Huifang M. Zhang

Inhibition of Coxsackievirus B3 Replication by Small Interfering RNAs Requires Perfect Sequence Match in the Central Region of the Viral Positive Strand

ABSTRACT Coxsackievirus B3 (CVB3) is the most common causal agent of viral myocarditis, but existing drug therapies are of limited value. Application of small interfering RNA (siRNA) in knockdown of gene expression is an emerging technology in antiviral gene therapy. To investigate whether RNA interference (RNAi) can protect against CVB3 infection, we evaluated the effects of RNAi on viral replication in HeLa cells and murine cardiomyocytes by using five CVB3-specific siRNAs targeting distinct regions of the viral genome. The most effective one is siRNA-4, targeting the viral protease 2A, achieving a 92% inhibition of CVB3 replication. The specific RNAi effects could last at least 48 h, and cell viability assay revealed that 90% of siRNA-4-pretreated cells were still alive and lacked detectable viral protein expression 48 h postinfection. Moreover, administration of siRNAs after viral infection could also effectively inhibit viral replication, indicating its therapeutic potential. Further evaluation by combination found that no enhanced inhibitory effects were observed when siRNA-4 was cotransfected with each of the other four candidates. In mutational analysis of the mechanisms of siRNA action, we found that siRNA functions by targeting the positive strand of virus and requires a perfect sequence match in the central region of the target, but mismatches were more tolerated near the 3′ end than the 5′ end of the antisense strand. These findings reveal an effective target for CVB3 silencing and provide a new possibility for antiviral intervention.

Overexpression of Interferon-γ-inducible GTPase Inhibits Coxsackievirus B3-induced Apoptosis through the Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway and Inhibition of Viral Replication

Our previous studies using differential mRNA display have shown that interferon-γ-inducible GTPase (IGTP), was up-regulated in coxsackievirus B3 (CVB3)-infected mouse hearts. In order to explore the effect of IGTP expression on CVB3-induced pathogenesis, we have established a doxycycline-inducible Tet-On HeLa cell line overexpressing IGTP and have analyzed activation of several signaling molecules that are involved in cell survival and death pathways. We found that following IGTP overexpression, protein kinase B/Akt was strongly activated through phosphorylation, which leads to phosphorylation of glycogen synthase kinase-3 (GSK-3). Furthermore, in the presence of CVB3 infection, the intensity of the phosphorylation of Akt was further enhanced and associated with a delayed activation of caspase-9 and caspase-3. These data indicate that IGTP expression appears to confer cell survival in CVB3-infected cells, which was confirmed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt cell viability assay. However, the ability of IGTP to induce phosphorylation of Akt and to promote cell survival was attenuated by the phosphotidylinositol-3 kinase (PI3-K) inhibitor LY294002. Transient transfection of the cells with a dominant negative Akt construct followed by doxycycline induction and CVB3 infection reversed Akt phosphorylation to basal levels and returned caspase-3 activity to levels similar to those when the PI3-K inhibitor LY294002 was added. Moreover, IGTP expression inhibited viral replication and delayed CVB3-induced cleavage of eukaryotic translation initiation factor 4G, indicating that IGTP-mediated cell survival relies on not only the activation of PI3-K/Akt, inactivation of GSK-3 and suppression of caspase-9 and caspase-3 but also the inhibition of viral replication.

Ubiquitin-Dependent Proteolysis of Cyclin D1 Is Associated with Coxsackievirus-Induced Cell Growth Arrest

