[Abstract unavailable

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.

We report the NMR solution structure of the intramolecular G-quadruplex formed in human telomeric DNA in K+. The hybrid-type telomeric G-quadruplex consists of three G-tetrads linked with mixed parallel–antiparallel G-strands, with the bottom two G-tetrads having the same G-arrangement (anti:anti:syn:anti) and the top G-tetrad having the reversed G-arrangement (syn:syn:anti:syn). The three TTA loop segments adopt different conformations, with the first TTA assuming a double-chain-reversal loop conformation, and the second and third TTA assuming lateral loop conformations. The NMR structure is very well defined, including the three TTA loops and the two flanking sequences at 5′- and 3′-ends. Our study indicates that the three loop regions interact with the core G-tetrads in a specific way that defines and stabilizes the unique human telomeric G-quadruplex structure in K+. Significantly, a novel adenine triple platform is formed with three naturally occurring adenine residues, A21, A3 and A9, capping the top tetrad of the hybrid-type telomeric G-quadruplex. This adenine triple is likely to play an important role in the formation of a stable human telomeric G-quadruplex structure in K+. The unique human telomeric G-quadruplex structure formed in K+ suggests that it can be specifically targeted for anticancer drug design.

Posttranslational neddylation of cullins in the Cullin-Ring E3 ligase (CRL) complexes is needed for proteolytic degradation of CRL substrates, whose accumulation induces cell-cycle arrest, apoptosis, and senescence. The Nedd8-activating enzyme (NAE) is critical for neddylation of CRL complexes and their growth-promoting function. Recently, the anticancer small molecule MLN4924 currently in phase I trials was determined to be an inhibitor of NAE that blocks cullin neddylation and inactivates CRL, triggering an accumulation of CRL substrates that trigger cell-cycle arrest, apoptosis, and senescence in cancer cells. Here, we report that MLN4924 also triggers autophagy in response to CRL inactivation and that this effect is important for the ability of MLN4924 to suppress the outgrowth of liver cancer cells in vitro and in vivo. MLN4924-induced autophagy was attributed partially to inhibition of mTOR activity, due to accumulation of the mTOR inhibitory protein Deptor, as well as to induction of reactive oxygen species stress. Inhibiting autophagy enhanced MLN4924-induced apoptosis, suggesting that autophagy is a survival signal triggered in response to CRL inactivation. In a xenograft model of human liver cancer, MLN4924 was well-tolerated and displayed a significant antitumor effect characterized by CRL inactivation and induction of autophagy and apoptosis in liver cancer cells. Together, our findings support the clinical investigation of MLN4924 for liver cancer treatment and provide a preclinical proof-of-concept for combination therapy with an autophagy inhibitor to enhance therapeutic efficacy. Cancer Res; 72(13); 3360-71. Ó2012 AACR.

Although the Chlamydia trachomatis genome is predicted to encode 50 inclusion membrane proteins, only 18 have been experimentally localized in the inclusion membrane of C. trachomatis-infected cells. Using fusion proteins and anti-fusion protein antibodies, we have systematically evaluated all 50 putative inclusion membrane proteins for their localization in the infected cells, distribution patterns, and effects on subsequent chlamydial infection when expressed ectopically, as well as their immunogenicity during chlamydial infection in humans. Twenty-two of the 50 proteins were localized in the inclusion membrane, and 7 were detected inside the inclusions, while the location of the remaining 21 was not defined. Four (CT225, CT228, CT358, and CT440) of the 22 inclusion membrane-localized proteins were visualized in the inclusion membrane of Chlamydiainfected cells for the first time in the current study. The seven intra-inclusion-localized proteins were confirmed to be chlamydial organism proteins in a Western blot assay. Further characterization of the 50 proteins revealed that neither colocalization with host cell endoplasmic reticulum nor inhibition of subsequent chlamydial infection by ectopically expressed proteins correlated with the inclusion membrane localization. Interestingly, antibodies from women with C. trachomatis urogenital infection preferentially recognized proteins localized in the inclusion membrane, and the immunodominant regions were further mapped to the region predicted to be on the cytoplasmic side of the inclusion membrane. These observations suggest that most of the inclusion membrane-localized proteins are both expressed and immunogenic during C. trachomatis infection in humans and that the cytoplasmic exposure may enhance the immunogenicity.

Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs exert anti-viral functions due to their involvement in protein synthesis shut off and recruitment of innate immune signaling intermediates. The largest RNA viruses, coronaviruses, impose great threat to public safety and animal health; however, the significance of SGs in coronavirus infection is largely unknown. Infectious Bronchitis Virus (IBV) is the first identified coronavirus in 1930s and has been prevalent in poultry farm for many years. In this study, we provided evidence that IBV overcomes the host antiviral response by inhibiting SGs formation via the virus-encoded endoribonuclease nsp15. By immunofluorescence analysis, we observed that IBV infection not only did not trigger SGs formation in approximately 80% of the infected cells, but also impaired the formation of SGs triggered by heat shock, sodium arsenite, or NaCl stimuli. We further demonstrated that the intrinsic endoribonuclease activity of nsp15 was responsible for the interference of SGs formation. In fact, nsp15-defective recombinant IBV (rIBV-nsp15-H238A) greatly induced the formation of SGs, along with accumulation of dsRNA and activation of PKR, whereas wild type IBV failed to do so. Consequently, infection with rIBV-nsp15-H238A strongly triggered transcription of IFN-β which in turn greatly affected rIBV-nsp15-H238A replication. Further analysis showed that SGs function as antiviral hub, as demonstrated by the attenuated IRF3-IFN response and increased production of IBV in SG-defective cells. Additional evidence includes the aggregation of pattern recognition receptors (PRRs) and signaling intermediates to the IBV-induced SGs. Collectively, our data demonstrate that the endoribonuclease nsp15 of IBV interferes with the formation of antiviral hub SGs by regulating the accumulation of viral dsRNA and by antagonizing the activation of PKR, eventually ensuring productive virus replication. We further demonstrated that nsp15s from PEDV, TGEV, SARS-CoV, SARS-CoV-2 harbor the conserved function to interfere with the formation of chemically-induced SGs. Thus, we speculate that coronaviruses employ similar nsp15-mediated mechanisms to antagonize the host anti-viral SGs formation to ensure efficient virus replication.

The primate lentiviral accessory protein Nef downregulates CD4 and major histocompatibility complex class I (MHC-I) from the cell surface via independent endosomal trafficking pathways to promote viral pathogenesis. In addition, Nef antagonizes a novel restriction factor, SERINC5 (Ser5), to increase viral infectivity. To explore the molecular mechanism of Ser5 antagonism by Nef, we determined how Nef affects Ser5 expression and intracellular trafficking in comparison to CD4 and MHC-I. We confirm that Nef excludes Ser5 from human immunodeficiency virus type 1 (HIV-1) virions by downregulating its cell surface expression via similar functional motifs required for CD4 downregulation. We find that Nef decreases both Ser5 and CD4 expression at steady-state levels, which are rescued by NHCl or bafilomycin A1 treatment. Nef binding to Ser5 was detected in living cells using a bimolecular fluorescence complementation assay, where Nef membrane association is required for interaction. In addition, Nef triggers rapid Ser5 internalization via receptor-mediated endocytosis and relocalizes Ser5 to Rab5 early, Rab7 late, and Rab11 recycling endosomes. Manipulation of AP-2, Rab5, Rab7, and Rab11 expression levels affects the Nef-dependent Ser5 and CD4 downregulation. Moreover, although Nef does not promote Ser5 polyubiquitination, Ser5 downregulation relies on the ubiquitination pathway, and both K48- and K63-specific ubiquitin linkages are required for the downregulation. Finally, Nef promotes Ser5 colocalization with LAMP1, which is enhanced by bafilomycin A1 treatment, suggesting that Ser5 is targeted to lysosomes for destruction. We conclude that Nef uses a similar mechanism to downregulate Ser5 and CD4, which sorts Ser5 into a point-of-no-return degradative pathway to counteract its restriction. Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) express an accessory protein called Nef to promote viral pathogenesis. Nef drives immune escape through downregulation of CD4 and MHC-I from the host cell surface. Recently, Nef was reported to counteract a novel host restriction factor, Ser5, to increase viral infectivity. Nef downregulates cell surface Ser5, thus preventing its incorporation into virus particles, resulting in disruption of its antiviral activity. Here, we report mechanistic studies of Nef-mediated Ser5 downregulation in comparison to CD4 and MHC-I. We demonstrate that Nef binds directly to Ser5 in living cells and that Nef-Ser5 interaction requires Nef association with the plasma membrane. Subsequently, Nef internalizes Ser5 from the plasma membrane via receptor-mediated endocytosis, and targets ubiquitinated Ser5 to endosomes and lysosomes for destruction. Collectively, these results provide new insights into our ongoing understanding of the Nef-Ser5 arms race in HIV-1 infection.

bNewcastle disease virus (NDV) is an important avian pathogen. We previously reported that NDV triggers autophagy in U251 glioma cells, resulting in enhanced virus replication. In this study, we investigated whether NDV triggers autophagy in chicken cells and tissues to enhance virus replication. We demonstrated that NDV infection induced steady-state autophagy in chickenderived DF-1 cells and in primary chicken embryo fibroblast (CEF) cells, evident through increased double-or single-membrane vesicles, the accumulation of green fluorescent protein (GFP)-LC3 dots, and the conversion of LC3-I to LC3-II. In addition, we measured autophagic flux by monitoring p62/SQSTM1 degradation, LC3-II turnover, and GFP-LC3 lysosomal delivery and proteolysis, to confirm that NDV infection induced the complete autophagic process. Inhibition of autophagy by pharmacological inhibitors and RNA interference reduced virus replication, indicating an important role for autophagy in NDV infection. Furthermore, we conducted in vivo experiments and observed the conversion of LC3-I to LC3-II in heart, liver, spleen, lung, and kidney of NDV-infected chickens. Regulation of the induction of autophagy with wortmannin, chloroquine, or starvation treatment affects NDV production and pathogenesis in tissues of both lung and intestine; however, treatment with rapamycin, an autophagy inducer of mammalian cells, showed no detectable changes in chicken cells and tissues. Moreover, administration of the autophagy inhibitor wortmannin increased the survival rate of NDV-infected chickens. Our studies provide strong evidence that NDV infection induces autophagy which benefits NDV replication in chicken cells and tissues.