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We report here an isatin derivative 45 (ID45) against coxsackievirus B3 (CVB3) replication, which was synthesized based on a high-throughput screen of a unique natural product library. ID45 showed the most potent anti-CVB3 activity among the four synthesized compounds. Treatment of cells with ID45 before or after infection significantly reduced viral particle formation, resulting in protection of cells from virus-induced apoptosis. In addition, ID45 treatment caused remarkable up-regulation of glucose-regulated protein 78 (GRP78), a hallmark of endoplasmic reticulum (ER) stress and an indicator of enhanced cell viability. In identifying the ER stress response pathway induced by ID45, we found that ID45 activated PKR-like ER protein kinase (PERK) but failed to up-regulate eIF2α phosphorylation. Instead ID45 activated transcription factor Nrf2 (NF-E2-related factor-2), which is evidenced by its nuclear translocation and upregulation of its downstream target genes NQO1 (NAD(P)H quinone-oxidoreductase 1) and GCLM (glutamate-cysteine ligase, modifier subunit). This observation was further verified by using siRNAs of GRP78 or Nrf2, which blocked both the translocation of Nrf2 and up-regulation of its target genes, leading to aggressive viral replication and enhanced cell apoptosis. Finally, we found that ID45-induced up-regulation of NQO1 protected eIF4GI, a eukaryotic cap-dependent translation initiation factor, from cleavage by CVB3 protease and degradation by proteasomes. Taken together, our findings established that a novel antiviral mechanism of isatin derivative ID45 inhibits CVB3 replication by promoting cell survival through a PERK/Nrf2-dependent ER stress pathway, which benefits host cap-dependent translation but suppresses CVB3 cap-independent translation.

1. Myocarditis may be an early indicator of or may subsequently lead to dilated cardiomyopathy in humans. This hypothesis has evolved from research on viruses that induce myocarditis, wherein the coxsackie B group viruses (CVB) in the family Picornaviridae are the most common known viral infectants of heart muscle. 2. Many competing hypotheses exist as to the pathogenesis of CVB3-induced myocarditis, including direct virus-induced myocyte damage and immunopathological disease with autoimmune sequelae. Evidence to support the direct-damage and viral RNA-persistence hypothesis is derived from in situ hybridization and gene amplification studies. 3. Recent use of terminal deoxynucleotidyl transferase-mediated nick-end labelling indicates that this injury in target organs is largely non-apoptotic in nature. Most apoptotic bodies in cardiac tissue are derived from immune cells. 4. Beyond infection of heart muscle, CVB3 can also associate with, infect and persist in cells of immune origin. The CVB3 localizes to follicles in spleens and lymph nodes of the murine host and this particular localization may continue in mice susceptible to more aggressive myocarditis. Whether virus-immune cell association in these compartments is advantageous (or essential) to the host in the evolution of anti-viral immune responses or whether it is more advantageous to the virus in immunosuppression of the host is not known. 5. We suggest that CVB3 can directly perturb or alter the immune response, thereby delaying viral clearance from vulnerable systemic organs. Both host and viral genetic factors can influence susceptibility, persistence and disease progression. 6. Picornaviruses use a unique method for the initiation of translation, involving the internal binding of the ribosome on a sequence element of the 5' untranslated region, termed an internal ribosome entry site (IRES). 7. The IRES of CVB3 is located at approximately stem loops G, H and I, spanning nucloetides 530 and 630. Arrest of host translation is also a feature of picornavirus infection. Such regulation of host cell translation machinery no doubt fosters viral replication at the expense of the host cell. 8. Differences between cell types in the mechanisms, along with those at other key steps in the viral life cycle and in signalling via kinase pathways, may determine viral tropism and cellular destruction and the physiological outcome of neighbouring cells.

The 5′ untranslated region (5′UTR) of coxsackievirus B3 (CVB3) RNA forms a highly ordered secondary structure that has been implicated in controlling initiation of viral translation by internal ribosomal entry. To test this hypothesis, synthetic bicistronic RNAs, with all or part of the 5′UTR in the intercistronic space, were translated in rabbit reticulocyte lysates. In the presence of an upstream cistron, the chloramphenicol acetyltransferase gene, designed to block ribosomal scanning, the CVB3 5′UTR was capable of directing the internal initiation of translation of the downstream reporter gene (P1), confirming the presence of an internal ribosomal entry site (IRES). This finding was further supported by the data on predicted secondary structures within the 5′UTR. Of special note, analysis of various deletion mutants demonstrated that the IRES of CVB3 is located roughly at stem–loops G, H, and I spanning nucleotides (nt) 529 and 630. The region from nt 1 to 63 (stem–loop A) also appears important, and it may be an essential binding site for translation initiation factors. Based on these findings,in vitrotranslation inhibition assays using RNA fragments of the 5′UTR as inhibitor were performed. Both antisense and sense RNA segments transcribed from these twocis-acting regions and the surrounding sequence of the initiation codon AUG showed strong inhibition of viral protein synthesis. Antisense molecules may inhibit translation by blocking ribosome and initiation factor binding within the 5′UTR via specific hybridization to their viral RNA target sequences, while sense sequences may function by competing with viral RNA for ribosomes and/or translation initiation factors. Thesecis-acting translational elements may serve as potential targets for the antiviral action of oligomers.

Infection by foot-and-mouth disease virus (FMDV) is triggered by the acidic pH in endosomes after virus uptake by receptor-mediated endocytosis. However, dissociation of the FMDV 146S particle in mildly acidic conditions renders inactivated foot-and-mouth disease (FMD) vaccines much less effective. Type Asia1 FMDV mutants with increased resistance to acid inactivation were selected to study the molecular basis of viral resistance to acid-induced disassembly and improve the acid stability of FMDV. Sequencing of capsid-coding regions revealed four amino acid replacements (VP1 N17D, VP2 H145Y, VP2 G192D, and VP3 K153E) in the viral population of the acid-selected 10th passage. We performed single or combined mutagenesis using a reverse genetic system, and our results provide direct experimental evidence that VP2 H145Y or VP1 N17D substitution confers an acid-resistant phenotype to type Asia1 FMDV.

Viral myocarditis is caused by a variety of viruses of more than 10 genera, such as coxsackievi‐ rus, adenovirus, parvovirus, hepatitis c virus, herpes virus, influenza virus, HIV, etc. [1]. How‐ ever, the most frequently reported and extensively studied one is coxsackievirus B3 (CVB3), which causes ~30% of all viral myocarditis cases [2]. Thus, in this chapter the review will main‐ ly focus on CVB3-induced myocarditis. This virus can infect multiple organs of human such as heart, pancreas, brain, liver, lung, spleen, etc. and cause myocarditis, pancreatitis, meningitis, hepatitis, etc. However, the most fatal disease is myocarditis, particular in children and young people [3]. Viral myocarditis is characterized by inflammatory infiltration of immune cells in the heart muscle after viral infection. This viral infection can cause direct damage of cardio‐ myocytes as well as immune-mediated destructions of the myocardium, leading to cardiac dysfunction. In addition, viral myocarditis often progresses into dilated cardiomyopathy (DCM), an end-stage heart dysfunction. Patients with DCM usually require heart transplanta‐ tion [4]. There is no other treatment option at the present. Viral myocarditis is one of the major life-threatening diseases in children. It is the cause of ~ 20% of sudden unexpected death in young people [5]. To date, there is no specific treatment for this viral infection.

