Numerous genetic and environmental insults impede the ability of cells to properly fold and posttranslationally modify secretory and transmembrane proteins in the endoplasmic reticulum (ER), leading to a buildup of misfolded proteins in this organelle—a condition called ER stress. ER-stressed cells must rapidly restore protein-folding capacity to match protein-folding demand if they are to survive. In the presence of high levels of misfolded proteins in the ER, an intracellular signaling pathway called the unfolded protein response (UPR) induces a set of transcriptional and translational events that restore ER homeostasis. However, if ER stress persists chronically at high levels, a terminal UPR program ensures that cells commit to self-destruction. Chronic ER stress and defects in UPR signaling are emerging as key contributors to a growing list of human diseases, including diabetes, neurodegeneration, and cancer. Hence, there is much interest in targeting components of the UPR as a therapeutic strategy t...

The Role of Endoplasmic Reticulum Stress in Human Pathology

CXCR3 is a chemokine receptor that is rapidly induced on naive T cells following activation, and preferentially remains highly expressed on type-1 helper (Th1)-type CD4(+) T cells, effector CD8(+) T cells and innate-type lymphocytes, such as natural killer (NK) and NKT cells. CXCR3 is activated by three interferon (IFN)-γ-inducible ligands CXCL9 (monokine induced by gamma-interferon), CXCL10 (interferon-induced protein-10) and CXCL11 (interferon-inducible T-cell alpha chemoattractant). Although some studies have revealed that these ligands have redundant functions in vivo, other studies have demonstrated that the three CXCR3 ligands can also collaborate and even compete with each other. Differential regulation of the three ligands at specific times in defined anatomically restricted locations in vivo likely participates in the fine control of T-cell trafficking over the course of an immune response. Among the differences in regulation, CXCL10 is induced by a variety of innate stimuli that induce IFN-α/β as well as the adaptive immune cell cytokine IFN-γ, whereas CXCL9 induction is restricted to IFN-γ. In this review, we will discuss how the balance, timing and pattern of CXCR3 ligand expression appears to regulate the generation of effector T cells in the lymphoid compartment and subsequent migration into peripheral sites of Th1-type inflammation in which the CXCR3 ligands also then regulate the interactions and migratory behavior of effector T cells in an inflamed peripheral tissue.

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CXCR3 ligands: redundant, collaborative and antagonistic functions.

CXCR3 in T cell function.

Like DNA, RNA can be designed and manipulated to produce a variety of different nanostructures. Moreover, RNA has a flexible structure and possesses catalytic functions that are similar to proteins. Although RNA nanotechnology resembles DNA nanotechnology in many ways, the base-pairing rules for constructing nanoparticles are different. The large variety of loops and motifs found in RNA allows it to fold into numerous complicated structures, and this diversity provides a platform for identifying viable building blocks for various applications. The thermal stability of RNA also allows the production of multivalent nanostructures with defined stoichiometry. Here we review techniques for constructing RNA nanoparticles from different building blocks, we describe the distinct attributes of RNA inside the body, and discuss potential applications of RNA nanostructures in medicine. We also offer some perspectives on the yield and cost of RNA production.

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The emerging field of RNA nanotechnology

Apoptosis is a form of cell death by which the body maintains the homeostasis of the internal environment. Apoptosis is an initiative cell death process that is controlled by genes and is mainly divided into endogenous pathways (mitochondrial pathway), exogenous pathways (death receptor pathway), and apoptotic pathways induced by endoplasmic reticulum (ER) stress. The homeostasis imbalance in ER results in ER stress. Under specific conditions, ER stress can be beneficial to the body; however, if ER protein homeostasis is not restored, the prolonged activation of the unfolded protein response may initiate apoptotic cell death via the up-regulation of the C/EBP homologous protein (CHOP). CHOP plays an important role in ER stress-induced apoptosis and this review focuses on its multifunctional roles in that process, as well as its role in apoptosis during microbial infection. We summarize the upstream and downstream pathways of CHOP in ER stress induced apoptosis. We also focus on the newest discoveries in the functions of CHOP-induced apoptosis during microbial infection, including DNA and RNA viruses and some species of bacteria. Understanding how CHOP functions during microbial infection will assist with the development of antimicrobial therapies.

The C/EBP Homologous Protein (CHOP) Transcription Factor Functions in Endoplasmic Reticulum Stress-Induced Apoptosis and Microbial Infection

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.

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

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-gamma 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-gamma 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.

CXCL10 Inhibits Viral Replication Through Recruitment of Natural Killer Cells in Coxsackievirus B3-Induced Myocarditis

Targeted Delivery of Anti-coxsackievirus siRNAs Using Ligand-conjugated Packaging RNAs

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.

MicroRNAs-Based Therapeutic Strategy for Virally Induced Diseases

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.

Yue Su

Papers

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

CXCL10 Inhibits Viral Replication Through Recruitment of Natural Killer Cells in Coxsackievirus B3-Induced Myocarditis

Targeted Delivery of Anti-coxsackievirus siRNAs Using Ligand-conjugated Packaging RNAs

MicroRNAs-Based Therapeutic Strategy for Virally Induced Diseases

Antisense DNA and RNA agents against picornaviruses.