-HT treatment in a time-dependent fashion. This data shows strong evidence that activation of the IRF-3ER fusion protein not only inhibits JFH-1 viral RNA replication but also inhibits Cap-dependent and HCV IRES-mediated translation.

Expression of ISGs in Huh7.5-IRF3ER cells

All of the IFN types activate JAK/STAT pathways, regulating the expression of over 300 ISGs in order to achieve their anti-viral effects. In our previous studies, we demonstrated a novel pathway by which IFN inhibits HCV IRES-mediated translation through up-regulating 1-8U gene expression and down-regulating expression of the hnRNP M gene (unpublished data). In this study, we measured 1-8U and hnRNP M expression in Huh7.5-IRF3ER cells with and without 4-HT treatment. The 1-8U protein was detected by Western blotting and was up-regulated in Huh7.5-IRF3ER cells after 4-HT treatment. Due to auto-dimerization of IRF-3ER fusion protein in Huh7.5-IRF3ER cells, the fold-induction of 1-8U protein is not as robust as described in our previous report in which the STAT1 gene was activated. Real-time quantitative reverse-transcription PCR was used to detect and measure hnRNP M mRNA expression in Huh7.5-IRF3ER cells. After 4-HT treatment, hnRNP M mRNA levels were down-regulated in a time-dependent fashion. This data confirms that activation of the IRF-3ER fusion protein triggers a cellular anti-HCV state through inducing IFNs production and regulating ISG expression.

Dimerization of IRF-3ER fusion protein induced by 4-HT in Huh7.5-IRF3ER cells

Activation of IRF-3 or IRF-7 is a critical step during virus infection, promoting the most potent type I IFN production. Previous studies showed the constitutively active forms (serines replaced by phosphomimetic aspartate amino acids) of human IRF-3 protein exerts the ability to modulate the apoptotic and anti-tumor properties after being delivered by recombinant adenovirus into macrophages. In our studies, a fusion protein of IRF-3 and C-terminal sequences (310 a.a.) of mouse estrogen receptor was used to establish the stable Huh7.5-IRF3ER cell line. In previous studies, mouse estrogen receptor was effective at inducing dimerization of STAT1 and STAT3 fusion proteins after 4-HT treatment. In these studies from us and others, 4-HT was titrated to a concentration of 1 μM that achieved the highest expression of STAT1ER and STAT3ER dimerization and the strongest inhibitory effects on HCV RNA replication. In our studies, 4-HT-treatment alone was also demonstrated to have no anti-HCV effects. In this study, similar sequences from the mouse ER C-terminal domain were fused to the C-terminus of the IRF-3 gene. In Figure 1, Western blotting with anti-IRF-3 antibody was used to detect the IRF-3 as well as the IRF-3ER monomer and dimer proteins. Lane 1 shows endogenous IRF3 protein (56.1 kd) but no IRF-3ER fusion protein in Huh 7.5 cells treated with 4-HT. Lane 2 shows both IRF-3 and IRF-3ER (monomer) (90 kd) in Huh7.5-IRF3ER cells without 4-HT treatment. Lane 3 shows that 4-HT treatment of Huh7.5-IRF3ER cells induces IRF-3ER fusion protein dimer formation (180 kd) in addition to IRF-3 protein and IRF-3ER monomers. The density of IRF-3ER dimers was less than the density of IRF-3ER monomers, which could be explained by the denaturing conditions used in the analysis as suggested in our previous report, including SDS-polyacrylamide gel electrophoresis, RIPA lysis buffer, and boiling during Western blotting. Interestingly, a small amount of IRF-3ER dimer formation was detected in Huh7.5-IRF3ER cells without 4-HT treatment. This may be due either to auto-dimerization of IRF-3ER or dimer formation induced by trace estrogen in the tissue culture medium. Multiple forms of the IRF-3ER fusion protein were also detected. Our data indicates the IRF-3ER fusion protein approach is an effective means to achieve IRF-3 homodimerization with 4-HT treatment.