ABSTRACT Coxsackievirus group B3 (CVB3) replication is influenced by host cell cycle status. However, the effect of CVB3 infection on cell cycle regulation and the mechanisms involved are not precisely defined. In this study, we examined cell cycle progression and regulation when the infection was initiated in late G1 phase of the cell cycle. Analysis of cellular DNA synthesis in infected cells by thymidine incorporation assays showed a significant reduction in [3H]thymidine uptake compared to that of sham-infected cells. To further clarify the effects of CVB3 on the host cell cycle, we examined the cell cycle regulatory proteins involved in G1 progression and G1/S transition. Infection resulted in dephosphorylation of retinoblastoma protein and reduced G1 cyclin-dependent kinase activities, accompanied by decreased levels of G1 cyclin protein expression (cyclin D1 and cyclin E). We further investigated the mechanisms by which CVB3 infection down-regulates cyclin D1 expression. Northern blotting showed that cyclin D1 mRNA levels were modestly increased following CVB3 infection, suggesting that cyclin D1 regulation occurs by a posttranscriptional mechanism. Viral infection resulted in only a 20 to 30% inhibition of cyclin D1 protein synthesis 3 h postinfection. However, the proteasome inhibitors MG132 and lactacystin prevent CVB3-induced cyclin D1 reduction, indicating that CVB3-induced down-regulation of cyclin D1 is facilitated by ubiquitin-proteasome proteolysis. Finally, using GSK3β pathway inhibitors, we showed that the reduction of cyclin D1 is GSK3β independent. Taken together, our results demonstrate that CVB3 infection disrupts host cell homeostasis by blocking the cell cycle at the G1/S boundary and induces cell cycle arrest in part through an increase in ubiquitin-dependent proteolysis of cyclin D1.

Coxsackievirus B3 Infection Activates the Unfolded Protein Response and Induces Apoptosis through Downregulation of p58IPK and Activation of CHOP and SREBP1

ABSTRACT Cardiomyocyte apoptosis is a hallmark of coxsackievirus B3 (CVB3)-induced myocarditis. We used cardiomyocytes and HeLa cells to explore the cellular response to CVB3 infection, with a focus on pathways leading to apoptosis. CVB3 infection triggered endoplasmic reticulum (ER) stress and differentially regulated the three arms of the unfolded protein response (UPR) initiated by the proximal ER stress sensors ATF6a (activating transcription factor 6a), IRE1-XBP1 (X box binding protein 1), and PERK (PKR-like ER protein kinase). Upon CVB3 infection, glucose-regulated protein 78 expression was upregulated, and in turn ATF6a and XBP1 were activated via protein cleavage and mRNA splicing, respectively. UPR activity was further confirmed by the enhanced expression of UPR target genes ERdj4 and EDEM1. Surprisingly, another UPR-associated gene, p58IPK, which often is upregulated during infections with other types of viruses, was downregulated at both mRNA and protein levels after CVB3 infection. These findings were observed similarly for uninfected Tet-On HeLa cells induced to overexpress ATF6a or XBP1. In exploring potential connections between the three UPR pathways, we found that the ATF6a-induced downregulation of p58IPK was associated with the activation of PKR (PERK) and the phosphorylation of eIF2α, suggesting that p58IPK, a negative regulator of PERK and PKR, mediates cross-talk between the ATF6a/IRE1-XBP1 and PERK arms. Finally, we found that CVB3 infection eventually produced the induction of the proapoptoic transcription factor CHOP and the activation of SREBP1 and caspase-12. Taken together, these data suggest that CVB3 infection activates UPR pathways and induces ER stress-mediated apoptosis through the suppression of P58IPK and induction/activation of CHOP, SREBP1, and caspase-12.

Gamma Interferon-Inducible Protein 10 Induces HeLa Cell Apoptosis through a p53-Dependent Pathway Initiated by Suppression of Human Papillomavirus Type 18 E6 and E7 Expression

ABSTRACT Gamma interferon-inducible protein 10 (IP10) is a member of the CXC family of chemokines. By differential mRNA display, we have demonstrated the upregulation of IP10 in coxsackievirus B3 (CVB3)-infected mouse hearts. Functional characterization of the IP10 gene in IP10-transfected Tet-On HeLa cells has found that IP10 induced cell apoptosis and inhibited viral replication. In the characterization of the IP10-induced apoptotic pathway, we found that overexpression of IP10 upregulated p53 and resulted in altered expression of p53-responsive genes such as the p21Cip1, p27kip1, NF-κB, Bax, and PUMA genes and the mitochondrial translocation of Bax. However, transduction of the IP10 cells with adenovirus expressing dominant negative p53 not only ablated p53-triggered gene expression but also abolished IP10-induced apoptosis and restored CVB3 replication to the control levels. These data suggest a novel mechanism by which IP10 inhibits viral replication through the induction of host cell death via a p53-mediated apoptotic pathway. We also found that constantly high-level expression of p53 in these tumor cells is attributed to the IP10-induced suppression of human papillomavirus E6 and E7 oncogene expression. Taken together, these data reveal not only a previously unrecognized link between chemokine IP10 and p53 in antiviral defense but also a mechanism by which IP10 inhibits tumor cell growth.