Previously we found that prolonged endoplasmic reticulum (ER) stress caused by coxsackievirus B3 (CVB3) infection led to p58(IPK) downregulation and subsequent cell apoptosis. This finding implies that p58(IPK) expression benefits cell survival and counteracts CVB3-induced apoptosis. In testing this hypothesis, we first found that PI3K/Akt survival pathway is more sensitive than ERK1/2 in response to p58(IPK) expression. This finding was further verified by silencing p58(IPK) with specific siRNAs, which led to the significant suppression of phosphorylation of Akt (p-Akt) but not ERK1/2. Further, using CVB3-infected cell line expressing dominant negative ATF6a (DN-ATF6a), we found that expression of p58(IPK) and p-Akt was significantly reduced, which led to the decreased cell viability. However, when the DN-ATF6a cells were transiently transfected with p58(IPK) , an opposite result was obtained. Finally, by CVB3 infection of cells stably expressing p58(IPK) , we found that CVB3-induced mitochondria-mediated apoptosis was suppressed, which was evidenced by the reduced cytochrome c release and upregulation of the mitochondrial membrane protein mitofusin 2. However, silencing p58(IPK) with either specific siRNAs or DN-ATF6a sensitized cells to CVB3-induced apoptosis. These results suggest that p58(IPK) suppresses CVB3-induced apoptosis through selective activation of PI3K/Akt pathway that requires activation of ATF6a and subsequently upregulates mitofusin 2.

Coxsackievirus B3 (CVB3) is one of the most prevalent causes of viral myocarditis and is associated with many other pathological conditions. CVB3 replication relies on host cellular machineries and causes direct damage to host cells. MicroRNAs have been found to regulate viral infections but their roles in CVB3 infection are still poorly understood. Here we describe a novel mechanism by which miR-126 regulates two signal pathways essential for CVB3 replication. We found that CVB3-induced ERK1/2 activation triggered the phosphorylation of ETS-1 and ETS-2 transcription factors, which induced miR-126 upregulation. By using both microRNA mimics and inhibitors, we proved that the upregulated miR-126 suppressed sprouty-related, EVH1 domain containing 1 (SPRED1) and in turn enhanced ERK1/2 activation. This positive feedback loop of ERK1/2–miR-126–ERK1/2 promoted CVB3 replication. Meanwhile, miR-126 expression stimulated GSK-3β activity and induced degradation of β-catenin through suppressing LRP6 and WRCH1, two newly identified targets in the Wnt/β-catenin pathway, which sensitized the cells to virus-induced cell death and increased viral progeny release to initiate new infections. Our results demonstrate that upregulated miR-126 upon CVB3 infection targets SPRED1, LRP6, and WRCH1 genes, mediating cross-talk between ERK1/2 and Wnt/β-catenin pathways, and thus promoting viral replication and contributes to the viral cytopathogenicity.

In vitro replication of coxsackievirus B3 (CVB3) in cells of the immune system derived from uninfected adolescent A/J and C57BL/6J mice and replication of CVB3 in and association with immune cells from spleens of infected animals in vivo were assessed. Nonstimulated or mitogen-stimulated spleen cells were minimally permissive for viral replication during an 8-h period. Three days postinfection (p.i.), CVB3 RNA was localized in vivo to B cells and follicular dendritic cells of germinal centers in both A/J and C57BL/6J mice; however, extrafollicular localization was greater in C57BL/6J mice (P = 0.0054). Although the pattern of CVB3 RNA localization was different, the total load of infections virus (PFU per milligram of tissue) was not different. Splenic CVB3 titers (PFU per milligram of tissue) in both strains were maximal at day 3 or 4 p.i. and were back to baseline by day 7 p.i., with most infectious virus being non-cell associated. CVB3 titers (PFU per milligram of tissue) correlated directly with in situ hybridization positivity in splenic follicles and extrafollicular regions in both murine strains; however, follicular hybridization intensity was greater in A/J mice at day 5 p.i. (P = 0.021). Flow cytometric analysis demonstrated that 50.4% of total spleen cells positive for CVB3 antigen were B cells and 69.6% of positive splenic lymphocytes were B cells. Myocardial virus load in C57BL/6J mice was significantly lower than that in A/J mice at days 4 and 5 p.i. These data indicate that CVB3 replicates in murine splenocytes in vitro and in B cells and extrafollicular cells in vivo.

In rubella virus-infected cells, genomic 40S and subgenomic 24S RNAs are present in the cytoplasm of infected cells. However, encapsidation by rubella virus capsid protein is specific for 40S genomic RNA. As a first step toward understanding the assembly of rubella virus nucleocapsid at the molecular level, the interaction between capsid protein and genomic RNA was studied by Northwestern (RNA-protein) blot analysis. RNA probes prepared by in vitro transcription were used to localize the RNA sequence that participates in binding to the capsid protein. We have identified a 29-nucleotide RNA sequence (nucleotides 347 to 375) that is essential for the binding. By using overlapping synthetic peptides of capsid protein, a peptide domain (residues 28 to 56) that displays specific RNA-binding activity of capsid protein has been located. This result suggests that the specific recognition of viral RNA during rubella virus assembly involves, at least in part, the nucleocapsid protein.

© 2013 Yang et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Viral Replication Strategies: Manipulation of ER Stress Response Pathways and Promotion of IRES-Dependent Translation

Bacteriophage 29 (Phi29) packaging RNA (pRNA) is one of the key components in the viral DNA-packaging motor. It contains two functional domains facilitating the translocation of DNA into the viral capsid by interacting with other elements in the motor and promoting adenosine triphosphates hydrolysis. Through the connection between interlocking loops in adjacent pRNA monomers, pRNA functions in the form of multimer ring in the motor. Previous studies have addressed the unique structure and conformation of pRNA. However, there are different DNA-packaging models proposed for the viral genome transportation mechanism. The DNA-packaging ability and the unique features of pRNA have been attracting efforts to study its potential applications in nanotechnology. The pRNA has been proved to be a promising tool for delivering nucleic acid-based therapeutic molecules by covalent linkage with ribozymes, small interfering RNAs, aptamers, and artificial microRNAs. The flexibility in constructing dimers, trimers, and hexamers enables the assembly of polyvalent nanoparticles to carry drug molecules for therapeutic purposes, cell ligands for target delivery, image detector for drug entry monitoring, and endosome disrupter for drug release. Besides these fascinating pharmacological advantages, pRNA-based drug delivery has also been demonstrated to prolong the drug half life with minimal induction of immune response and toxicity.