Expression of IFNs after activation of the IRF-3ER fusion protein

Due to deficient RIG-I gene function in Huh 7.5 cells, virus infection will not lead to IRF-3 activation and IFN secretion. This phenomenon allows us to study IRF-3 gene function against HCV infection by establishing a stable Huh7.5-IRF3ER cell line. Fusion proteins of STAT1 and STAT3 with the mouse estrogen receptor provided a useful means to study dimerization of those proteins and resulting in anti-HCV status. In this study, the IRF-3 gene was fused with same C-terminal sequences of mouse estrogen receptor as reported for inducing IRF-3ER dimerization by 4-HT treatment. Expression of type I IFNs (α and β) was examined after 4-HT treatment by real-time PCR. IFN-α and IFN-β increased and peaked 24 hours after 4-HT induction. To further demonstrate the biological activities of IFN-α and IFN-β after IRF-3ER dimerization, Western blotting was used to detect phosphorylated STAT1 and STAT3. In Figure 3A, phosphorylated STAT1 was detected with an antibody against STAT1 (S727) in Huh 7.5 and Huh7.5-IRF3ER cells. Different amounts of phosphorylated STAT1 were observed in both Huh 7.5 cells and Huh7.5-IRF3ER cells. There were no appreciable time-dependent differences in phosphorylated STAT1 in Huh7.5-IRF3ER cells with or without 4-HT treatment. This observation is consistent with the auto-dimerization of IRF-3ER fusion protein to produce IFNs. In Figure 3B, phosphorylated STAT3 was examined; there was no difference between Huh 7.5, Huh7.5-IRF3ER cells with or without 4-HT treatment. This phenomenon could be explained by the constant activation of IRF-7 to induce expression of IFN-α which activates the type I IFN pathway through STAT3 phosphorylation. Total STAT1 and STAT3 proteins was used as internal controls and demonstrated no differences with or without 4-HT treatment on Huh7.5-IRF3ER cells or Huh 7.5 cells.

HCV JFH-1 stocks and HCV infection

Preparation and titration of HCV JFH-1 virus was reported previously [24]. For examining anti-HCV effects, Huh7.5-IRF3ER cells were incubated with 0.5 MOI JFH-1 HCV for 14 days to achieve fully infected Huh7.5-IRF3ER monolayer cells [28]. The Huh7.5-IRF3ER cells were then treated with 4-HT for 72, 48 and 24 hours prior to collecting total cellular RNA. Huh7.5-IRF3ER cells without 4-HT treatment for 72 hours were used as control. Total RNA was isolated for detecting HCV RNA by real-time PCR.

Detection of IFN-α and IFN-β in Huh7.5-IRF3ER cells

Huh7.5-IRF3ER cells were treated with 4-HT for 72, 48, and 24 hours prior to collecting cellular lysates. Control is Huh7.5-IRF3ER cells that did not receive 4-HT treatment for 72 hours. Total cellular RNA was isolated for detecting IFN-α or IFN-β RNA by real-time PCR.

Real-Time PCR assay

Total cellular RNA was isolated from infected Huh7.5-IRF3ER monolayers by Trizol (Invitrogen). First-strand cDNA were synthesized from 1 μg total cellular RNA by reverse transcription (20 μl of reaction volume). Superscript II (200 U reverse transcriptase per reaction) and a RT-PCR kit (Invitrogen) was used to prime with oligo (dT) 12-18 for first-strand synthesis according to the manufacturer’s instructions. Taqman primers were obtained from Applied BioSystems. Reactions were conducted in a 96-well MyiQ cycler (Bio-Rad, Hercules, CA). Fluorescence was monitored during every PCR cycle at the annealing step. The primers for HCV JFH-1 are: forward, 5′-CGGAATTGCCGGGAAGAC-3′; reverse, 5′-CAAATGGCCGGGCATAG AG-3′; FAM probe, 5′-CTTTCTTGGATAAACCC-3′. The primers for IFN-α are: forward, 5′- GGGATGAGGACCTCCTAGACAAATT-3′; reverse, 5′- ACACAGGCTTCCAAGTCA TTC-AG-3′; FAM probe, 5′- CTGCACCGAACTCTAC-3′. The primers for IFN-β are: forward, 5′-TGGCTGGAATGAGACTATTGTTGAG-3′; reverse, 5′-CAGGACTGTCTTCA GATGG-TTTATCT-3′; FAM probe, 5′-CCTCCTGGCTAATGTC-3′. GADPH primers were purchased from the Applied Biosystems. PCR was performed with the following conditions: 50°C, 2 min; 95°C, 10 min; (95°C, 15s; 60°C, 1 min) for 40 cycles. Relative RNA level indicates statistical quantification of altered RNA levels from these cellular lysates with different primers. Samples were run in triplicate and the results were analyzed using the Bio-Rad iQ5 software; means ± the standard error of the mean are shown.