Coxsackievirus B3-Associated Myocardial Pathology and Viral Load Reduced by Recombinant Soluble Human Decay-Accelerating Factor in Mice

Coxsackievirus B3 (CVB3) infection can result in myocarditis, which in turn may lead to a protracted immune response and subsequent dilated cardiomyopathy. Human decay-accelerating factor (DAF), a binding receptor for CVB3, was synthesized as a soluble IgG1-Fc fusion protein (DAF-Fc). in vitro, DAF-Fc was able to inhibit complement activity and block infection by CVB3, although blockade of infection varied widely among strains of CVB3. To determine the effects of DAF-Fc in vivo, 40 adolescent A/J mice were infected with a myopathic strain of CVB3 and given DAF-Fc treatment 3 days before infection, during infection, or 3 days after infection; the mice were compared with virus alone and sham-infected animals. Sections of heart, spleen, kidney, pancreas, and liver were stained with hematoxylin and eosin and submitted to in situ hybridization for both positive-strand and negative-strand viral RNA to determine the extent of myocarditis and viral infection, respectively. Salient histopathologic features, including myocardial lesion area, cell death, calcification and inflammatory cell infiltration, pancreatitis, and hepatitis were scored without knowledge of the experimental groups. DAF-Fc treatment of mice either preceding or concurrent with CVB3 infection resulted in a significant decrease in myocardial lesion area and cell death and a reduction in the presence of viral RNA. All DAF-Fc treatment groups had reduced infectious CVB3 recoverable from the heart after infection. DAF-Fc may be a novel therapeutic agent for active myocarditis and acute dilated cardiomyopathy if given early in the infectious period, although more studies are needed to determine its mechanism and efficacy.

Nip21 Gene Expression Reduces Coxsackievirus B3 Replication by Promoting Apoptotic Cell Death via a Mitochondria-Dependent Pathway

Our previous studies, using differential mRNA display, suggested that the mouse Nip21 gene may be involved in myocarditis development in the coxsackievirus B3 (CVB3)–infected mouse heart. Sequence comparison indicated that the mouse Nip21 gene shares high sequence homology to human Nip2. This human protein is known to interact with both the apoptosis inhibitor Bcl-2 and a homologous protein, the adenovirus E1B 19-kDa protein. Such interactions implicate Nip21 gene in cell death pathways. To study the function of this gene, we have cloned Nip21 from mouse hearts and established a Tet-On doxycycline-inducible HeLa cell line and a cardiomyocyte H9c2 cell line expressing Nip21 to characterize gene function in relation to apoptosis. We demonstrated that Nip21 expression could induce apoptosis via caspase-depended mitochondria activation. To further determine the function of Nip21 in CVB3-induced apoptosis, the Tet-On/Nip21 HeLa cell line was induced by doxycycline followed by CVB3 infection. We found that activation of caspase-3 and cleavage of poly-(ADP-ribose) polymerase occurred 2 hours earlier than in vector-transfected control cells, suggesting that Nip21 expression enhances CVB3-induced apoptosis. We also demonstrated a significant decrease in HeLa cell and H9c2 cell viability. Particularly, as illustrated by viral plaque assay, CVB3 replication was dramatically reduced in Tet-On HeLa cells, due at least in part to the earlier killing of the host cells by Nip21 overexpression.