The objective of this study was to explore the immunogenicity of an adenovirus construction expressing a type O foot and mouth disease virus neutralising epitope (8E8) in the context of heterologous capsid proteins. Adenoviruses expressing four chimeric type Asia1 FMDV capsid proteins were constructed by inserting the type O FMDV 8E8 epitope into the G-H loop from the type Asia1 VP1 at amino acid residues 139/140, 150/151, 134/140 or at both 139/140 and 150/151. These recombinant proteins were recognised by antibodies against the type O 8E8 epitope and type Asia1 FMDV. When inoculated in mice, all of the recombinant chimeric capsid proteins for each single epitope insertion induced the production of anti-type O FMDV neutralising antibodies. The recombinant chimeric capsid proteins with a foreign insertion at position 139/140 or 150/151 induced high levels of anti-type Asia1 FMDV neutralising antibodies as the recombinant type Asia1 capsid proteins without any foreign epitope, suggesting that the foreign insertion did not affect the immunogenicity of the type Asia1 FMDV capsid proteins. This study suggests that a foreign epitope displayed on the surface of the FMDV capsid proteins could induce an epitope-specific response. Therefore, the adenovirus-vectored FMDV capsid proteins could be used as a vehicle for the development of an epitope-based vaccine.

Many virus infections and stresses can induce endoplasmic reticulum (ER) stress response, a host self-defense mechanism against viral invasion and stress. During this event, viral and cellular gene expression is actively regulated and often encounters a switching of the translation initiation from cap-dependent to internal ribosome-entry sites (IRES)-dependent. This switching is largely dependent on the mRNA structure of the 5' untranslated region (5' UTR) and on the particular stress stimuli. Picornaviruses and some other viruses contain IRESs within their 5' UTR of viral genome and employ an IRES-driven mechanism for translation initiation. Recently, a growing number of cellular genes involved in growth control, cell cycle progression and apoptosis were also found to contain one or more IRES within their long highly structured 5' UTRs. These genes initiate translation usually by a cap-dependent mechanism under normal physiological conditions; however, in certain environments, such as infection, starvation, and heat shock they shift translation initiation to an IRES-dependent modality. Although the molecular mechanism is not entirely understood, a number of studies have revealed that several cellular biochemical processes are responsible for the switching of translation initiation to IRES-dependent. These include the cleavage of translation initiation factors by viral and/or host proteases, phosphorylation (inactivation) of host factors for translation initiation, overproduction of homologous proteins of cap-binding protein eukaryotic initiation factors (eIF)4E, suppression of cap-binding protein eIF4E expression by specific microRNA, activation of enzymes for mRNA decapping, as well as others. Here, we summarize the recent advances in our understanding of the molecular mechanisms for the switching of translation initiation, particularly for the proteins involved in cell survival and apoptosis in the ER stress pathways during viral infections.

Viral myocarditis is the most common heart disease in infants, children, young adults and pregnant women. Although a number of viruses from different genera, such as adenovirus, hepatitis C virus (HCV), parvoviruses and cytomegalovirus have been reported to cause myocarditis (Bowles et al., 2003; Kindermann et al., 2008; Kuhl et al., 2005a; Kuhl et al., 2005b; Kyto et al., 2005; Mahrholdt et al., 2006; Matsumori, 2005; Matsumori et al., 2006), coxsackievirus, particularly coxsackievirus B3 (CVB3), is generally considered the primary etiological agent of myocarditis (Blauwet, 2010; Kuhl et al., 2005a; Mahrholdt et al., 2006). CVB3 infection of the heart is often persistent and enters the chronic phase, leading to dilated cardiomyopathy (DCM)(Andreoletti et al., 2009; L. T. Cooper, Jr., 2009; Kuhl et al., 2005b; Yajima& Knowlton, 2009), a squelae of viral myocarditis characterized by ventricular chamber dilation, increased wall thickness, weaker beating and abnormal heart function. Patients with DCM eventually develop into congestive heart failure. To date, there is no clinically proven specific treatment available for viral myocarditis and DCM. Patients with DCM eventually need heart transplantation as the final treatment (Schultz et al., 2009). The managements for viral myocarditis are usually supportive therapies, such as improvements in cardiophysiology with medicine used to treat other kinds of heart diseases, and application of non-specific antiviral agents to decrease the viral load. The former measurements include administration of angiotensin-converting enzyme inhibitor or angiotensin receptor blockade, beta-adrenergic blockade, diuretics, etc (Dennert et al., 2008; Rose, 2009; Schultz et al., 2009); the later measurements include application of type I interferons or nucleotide analogs such as ribavirin, which is reviewed elsewhere (Blauwet, 2010; Dennert& Crijns& Heymans, 2008; Schultz et al., 2009). If myocarditis was caused by an autoimmune disorder, it would be appropriately treated by immunosuppression (Rose, 2009; Schultz et al., 2009). However, the effectiveness of treatment with immunosuppressive therapies has not reached a consensus amongst different studies. This can probably be attributed to the difficulty of confirmation and diagnosis of the etiology and pathogenesis of myocarditis. Thus it is very important to distinguish infectious and autoimmune disease since the same methods of treatment will not be optimal for both forms of heart muscle diseases. The diagnostic gold standard is endomyocardial biopsies with the histological Dallas criteria, in association with new

Foot-and-mouth disease virus (FMDV) serotype O remains an important threat to animal husbandry worldwide, and the variability of the virus presents a major problem for FMDV vaccine design. High-affinity neutralising antibodies against a conserved epitope could provide protective immunity against diverse subtypes of FMDV serotype O and protect against future pandemics. We generated a novel monoclonal antibody (MAb) 8E8 that potently neutralised infection of FMDV O/YS/CHA/05 both in vitro and in vivo. Screening of a phage-displayed random 12-peptide library revealed that MAb 8E8 bound to phages displaying a consensus motif GDLNVRT, which is highly homologous to (146)GDLQVLT(152) of the FMDV VP1 protein. Given that MAb 8E8 showed reactivity with the (146)GDLQVLT(152) motif, we proposed that this motif represented a linear B-cell epitope of the VP1 protein. Western blot analysis revealed that the epitope peptide could be recognised by the positive sera from serotype O FMDV-infected pigs. The (147)DLQVLT(152) motif was the minimal requirement for reactivity as demonstrated by reactivity of MAb 8E8 with several truncated peptides derived from the motif. For further mapping, a set of different extended motifs derived from the minimally reactive epitope was expressed with a GST-tag and subjected to western blot. The results showed that a 10-aa peptide (145)RGDLQVLTPK(154) was the minimal unit with maximal binding activity to MAb 8E8. Subsequent alanine scanning mutagenesis studies revealed that D(147), Q(149) and V(150) are crucial for MAb 8E8 binding. Furthermore, the epitope was found to be highly conserved among different topotypes of serotype O FMDV through sequence alignment analysis and detection of MAb 8E8 for affinity to some isolates collected in China. Thus, the 8E8 epitope identified here should be helpful for designing epitope-based, intra-typic, cross-protective vaccines of serotype O FMDV.