Luciferase assays

Huh7.5-IRF3ER cells were cultured in 6-well plates and transfected with the plasmid pRL-HL and lipofectamine 2000 (Invitrogen). After 24-hours of transfection, Huh7.5-IRF3ER cells were treated with 4-HT for 96, 72, and 48 hours prior to preparing cell lysates. Control Huh7.5-IRF3ER cells were incubated for 96 hours in the absence of 4-HT. All samples were analyzed for luciferase activity using the Dual-Luciferase Reporter Assay System Kit (Promega, Madison, WI) in triplicate. The translation efficiency was calculated as a proportion of control (100%).

Statistical analysis

Different cellular lysates were collected for analysis of luciferase activity or relative RNA level from Huh7.5-IRF3ER cells with special treatment. Results of these studies are expressed as means ± standard deviation (SD).

Expression of an IRF-3 fusion protein and mouse estrogen receptor, inhibits hepatitis C viral replication in RIG-I-deficient Huh 7.5 cells. Part 2

In order to study the direct anti-HCV response of IRF-3 activation, an inducible Huh7.5-IRF3ER cell line was established in RIG-I deficient Huh 7.5 cells that allow IRF-3 protein homodimer formation in a cytokine/receptor-independent fashion. Huh 7.5 cells are a highly adapted and poorly differentiated hepatoma cell line that lacks the ability to produce detectable interferon-α/β when infected with HCV JFH-1 virus. Therefore, Huh7.5-IRF3ER cells is an adequate system to study the downstream molecular events of IRF-3 activation and establishment of a subsequent anti-HCV state without RIG-I activation in Huh 7.5 cells.


A mammalian expression vector, pTIRF3ER, was constructed as a fusion protein of the IRF3 gene (51.6 Kd) [22] and C-terminal sequences of the mouse estrogen receptor (310 a.a.) in the pEF6/V5-His TOPO® TA vector (Invitrogen, Carlsbad, CA). The plasmid pJFH-1 contains a full-length HCV genomic cDNA. The plasmid pRL-HL is a dicistronic construct that mediates Cap-dependent and HCV IRES-dependent translation. Synthetic 4-hydroxytamoxifen (4-HT) was purchased from Sigma (Saint Louis, MO) and dissolved in ethanol as a 5 mM stock solution.

Cell lines

Human hepatoma Huh 7.5 cells were grown in Dulbecco’s modified Eagle’s medium (Invitrogen). To establish the Huh7.5-IRF3ER cell line, Huh 7.5 cells were transfected with the plasmid pTIRF3ER and Lipofectin (Invitrogen). Blasticidin (Invitrogen) (10 μg/ml) was used for the clone selection 24-hours after transfection. Medium was changed every 3 days with fresh Blasticidin until day 14, at which time, positive clones were propagated. The clones were amplified and IRF-3ER dimer formation was measured by Western blotting after 4-HT treatment.