Targeted delivery of anti-coxsackievirus siRNAs using ligand-conjugated packaging RNAs

Abstract Coxsackievirus B3 (CVB3) is a common pathogen of myocarditis. We previously synthesized a siRNA targeting the CVB3 protease 2A (siRNA/2A) gene and achieved reduction of CVB3 replication by 92% in vitro. However, like other drugs under development, CVB3 siRNA faces a major challenge of targeted delivery. In this study, we investigated a novel approach to deliver CVB3 siRNAs to a specific cell population (e.g. HeLa cells containing folate receptor) using receptor ligand (folate)-linked packaging RNA (pRNA) from bacterial phage phi29. pRNA monomers can spontaneously form dimers and multimers under optimal conditions by base-pairing between their stem loops. By covalently linking a fluorescence-tag to folate, we delivered the conjugate specifically to HeLa cells without the need of transfection. We further demonstrated that pRNA covalently conjugated to siRNA/2A achieved an equivalent antiviral effect to that of the siRNA/2A alone. Finally, the drug targeted delivery was further evaluated by using pRNA monomers or dimers, which carried both the siRNA/2A and folate ligand and demonstrated that both of them strongly inhibited CVB3 replication. These data indicate that pRNA as a siRNA carrier can specifically deliver the drug to target cells via its ligand and specific receptor interaction and inhibit virus replication effectively.

MicroRNA-203 enhances coxsackievirus B3 replication through targeting zinc finger protein-148.

Coxsackievirus B3 (CVB3) is the primary causal agent of viral myocarditis. During infection, it hijacks host genes to favour its own replication. However, the underlying mechanism is still unclear. Although the viral receptor is an important factor for viral infectivity, other factors such as microRNAs (miRNA) may also play an essential role in its replication after host cell entry. miRNAs are post-transcriptional gene regulators involved in various fundamental biological processes as well as in diseases. To identify miRNAs involved in CVB3 pathogenesis, we performed microarray analysis of miRNAs using CVB3-infected murine hearts and identified miR-203 as one of the most upregulated candidates. We found that miR-203 upregulation is through the activation of protein kinase C/transcription factor AP-1 pathway. We further identified zinc finger protein-148 (ZFP-148), a transcription factor, as a novel target of miR-203. Ectopic expression of miR-203 downregulated ZFP-148 translation, increased cell viability and subsequently enhanced CVB3 replication. Silencing of ZFP-148 by siRNA showed similar effects on CVB3 replication. Finally, analyses of the signalling cascade downstream of ZFP-148 revealed that miR-203-induced suppression of ZFP-148 differentially regulated the expression of prosurvival and proapoptotic genes of the Bcl-2 family proteins as well as the cell cycle regulators. This altered gene expression promoted cell survival and growth, which provided a favourable environment for CVB3 replication, contributing to the further damage of the infected cells. Taken together, this study identified a novel target of miR-203 and revealed, for the first time, the molecular link between miR-203/ZFP-148 and the pathogenesis of CVB3.

Focal adhesion kinase mediates the interferon-γ-inducible GTPase-induced phosphatidylinositol 3-kinase/Akt survival pathway and further initiates a positive feedback loop of NF-κB activation

Interferon‐γ‐inducible GTPase (IGTP) expression is upregulated in coxsackievirus B3 (CVB3)‐infected murine heart and inhibits CVB3‐induced apoptosis through activation of the PI3 kinase/Akt pathway. However, the mechanism of this pathway activation is unknown. In this study, using doxcycycline‐inducible Tet‐On HeLa cells that overexpress IGTP, we have demonstrated that focal adhesion kinase (FAK) is phosphorylated in response to IGTP expression and that transfection of the Tet‐On HeLa cells with a dominant negative FAK (FRNK) blocks Akt activation. Furthermore, induction of IGTP also promoted the NF‐κB activation as evidenced by its enhanced nuclear translocation, binding to transcriptional promoters and increased transcriptional activity. However, FRNK transfection and phosphatidylinositol 3‐kinase (PI3K) inhibitor LY294002 both blocked the IGTP‐induced translocation and NF‐κB activation. Furthermore, silencing NF‐κB with siRNAs significantly inhibited the phosphorylation of FAK and Akt, but not their total expression levels, indicating that NF‐κB activation is required for the IGTP‐induced activation of FAK and PI3K/Akt. Finally, blocking this survival pathway by transfection of FRNK or silencing of NF‐κB reduced CVB3 replication and enhanced cell death during CVB3 infection. Taken together, these results suggest that FAK is a mediator upstream of PI3K/Akt and NF‐κB functions as a downstream effector and also positively regulates the activity of upstream kinases.