Coxsackievirus B3 (CVB3) is a single-stranded positive-sense RNA virus of the genus Enterovirus in the family Picornaviridae and can cause acute or chronic viral myocarditis. Epidemiological studies reveal that viral myocarditis is one of the major heart diseases worldwide, particularly in infants, children, and adolescents (17). Further, CVB3-induced myocarditis can result in dilated cardiomyopathy, a condition for which the only treatment is heart transplantation (12). CVB3 infection has been studied for decades in various systems, but the mechanisms of pathogenesis underlying CVB3-induced myocarditis in humans remain poorly defined. Cumulative evidence suggests that both direct viral injury and subsequent inflammatory responses contribute to the damage of cardiac myocytes, and that the extent of such damage determines the severity of late-stage heart dysfunction (10, 35). Previous studies have documented that apoptosis in cardiomyocytes can result in damaged myocardial tissue and is a hallmark of CVB3-induced myocarditis (1, 45). Although it has been shown that apoptosis facilitates the release of viral progeny during CVB3 infection (9, 48), the molecular events leading to apoptosis in CVB3-infected cells have not been well characterized. The endoplasmic reticulum (ER) system, a primary site for protein synthesis and folding, is a major site of signal initiation and transduction in response to a variety of stimuli, including virus infections (24, 66). Endogenous imbalances in cells, such as the overproduction of proteins, the accumulation of mutant proteins, or the loss of calcium homeostasis, can cause a malfunction of cellular processes and stress to the ER system (26). In response to ER stress, a coordinated adaptive program called the unfolded protein response (UPR) is activated and serves to minimize the accumulation and aggregation of misfolded proteins by increasing the capacity of the ER machinery to fold proteins correctly and activate the degradation of aberrant proteins. The UPR program represents a network of signal transduction from the ER to various locations within the cytoplasm and the nucleus, resulting in either the enhancement of cell survival or the induction of apoptosis (5). Glucose-regulated protein 78 (GRP78) functions as a master regulator of the UPR, and its upregulation indicates the activation of the UPR program (18, 27, 44). Under normal conditions, GRP78 is associated with stress sensor proteins in the ER luminal domain. Under stress conditions, GRP78 is released and binds to misfolded proteins, resulting in the activation of stress sensors (22). The UPR network of interactions is considered to have three major arms, each activated by a characteristic sensor, PERK (PKR-like ER protein kinase), IRE1 (inositol-requiring enzyme 1), and ATF6a (activating transcription factor 6a) (59). The activation of the PERK pathway results in the phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α) subunit, leading to translation attenuation (18). PERK also activates the expression of ATF4, a transcription factor, leading to an upregulation of the proapoptotic genes CHOP (c/EBP homologous protein) and GADD34 (growth arrest and DNA damage-inducible protein-34) (38). IRE1 is a bifunctional ER transmembrane protein with both serine-threonine kinase and RNase activities (52, 56). Upon activation, IRE1 can remove a 26-nucleotide (nt) intron from unspliced X box binding protein 1 (XBP1) mRNA (XBP1u) by RNase activity, resulting in a translational frameshift. The spliced form of XBP1 mRNA (XBP1s) encodes a protein with a novel C terminus and acts as a potent transcriptional activator of many genes involved in the UPR (42). ATF6a is an ER transmembrane protein residing in the cytosol under normal physiological conditions. Upon the accumulation of misfolded proteins in the ER, ATF6a migrates to the Golgi apparatus, where it is cleaved by S1P and S2P proteases, releasing a soluble fragment that enters the nucleus and activates the transcription of ER chaperones and other genes responsible for correct protein folding (20). A number of viruses have been shown to trigger ER stress upon infection. However, the pattern of molecular interactions that occurs within the UPR program differs depending on virus identity and type of host cell. Many viruses apparently activate only one or a subset of UPR pathways, and interestingly, some viral infections activate one pathway yet suppress others. For example, the expression of hepatitis C virus (HCV) proteins activates the PERK- and ATF6a-initiated pathways (4, 8, 40) yet suppresses the IRE1-XBP1 pathway (51). Similarly, human cytomegalovirus (CMV) activates PERK and IRE1-XBP1 but suppresses the ATF6a pathway (23, 53). In this study, with the use of mouse cardiomyocytes and unmodified HeLa cells, as well as HeLa cell lines engineered to inducibly express genes integral to the UPR, we focus on the mechanisms of linkage between the ER stress response to CVB3 infection and the induction of apoptosis. We found that CVB3 infection activates ER stress effectors and differentially regulates the three arms of the UPR. CVB3 infection produced a downregulation of p58IPK and associated enhancement of PKR (PERK) phosphorylation activity, and these alterations affected the other two arms of the UPR. Subsequently, the proapoptotic proteins CHOP, SREBP1 (sterol regulatory element binding protein 1), and caspase-12 can become induced. Taken together, these activities appear to participate in a coordinated shifting of the ER stress response to an apoptotic program in CVB3-infected cells.

Our previous study reported that mouse BNIP-21 (mBNIP-21) induces apoptosis through a mitochondria-dependent pathway. To map the functional domains of mBNIP-21, we performed mutational analyses and demonstrated that the BNIP-2 and Cdc42GAP homology (BCH) domain is required for apoptosis induction by mBNIP-21 targeting the mitochondria and inducing cytochrome c release. This pro-apoptotic activity was enhanced by coxsackievirus infection. However, deletion of the Bcl-2 homology 3 (BH3)-like domain, a well-known cell 'death domain' in proapoptotic Bcl-2 family proteins, did not affect the activity of mBNIP-21. These data were further supported by transfection of a mouse Bax (mBax) mutant, whose BH3 was replaced by the mBNIP-21 BH3-like domain. This replacement significantly reduced the pro-apoptotic activity of mBax. We also found that the predicted calcium binding domain has no contribution to the mBNIP-21-induced apoptosis. Further mapping of the motifs of BCH domain demonstrated that deletion of the hydrophobic motif proximal to the C-terminal of the BCH significantly reduced its proapoptotic activity. These findings suggest that mBNIP-21, as a member of the BNIP subgroup of the Bcl-2-related proteins, functions without need of BH3 but its BCH domain is critical for its activity in inducing cell elongation, membrane protrusions and apoptotic cell death.

Targeted drug delivery system, consisting of a guidance component, a transport vehicle and the drug, is aiming at distributing the drugs to a specific organ or cell type in a sufficient concentration to achieve the high efficiency and alleviate the side-effects of the drugs. During the past decade, besides physical and chemical methods, biological strategies have been developing at an amazing speed improving the drug targeting system. These include: i) searching for guidance components, which include integrin ligands, extracellular matrix proteins, carbohydrates, vitamins, antibodies, nucleic acid aptamers and peptides, leading the whole system to the targeting sites; ii) development of the transport vehicles, such as polymers and plasmid/virus vectors that play the role in carrying drugs and protecting them on their way to the destination; and iii) the design or selection of an effective drug, the "final bullet" that functions on the targeting site, to test the efficiency of the system. Here, we briefly review the recent advances on the development of the most essential targeted drug delivery systems and strategies, and particularly focusing on the progress in discovery of the ligand components and construction of carrying vehicles for drug targeted delivery systems. In addition, we briefly discuss the prospect of this field.