Detection of IRF-3ER dimers, p-STAT1 (S727), p-STAT3 (Y705), and 1-8U protein by Western blotting

Huh7.5-IRF3ER cell monolayers were washed in phosphate buffered saline (PBS) post 4-HT treatment with protease inhibitor cocktail (Sigma). Preparation of Huh7.5-IRF3ER cell lysates was performed as reported previously. Cellular lysates were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) (6% gel for IRF-3ER dimers; 8% gel for p-STAT1 (S727) and p-STAT3 (Y705)). Western blotting was carried out as previously reported with antibodies for actin (Santz Cruz Biotechnology, Inc., Santa Cruz, CA), p-STAT3 (Y705) (Cell Signaling, Boston, MA), p-STAT3 (Y705) (Cell Signaling), and STAT1 (Santz Cruz). Western blotting of STAT1 and STAT3 proteins were performed with the same PVDF membrane used for detection of p-STAT1 (S727) and p-STAT3 (Y705) after stripping the blot (Bio-rad stripping buffer).

Expression of an IRF-3 fusion protein and mouse estrogen receptor, inhibits hepatitis C viral replication in RIG-I-deficient Huh 7.5 cells


Interferon Regulatory Factor-3 (IRF-3) plays a central role in the induction of interferon (IFN) production and succeeding interferon-stimulated genes (ISG) expression en route for restraining hepatitis C virus (HCV) infection. Here, we established a stable Huh7.5-IRF3ER cell line expressing a fusion protein of IRF-3 and mouse estrogen receptor (ER) to examine IFN production and anti-HCV effects of IRF-3 in retinoic acid inducible-gene-I (RIG-I) deficient Huh 7.5 cells. Homodimerization of the IRF-3ER fusion protein was detected by Western blotting after treatment with the estrogen receptor agonist 4-hydrotamoxifen (4-HT) in Huh7.5-IRF3ER cells. Expression of IFN-α, IFN-β, and their inhibitory effects on HCV replication were demonstrated by real-time polymerase chain reaction (PCR). Peak expression of IFN-α and IFN-β was achieved 24-hours post 4-HT treatment, coinciding with the appearance of phosphorylated signal transducer and activator of transcription (STAT) proteins. Additionally, HCV viral replication declined in time-dependent fashion. In previous studies, a novel IFN-mediated pathway regulating expression of 1-8U and heterogeneous nuclear ribonucleoprotein M (hnRNP M) inhibited HCV internal ribosomal entry site (IRES)-dependent translation. When expression of ISGs such as 1-8U and hnRNP M were measured in 4-HT-treated Huh7.5-IRF3ER cells, both genes were positively regulated by activation of the IRF-3ER fusion protein. In conclusion, the anti-HCV effects of IRF-3ER homodimerization inhibited HCV RNA replication as well as HCV IRES-dependent translation in Huh7.5-IRF3ER cells. The results of this study indicate that IRF-3ER homodimerization is a key step to restore IFN expression in Huh7.5-IRF3ER cells and in achieving its anti-HCV effects.


Hepatitis C virus infection causes chronic liver diseases, cirrhosis, and hepatic cellular carcinoma (HCC) with 170 million people worldwide and 4 million people in the United States reportedly infected (CDC, 1998). In addition to its global health problem, future projections suggest that HCV related mortality will increase 2-3-fold over the next decade with more than 180 billion US dollars estimated total social economic cost in the United States. The standard treatment of chronic HCV is anti-viral therapy with IFN and ribavirin (RBV) but no HCV vaccine available. Despite additional chemotherapeutics is on hand for treatment of genotype I HCV patients recently, the anti-viral mechanisms of IFN-based therapies are not well defined, but most likely involve the activation of host innate immunity to limit HCV replication.

During microbial infection, the recognition of microbial components is mediated by host-specific cellular pathogen-recognition receptors (PPRs). PPRs are members of the toll-like receptor (TLRs) family and are localized either to cellular plasma (TLR4 for lipopolysaccharide (LPS) and viral envelops) or endosomal membranes (TLR3 for dsRNA, TLR7/8 for ssRNA and TLR9 for DNA). Conversely, intracellular dsRNA is also recognized by the RIG-I cytosolic RNA helicase or melanoma differentiation associated gene (MDA)-5. RIG-I RNA helicase was found to be an essential mediator of anti-HCV effects due to its binding to un-capped 5′-end and 3′-end HCV dsRNA, triggering host innate immunity.