In humans, coxsackievirus B3 is the primary etiological agent of viral myocarditis, an inflammatory disease process involving the heart muscle. Specific therapy is currently unavailable. Viral myocarditis is a complex, multiphasic infectious-inflammatory-reparative process. To address the temporal dimensionality of myocarditis, array- and nonarray-based molecular techniques, and histological and functional assays were used to help define enteroviral pathogenesis and its relation to heart failure. The application of high throughput genomic strategies and bioinformatics tools – coupled with established molecular techniques – have allowed us to perform a large-scale analysis of gene expression to better understand the host response to viral infection. Differential messenger RNA display, spotted complementary DNA arrays and Affymetrix Gene Chips (Affymetrix, United States) were used to study murine hearts during acute viremic, inflammatory and reparative stages. The observed global decreases in expression of metabolic and mitochondrial genes were focused on. The authors have previously characterized the role of mitochondria-triggered apoptosis, and pro- and anti-apoptotic Bcl-2 family proteins in enteroviral infections. The impact of altered mitochondrial transcripts on such host cell death and on metabolic injury to the heart is currently under study. In the authors’ experience, the experimental progression from high throughput, unbiased analysis to biological validation has been only partially systematic. Insights are offered into the logic behind the selection of genes of potential interest for further investigation in the myocarditis model. A series of criteria for validatory decision-making, which the authors have developed based on their experiences, is described. Such criteria reflect known or putative gene function and expression patterns, as well as pragmatic considerations in the determination of steps toward investigation. This approach may help other investigators who need to dissect large genomic data sets to find targets for biological confirmation. Together, the authors’ genomic studies have generated new, testable hypotheses regarding the interaction between host and enterovirus.

Coxsackievirus (CV)B3 is the primary cause of viral myocarditis. We previously observed CXC chemokine ligand 10 (CXCL10) upregulation in the myocardium early in infection. However, the impact of CXCL10 in CVB3-induced myocarditis is unknown. Using isolated primary mouse cardiomyocytes we demonstrated for the first time that cardiomyocytes can express CXCL10 on interferon-γ stimulation. To explore the role of CXCL10 in CVB3-induced myocarditis, both CXCL10 transgenic and knockout mice were used. Following CVB3 challenges, the viral titer in the hearts inversely correlated with the levels of CXCL10 at early phase of infection before visible immune infiltration. Furthermore, as compared with the control mice, the decreased virus titers in the CXCL10 transgenic mouse hearts led to less cardiac damage and better cardiac function and vice verse in the knockout mice. This antiviral ability of CXCL10 might be through recruitment of natural killer (NK) cells to the heart and increased interferon-γ expression early in infection. At day 7 postinfection, with massive influx of mononuclear cells the expression of CXCL10 enhanced the infiltration of CXCR3+cells, CD4+, and CD8+T cells, as well as the expression of associated inflammatory cytokines. However, the augmented accumulation of these immune cells and associated cytokines failed to alter the viral clearance and mice survival. These results suggest the protective role of CXCL10 during the early course of CVB3 infection, which is attributed to the recruitment of NK cells. Nonetheless, CXCL10-directed chemoattractant effect is not sufficient for host to clear the virus in the heart.

Retrovirus: Efforts to Characterize Host Response to HIV-1 Infection (M Montano & P Sebastiani) Host Immune Responses in HIV Infection (R D Allison & S Kottilil) Negative Single-Stranded RNA Virus: Host Immune Response to Influenza Virus (T M Moran & C B Lopez) Innate Recognition of Viral Infection and the Involvement of Autophagy (B Ramanathan & A Iwasaki) Hantavirus Infection and Innate Immunity (N Sen et al.) Positive Single-Stranded RNA Virus: Impact of Filovirus Infection upon Cellular Signaling Pathways (C F Basler) Host Signaling Responses to Coxsackievirus Infection (G Gao et al.) Double-Stranded RNA Virus: Host Signaling Responses to Reovirus Infection (D Pan et al.) Rotavirus Antagonism of the Host Innate Immune Response (J T Patton & M Barro) and other papers.

MicroRNAs (miRNAs) are endogenous, short, double-stranded and noncoding RNA molecules that have been identified in a variety of organisms and certain viruses. This group of new molecules is transcribed mainly from the introns and/or exons or intergenic regions and plays important regulatory roles in development and gene expression. Mature miRNAs are typically 20-24 nucleotides in length and regulate target mRNAs post transcriptionally by interactions with partially mismatched sequences in the 3 ’ untraslated regions of these messengers. These interactions result in the suppression of translation or degradation of target mRNAs. At the present, although the biological functions of miRNAs are not completely revealed, a growing body of evidence implicates that miRNA pathway is a new mechanism of gene regulation in both normal and diseased conditions and therefore investigation of miRNA biogenesis and function may add new tools for gene functional study and drug development. In this article, we will briefly review the structure, biogenesis and basic mechanism of action of miRNAs identified in higher organisms and viruses and then focus on the recent progress in research for drug development using the miRNA pathway as a strategy. Particularly, we will discuss the advance, challenge and future directions on antiviral drug development using miRNA as a target or a gene silencing tool for the treatment of viral infections.

Anti-picornaviral antisense agents are part of a broader group of nucleic acid-based molecules developed for sequence-specific inhibition of translation and/or transcription of the target sequence through induced nuclease activity or physical hindrance. Three types of nucleic acid-based gene silencing molecules can be distinguished, including DNA-base antisense oligonucleotides (ASO), nucleic acid enzymes (ribozyme and DNAzyme) and double-stranded small interfering RNA (siRNA or microRNA). These antisense DNA and RNA molecules have been widely studied for gene functional studies and therapeutic purposes. In this review, we focus on drug development using ASO and siRNA strategies to inhibit picornavirus infections. The picornavirus genome organization and life cycle is described, followed by discussion of design considerations, chemical modifications and drug delivery approaches. Recent studies using antisense against picornavirus are reviewed. Finally, we compare the advantages and disadvantages of the antisense agents with those of other therapeutics, taking into consideration their limitations which need to be overcome to achieve the final goal of clinical application.

CVB3 myocarditis can lead to dilated cardiomyopath (DCM). DCM is one of the leading causes of the need for heart transplantation, so it is important to understand the life cycle of CVB3 and its interactions with the host cell. Infection causes rapid death of host cardiomyocytes by altering normal cellular homeostasis for the efficient release of progeny virion. In this chapter, we will examine the impact that CVB3 replication has on host cell biology, from events that take place at receptor ligation to progeny virus release. The primary focus will be on the myriad of signalling pathways that are activated at all stages of virus replication and their downstream effects. We will also discuss some of the extracellular effects of infection as well as immune and matrixmetalloprotease activation. Interactions of host cell proteins with the 5' untranslated region (UTR) are required for translation and replication of CVB3. These interactions do not always benefit the virus since the interactions of a 28-kDa host protein with the 5' UTR are thought to be responsible for inhibitory activity against CVB3. Finally, we will discuss how the elucidation of the different stages of replication has provided the opportunity to develop novel strategies for combating CVB3 infection.

Coxsackievirus B3 (CVB3) is the primary human pathogen of viral myocarditis, a disease which causes sudden, unexpected death of infants and youth. Myocarditis was originally considered predominantly an inflammatory disease, but subsequent studies have revealed that direct myocardial injury by CVB3 prior to host immune responses contributes significantly to the progression of myocarditis. Heart transplantation is the only definitive treatment for serious myocarditis; thus, development of therapeutic intervention based on the pathogenesis of CVB3 becomes a preferred approach. Studies by our laboratory and others have shown that CVB3 infection triggers apoptosis of host cells through cytochrome C release and activation of multiple caspases. Furthermore, CVB3 infection activates several intracellular signaling pathways, such as the ERK pathway, the p38 MAPK pathway, and the PI3K/Akt pathway, for its efficient replication and release of progeny virions in order to complete its life cycle. Major cellular protein degradation systems, such as the ubiquitin–proteasome system, are also exploited by CVB3. These findings on the critical roles of host factors in pathogenesis of CVB3 thus provide us cellular targets for development of possible therapeutic interventions at molecular levels to restrain CVB3 infection.

Summary Ribosomal RNA sequence analysis has been used to show that four flexibacterial species, Flexibacter aurantiacus, Fx. canadensis, Fx. flexilis and Fx. sancti, are members of the eubacterial phylum defined by the flavobacteria, bacteroides and assorted other isolated species. However, within that phylum the flexibacterial species are not specifically related to one another. Each, instead, has a specific relative that is not a flexibacterium. The widespread distribution of flexibacterial species within the Flavobacterium-Bacteroides phylum suggests that gliding motility may have been an aboriginal property of this major group.

The Clostridium bryantii-Methanospirillum hungatei syntrophic coculture, grown on caproate, was adapted to grow on crotonate. Then, C. bryantii was isolated in pure culture from crotonate bottle plates. A 16S rRNA sequence analysis of the pure subculture revealed that, as a member of the gram-positive phylum, it was not closely related to any of the Clostridium species with which it was compared or to any of the other clusters in the gram-positive phylum with which it was compared. However, it was closely related to another syntrophic fatty acid-degrading bacterium, Syntrophomonas wolfei. On the basis of its phylogeny, physiology, and cell wall ultrastructure, we propose assignment of C. bryantii to Syntrophospora bryantii gen. nov., nov. comb.

Positive single-stranded RNA viruses constitute a broad and prevalent group of pathogens that threaten human health and life worldwide. While effective vaccines have been developed for some, such as poliovirus and hepatitis A, others such as coxsackievirus, severe acute respiratory syndrome coronavirus (SARS-CoV) and West Nile virus have no accredited drug treatments. Antisense technologies, which encompass small interfering RNA, antisense oligonucleotides, ribozymes and their chemically modified analogs, involve small sequence-specific nucleic-acid-based molecules that inhibit viral replication at the level of translation. Many antisense oligomers are proven antiviral agents in vitro. In this review, iwe provide an overview of the antiviral antisense field, highlighting specific studies of interest over the past several years, using our experience with coxsackievirus B3 as a reference point. Overall, both the challenges and successes of existing antisense therapies for positive single-stranded RNA viruses can be paralleled to those for other virus groups, and vice versa.

Microarrays have helped researchers gain much insight into gene expression profiles in the context of many diseases including those in the injured heart. Our genomic investigations have been focused on elucidation of host gene responses to enterovirus infection. We have gained valuable technical expertise in using Affymetrix oligonucleotide arrays, also known as GeneChips, and cDNA spotted arrays to probe differential gene expression in both cultured cells and in heart tissue. Here, we provide a technique-focused supplement to the Affymetrix GeneChip Expression Analysis Manual for sample preparation, processing, and array hybridization. We provide expanded explanations to highlight important points within the existing protocol and offer variations to standard procedures when appropriate. For investigators using myocardial tissues for microarray experiments, we further address the necessity of and methods for in situ flushing of the vasculature, tissue homogenization, and considerations for limits of expression detection in rare cells. It is our intention to provide useful technical information, based on our experience, to assist those researchers using Affymetrix GeneChips in their own genomic research.

Gamma interferon-inducible protein 10 (IP10) is a CXC chemokine in the chemokine superfamily. It is a chemoattractant for T cells, monocytes (33, 58, 59), and NK cells (34). It has an antiproliferative effect on endothelial cells (35), as well as angiostatic and antitumor activity (1, 31, 34). Recently, it has been reported that coexpression of IP10 and its receptor CXCR3 plays an important role in human cardiac allograft rejection (37). On the other hand, IP10 upregulation was shown in a mouse model of hepatectomy to play a fundamental role in hepatic repair and regeneration (28). Among the current expanding list of cellular activities discovered for IP10, there has been no study on virus-induced IP10 upregulation in infectious heart diseases. Therefore, we focused our investigation on the role of IP10 in coxsackievirus B3 (CVB3) infection, particularly on the characterization of the IP10-induced apoptotic pathway. CVB3 is the most common causative agent of viral myocarditis in humans (15). In addition, CVB3-induced acute myocarditis may develop into chronic infection leading to dilated cardiomyopathy, whose only treatment is heart transplantation (8). The pathogenesis of CVB3 infection has been studied for decades. However, it is only recently that the disease occurrence has been found to be determined by complex interactions among several variables, such as viral genome structure (6), host genetic background (6, 20, 68), and the age (30) and the immune status (17, 21) of the host. The molecular biology of CVB3 is well documented. However, the roles of host gene responses to CVB3 infection are poorly understood. Our previous studies using differential mRNA display identified 28 genes which were either up- or down-regulated in CVB3-infected mouse hearts, and five of these genes have been reported (68). In this paper, we report an additional upregulated gene, the IP10 gene, and focus on its functional analysis, which has demonstrated that this gene induces host cell apoptosis through a p53-dependent pathway. p53 is a sequence-specific transcription factor and plays a pivotal role in cellular responses to a variety of genotoxic stresses, which result in cell cycle arrest or apoptosis (12, 16). While the p53-dependent cell cycle checkpoints are well characterized, the actual mechanism whereby p53 activates apoptosis is still not fully understood. p53 can induce the expression of several apoptotic genes, including genes those for the death receptors CD95/Fas and KILLER/DR5 and the proapoptotic Bcl-2 family members Bax and PUMA (39, 40, 67). However, their roles in apoptotic pathways remain to be defined. Moreover, the expression of these proapoptotic genes induced by p53 is variable depending on the experimental system used (48, 62, 63). Thus, investigation of the differential regulation of gene expression in the IP10-induced p53-mediated apoptotic pathway and further correlation of these gene responses to the IP10-mediated antiviral activity are critically important for understanding the host defense mechanism in CVB3 infection. The p53 gene is a tumor suppressor gene, and its expression is down-regulated in certain tumor cell lines such as HeLa cells. This is because the cells harbor high-risk human papillomavirus type 18 (HPV-18) E6 and E7 oncogenes (75), which can mediate ubiquitin-dependent proteolytic degradation of tumor suppressors p53 and retinoblastoma protein (pRb), respectively (23, 46). Therefore, the objective of this study is to determine how p53 induces apoptosis in HeLa cells and what the function of IP10 is in this induction of apoptotic cell death. To achieve this goal, we have performed experiments in this study to investigate the function of IP10 by establishing a Tet-On HeLa cell line, which expresses IP10 upon induction using doxycycline (Dox). We demonstrated that IP10 expression activated a p53-dependent apoptosis pathway. In the further characterization of the cell death pathway and exploration of the mechanism of IP10-mediated antiviral activity, we analyzed the expression of a number of p53-responsive genes and several other related proteins. We found that several proapoptotic genes downstream of p53 had an altered expression, resulting in an increased rate of cell death and a corresponding inhibition of viral capsid protein synthesis. We propose that IP10 upregulation may benefit the host by an early induction of cell apoptosis through a p53-dependent pathway, thereby limiting viral replication and spread of infection. Moreover, in an attempt to determine why p53 can keep consistently high levels of expression in HeLa cells expressing IP10, we finally found that p53 upregulation is due, at least in part, to the suppression of HPV E6 and E7 expression by IP10.