IFNs bind to the IFN-α/β receptor (IFNAR) in either an autocrine and/or paracrine manner to initiate a positive feedback loop that results in the production of more type I IFNs. IFNARs trigger the activation of the JAK/STAT pathway to phosphorylate the STAT proteins. The STAT transcription factors associated with IRF-9 to form a heterotrimeric complex, IFN-stimulated gene factor 3 (ISGF3), initiating the transcription of IFN-stimulated genes (ISGs) and inhibiting the different stages of virus replication and eliciting an anti-viral state in the host. During HCV infection, these anti-viral effects include the inhibitory effects on host and HCV translation, regulation of cellular proliferation and apoptosis, regulation of adaptive immunity, and recruitment of NK cells to the site of infection to clear HCV infection by inhibiting HCV gene expression and HCV replication. Patients with cleared HCV infection without IFN-based treatment provides strong evidence for the host innate immune response during acute HCV infection.

OPV III immunization in all provinces of Pakistan

Following the successful eradication of small pox, World Health Assembly (WHA) decided to target polio virus eradication. By the efforts of World Health Organization (WHO) program Global Eradication of paralytic poliomyelitis has been eradicated from America, Western Europe, and many other regions of the world. Expended program on immunization began in Pakistan in 1976 and expanded countrywide by 1978. Both regionally and worldwide EPI has a significant impact on poliomyelitis eradication performance. Pakistan is achieving immunization targets set globally and has made progress towards achieving Millennium Development Goal 4. But lack of parent knowledge, limited access to immunization services and poor managements are present as hard barriers in front of immunization progress. Every year vaccines for approximately 5.8 million children are procured by the program. More than 30 million children are immunized in every round of polio supplemental immunization activities. EPI is the exclusive provider of immunization in Pakistan and about 3% of immunization is provided by private sector. Each year Over 6000 permanent centers and more than one million outreach and mobile vaccination sessions provides immunization services. Over 10,000 vaccinators and approximately 6000 lady health visitors (LHVs) are assigned in these centers. About 100,000 lady health workers (LHWs) also aid in routine and supplementary immunization activities.
Materials and methods

Several Government documents, survey reports and unpublished program documents were studied and online searches were made to find literature on EPI Pakistan. World Health Organization (WHO), United Nations Children’s Fund (UNICEF) and other websites were also explored. The EPI program official database was also analyzed for this study. SPSS 16 and Microsoft Excel 7 were used for the statistical analysis, tabulation and compiling of collected data.

OPV III immunization in all provinces of Pakistan

A “fully immunized child” is one who has received at least 1 dose of Bacilli Calmette-Guérin (BCG) vaccine, 3 doses of oral polio vaccine (OPV), DPT3 and measles1 vaccine. EPI programs target is to immunize children of 0-11 months against seven EPI target diseases. According to EPI surveys 2001, Khyber Pakhtunkhwa was the best performing province with 89% immunization (OPV III) in fighting against polio. The lowest immunization results were in Baluchistan and Gilgit Baldistan with 52% and 34.6% immunization respectively. The EPI surveys are regularly conducted in Pakistan by the Ministry of the Health in collaboration with WHO and other organizations that are active in fight against polio. The main aim of the surveys is to get data about the immunization, to identify the regions that are at the risk so that the special consideration should be given to these areas in planning the programs, in future.

The vaccination campaigns conducted from 2002-2004 shows approximately the same results as in previous exercise in 2001. Azad Jammu and Kashmir (AJK) and KPK were the best performing provinces with 88% and 74% immunization and Baluchistan and Gilgit Baldistan having the lowest immunization 54%, 58% respectively. The main reason for the lowest immunization in Baluchistan and Gilgit Baldistan are the poor infrastructure, low education rate, lack of political well and highly diverse and inaccessible population. In 2005, over all reported immunization was 65%, the immunization campaign highly affected due to severe earthquake in the northern area of the Pakistan, due to which a huge population migrated to the other areas of the country. The surveys conducted during 2006 to 2010 in all the province of Pakistan shows that vaccine immunization has been greatly improved in door-to-door immunization campaigns.