Coxsackievirus B3 (CVB3), a common human pathogen for viral myocarditis, induces a direct cytopathic effect (CPE) and apoptosis on infected cells. To elucidate the mechanisms that contribute to these processes, we studied the role of glycogen synthase kinase 3beta (GSK3beta). GSK3beta activity was significantly increased after CVB3 infection and addition of tyrosine kinase inhibitors blocked CVB3-triggered GSK3beta activation. Inhibition of caspase activity had no inhibitory effect on CVB3-induced CPE; however, blockage of GSK3beta activation attenuated both CVB3-induced CPE and apoptosis. We further showed that CVB3 infection resulted in reduced beta-catenin protein expression, and GSK3beta inhibition led to the accumulation and nuclear translocation of beta-catenin. Finally, we found that CVB3-induced CPE and apoptosis were significantly reduced in cells stably overexpressing beta-catenin. Taken together, our results demonstrate that CVB3 infection stimulates GSK3beta activity via a tyrosine kinase-dependent mechanism, which contributes to CVB3-induced CPE and apoptosis through dysregulation of beta-catenin.

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.

Microarrays have helped researchers gain much insight into gene expression profiles in the context of many diseases including those in the injured heart. Our genomic investigations have been focused on elucidation of host gene responses to enterovirus infection. We have gained valuable technical expertise in using Affymetrix oligonucleotide arrays, also known as GeneChips, and cDNA spotted arrays to probe differential gene expression in both cultured cells and in heart tissue. Here, we provide a technique-focused supplement to the Affymetrix GeneChip Expression Analysis Manual for sample preparation, processing, and array hybridization. We provide expanded explanations to highlight important points within the existing protocol and offer variations to standard procedures when appropriate. For investigators using myocardial tissues for microarray experiments, we further address the necessity of and methods for in situ flushing of the vasculature, tissue homogenization, and considerations for limits of expression detection in rare cells. It is our intention to provide useful technical information, based on our experience, to assist those researchers using Affymetrix GeneChips in their own genomic research.

Coxsackievirus is the most prevalent virus associated with the pathogenesis of myocarditis and its sequela dilated cardiomyopathy. We have previously shown that coxsackievirus infection facilitates the ubiquitin/proteasome processing of the cell-cycle protein cyclin D1 and the tumor suppressor p53, which raises the possibility that the ubiquitin/proteasome pathway may be used by virus to promote viral replication. In this study, we examined the interplay between coxsackievirus replication and the ubiquitin/proteasome pathway in murine cardiomyocytes. We found that treatment of cells with the proteasome inhibitors MG132 or lactacystin significantly decreased virus titers in the supernatant and prevented virus-induced cell death. We further examined the effects of proteasome inhibitor on different stages of coxsackievirus life-cycle. We showed that inhibition of the ubiquitin/proteasome pathway did not affect virus entry and had no influence on viral protease proteolytic activities. However, viral RNA transcription and protein translation were markedly reduced after addition of proteasome inhibitors. We further demonstrate that ubiquitin/proteasome pathway-mediated viral replication does not appear to be related to changes in proteasome activities. Taken together, our data suggest that proteasome inhibitor reduces coxsackievirus replication through inhibition of viral RNA transcription and protein synthesis. Thus, proteasome inhibition may represent a novel therapeutic approach against myocarditis.

Our previous studies using differential mRNA display have shown that interferon-gamma-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.

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.

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 G 1 phase of the cell cycle. Analysis of cellular DNA synthesis in infected cells by thymidine incorporation assays showed a significant reduction in [ 3 H]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 G 1 progression and G 1 /S transition. Infection resulted in dephosphorylation of retinoblastoma protein and reduced G 1 cyclin-dependent kinase activities, accompanied by decreased levels of G 1 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 G 1 /S boundary and induces cell cycle arrest in part through an increase in ubiquitin-dependent proteolysis of cyclin D1.

The lengthy 5' untranslated region (5'UTR) of coxsackievirus B3 (CVB3) forms a highly ordered secondary structure, which plays an important role in controlling viral transcription and translation. Our previous work has delineated the internal ribosome entry site (IRES) by mutation of mono- and bicistronic plasmids containing the 5'UTR and subsequent cell- free translation in rabbit reticular lysate (D. Yang, J. E. Wilson, D. R. Anderson, L. Bohunek, C. Cordeiro, R. Kandolf, and B. M. McManus. (1997). Virology 228, 63-73). To further identify the sequence elements responsible for viral translation and infectivity in tissue culture cells, >30 full-length mutants of CVB3 were constructed by mutations of the IRES and its flanking regions. Viral RNAs were transcribed from these constructs and transfected into HeLa cells. When the stem-loops G and H in the putative IRES were deleted, viral infectivity was abolished and viral protein translation was also undetectable by immunoblot analysis. However, when stem-loops A and B were deleted or stem-loop E was partially deleted, viral protein translation could be detected although cytopathic effect could not be observed. The data suggest that the crucial sequence of the IRES is located at stem-loops G and H. Further serial deletion mapping up and down stream of the crucial sequence defined more accurately the 5' and 3' boundaries of the IRES, located at nucleotides (nts) 309-432 and 639-670, respectively. These results indicate that the core sequence of the IRES should be located at nts 432-639. This IRES segment is much shorter and located closer to the initiation codon than that of poliovirus. To further define critical nucleotides within the IRES core, site-directed mutagenesis was conducted at the IRES core sequence by PCR. A 46-nt deletion in the pyrimidine-rich tract of stem-loop G abolished viral translation and infectivity. Interestingly, five single-nt substitutions in the pyrimidine-rich tract aimed at destabilizing the base pairing between the viral IRES and host 18S rRNA did not abolish CVB3 infectivity although viral protein translation was significantly reduced. This finding suggests that ribosomal internal initiation of translation and viral infectivity not only may require RNA secondary structure but also may need tertiary structure and perhaps the assistance of host protein factors.

The genes for nine ribosomal proteins, L24, L5, S14, S8, L6, L18, S5, L30, and L15, have been isolated and sequenced from the spc operon in the archaeon (Crenarchaeota) Sulfolobus acidocaldarius, and the putative amino acid sequence of the proteins coded by these genes has been determined. In addition, three other genes in the spc operon, coding for ribosomal proteins S4E, L32E, and L19E (equivalent to rat ribosomal proteins S4, L32, and L19), were sequenced and the structure of the putative proteins was determined. The order of the ribosomal protein genes in the spc operon of the Crenarchaeota kingdom of Archaea is identical to that present in the Euryarchaeota kingdom of Archaea and also identical to that found in bacteria, except for the genes for r-proteins S4E, L32E, and L19E, which are absent in bacteria. Although AUG is the initiation codon in most of the spc genes, GUG (val) and UUG (leu) are also used as initiation codons in S. acidocaldarius. Over 70% of the codons in the Sulfolobus spc operon have A or U in the third position, reflecting the low GC content of Sulfolobus DNA. Phylogenetic analysis indicated that the archaeal r-proteins are a sister group of their eucaryotic counterparts but did not resolve the question of whether the Archaea is monophyletic, as suggested by the L6P, L15P, and L18P trees, or the question of whether the Crenarchaeota is separate from the Euryarchaeota and closer to the Eucarya, as suggested by the S8P, S5P, and L24P trees. In the case of the three Sulfolobus r-proteins that do not have a counterpart in the bacterial ribosome (S4E, L32E, and L19E), the archaeal r-proteins showed substantial identity to their eucaryotic equivalents, but in all cases the archaeal proteins formed a separate group from the eucaryotic proteins.

In recent years, intense research has been directed towards understanding molecular mechanisms involved in viral pathogenesis. It is now known that many viruses manipulate host defense mechanisms to prevent apoptosis in order to maximize viral replication. Towards the end of their replication cycle, certain viruses direct the synthesis of proteins that induce apoptosis or cell lysis thereby facilitating viral release from the cell. The present review summarizes the current understanding of interactions between viral proteins and the host cell death machinery.

Rubella virus (RV) virions contain two glycosylated membrane proteins, E1 and E2, that exist as a heterodimer and form the viral spike complexes on the virion surface. Formation of an E1-E2 heterodimer is required for transport of E1 out of the endoplasmic reticulum lumen to the Golgi apparatus and plasma membrane. To investigate the nature of the E1-E2 interaction, we have introduced mutations in the internal hydrophobic region (residues 81 to 109) of E1. Substitution of serine at Cys82 (mutant C82S) or deletion of this hydrophobic domain (mutant dt) of E1 resulted in a disruption of the E1 conformation that ultimately affected E1-E2 heterodimer formation and cell surface expression of both E1 and E2. Substitution of either aspartic acid at Gly93 (G93D) or glycine at Pro104 (P104G) was found to impair neither E1-E2 heterodimer formation nor the transport of E1 and E2 to the cell surface. Fusion of RV-infected cells is induced by a brief treatment at a pH below 6.0. To test whether this internal hydrophobic domain is involved in the membrane fusion activity of RV, transformed BHK cell lines expressing either wild-type or mutant spike proteins were exposed to an acidic pH and polykaryon formation was measured. No fusion activity was observed in the C82S, dt, and G93D mutants; however, the wild type and the P104G mutant exhibited fusogenic activities, with greater than 60% and 20 to 40% of the cells being fused, respectively, at pH 4.8. These results suggest that it is likely that the region of E1 between amino acids 81 and 109 is involved in the membrane fusion activity of RV and that it may be important for the interaction of that protein with E2 to form the E1-E2 heterodimer.

The role of lymphocytes in the pathogenesis of viral-induced insulin dependent diabetes mellitus (IDDM) is controversial. To better understand how a virus-induced IDDM depends on the infiltrating lymphocytes, encephalomyocarditis virus (EMCV) was inoculated intraperitoneally into three kinds of mice; virus-susceptible C57BL/6, virus-resistant 129/SV and recombination activity gene-2 (Rag2) knockout 129/SV mice. Pancreatic inflammation and beta cell necrosis were evaluated after EMCV, D variant (10(3) pfu/mouse) inoculation. On post-inoculation day 14, the lethal rates of C57BL/6, 129/SV and Rag2 knockout mice were 52, 10 and 100%, respectively. The blood glucose in Rag2KO mice on day 8 was significantly elevated as compared with 129SV mice (231 +/- 49 vs 169 +/- 32 mg/dl, P0.05). In situ hybridization demonstrated the EMCV genome in the pancreas of Rag2 knockout and C57BL/6 mice, but not in 129/SV mice. Beta cell necrosis were more severe in Rag-2 knockout mice than in wild type 129/SV mice, but lymphocyte infiltration was less severe than C57BL/6. Pancreas in Rag2 knockout mice infected with virus were affected more severely than the virus-resistant strain of mice. Diabetogenic virus induced IDDM in virus-resistant mice without mature lymphocytes.

Innate immunity is central to the clearance of pathogens from hosts as well as to the definition of acquired immune responses (D. T. Fearon, and R. M. Locksley, Science 272:50-53, 1996). Coxsackievirus B3 (CVB3), a human cardiopathic virus, was evaluated for the ability to activate the alternative and classical pathway of complement. CVB3 proteins interact with complement component 3 (C3, a soluble protein effector of innate immunity) after either in vitro exposure to mouse serum or in vivo murine infection and activate the alternative pathway of complement. In addition, we demonstrate that viral antigen retention and localization in germinal centers is dependent on C3, while virus antigen retention in extrafollicular regions in the spleen is not. In vivo depletion of native C3 abolished the rapid formation of virus-specific germinal centers (by day 3 post-CVB3 infection) in the absence of serum anti-CVB3 antibodies. These studies demonstrate that innate immune mechanisms, such as C3 interaction with CVB3, are essential for splenic antiviral germinal center formation in naive (antigen nonsensitized) mice resistant (C57BL/6J strain) and susceptible (A/J strain) to CVB3-induced myocarditis.

Abstract Inflammatory cytokines play a key role in the myocardial injury produced by viral myocarditis. Although interleukin-6 (IL-6) reportedly possesses antiviral properties, its effect in viral myocarditis is unclear. To investigate the role of IL-6 in viral myocarditis induced by encephalomyocarditis virus (EMCV) in mice, we evaluated (1) the survival rate following IL-6 administration, (2) the viral titer in the heart, (3) viral replication in the heart by in situ hybridization, (4) histopathological changes using immunohistochemical staining, (5) neutralizing antibody against EMCV, (6) circulating interferon and tumor necrosis factor-α (TNF-α), (7) viral suppression in vitro by IL-6, and (8) natural killer (NK)–cell activity. Eight-week-old C3H/HeJ mice were injected intraperitoneally with EMCV (day 0) and were also injected subcutaneously twice daily for 4 consecutive days with 10 μg/0.1 mL of human IL-6 on day −4 (group A), day 0 (group B), or day +4 (group D) for 4 days. As a control, 0.1 mL PBS instead of IL-6 was injected on day 0 for 4 days (group C). Certain mice were killed on day 4. The myocardial virus titers, viral replication in situ, and NK-cell activity in the spleen were determined. Decreased viral titer and viral replication in the heart reduced the titer of circulating TNF-α, and lower NK-cell activity was observed in group B versus group C (control group). The titer of neutralizing antibodies against EMCV was significantly ( P