Herpesviruses and Autophagy: Catch Me If You Can!

Autophagy is an evolutionarily conserved cellular degradation pathway involving the digestion of intracellular components via the lysosomal pathway. The autophagic pathway constitutively maintains cellular homeostasis by recycling cytoplasmic organelles and proteins, but it is also stimulated by environmental stress conditions, such as starvation, oxidative stress, and the accumulation of misfolded proteins. It also acts as a cellular defense mechanism against microorganisms by contributing to both the innate and adaptive immunity, and by eliminating intracellular pathogens (xenophagy). There is growing evidence that host cells try to control Herpesvirus infections by activating the autophagic machinery. However, it is well-known that Herpesviruses are smart pathogens and several, such as HSV-1, HCMV and HHV-8, are known to have developed numerous defense strategies for evading the host’s immune response. Inhibition of the antiviral autophagic mechanism has also been reported. Autophagy has also been shown to enhance the major histocompatibility complex presentation of at least two viral proteins, the EBV-encoded EBNA-1 and the HSV-1 encoded gB. In this review, we present an overview of recent advances in our understanding of the complex interplay between autophagy and Herpesviruses

In vivo importance of heparan sulfate-binding glycoproteins for murid herpesvirus-4 infection

Many herpesviruses bind to heparan sulfate (HS). Murid herpesvirus-4 (MuHV-4) does so via its envelope glycoproteins gp70 and gH/gL. MuHV-4 gp150 further regulates an HS-independent interaction to make that HS-dependent too. Cell binding by MuHV-4 virions is consequently strongly HS-dependent. Gp70 and gH/gL show some in vitro redundancy: an antibody-mediated blockade of HS binding by one is well tolerated, whereas a blockade of both severely impairs infection. In order to understand the importance of HS binding for MuHV-4 in vivo, we generated mutants lacking both gL and gp70. As expected, gL−gp70− MuHV-4 showed very poor cell binding. It infected mice at high dose but not at low dose, indicating defective host entry. But once entry occurred, host colonization, which for MuHV-4 is relatively independent of the infection dose, was remarkably normal. The gL−gp70− entry deficit was much greater than that of gL− or gp70− single knockouts. And gp150 disruption, which allows HS-independent cell binding, largely rescued the gL−gp70− cell binding and host entry deficits. Thus, it appeared that MuHV-4 HS binding is important in vivo, principally for efficient host entry

RNA Binding Protein CUGBP2/CELF2 Mediates Curcumin-Induced Mitotic Catastrophe of Pancreatic Cancer Cells

Curcumin inhibits the growth of pancreatic cancer tumor xenografts in nude mice; however, the mechanism of action is not well understood. It is becoming increasingly clear that RNA binding proteins regulate posttranscriptional gene expression and play a critical role in RNA stability and translation. Here, we have determined that curcumin modulates the expression of RNA binding protein CUGBP2 to inhibit pancreatic cancer growth.In this study, we show that curcumin treated tumor xenografts have a significant reduction in tumor volume and angiogenesis. Curcumin inhibited the proliferation, while inducing G2-M arrest and apoptosis resulting in mitotic catastrophe of various pancreatic cancer cells. This was further confirmed by increased phosphorylation of checkpoint kinase 2 (Chk2) protein coupled with higher levels of nuclear cyclin B1 and Cdc-2. Curcumin increased the expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) mRNA, but protein levels were lower. Furthermore, curcumin increased the expression of RNA binding proteins CUGBP2/CELF2 and TIA-1. CUGBP2 binding to COX-2 and VEGF mRNA was also enhanced, thereby increasing mRNA stability, the half-life changing from 30 min to 8 h. On the other hand, silencer-mediated knockdown of CUGBP2 partially restored the expression of COX-2 and VEGF even with curcumin treatment. COX-2 and VEGF mRNA levels were reduced to control levels, while proteins levels were higher.Curcumin inhibits pancreatic tumor growth through mitotic catastrophe by increasing the expression of RNA binding protein CUGBP2, thereby inhibiting the translation of COX-2 and VEGF mRNA. These data suggest that translation inhibition is a novel mechanism of action for curcumin during the therapeutic intervention of pancreatic cancers

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Caractérisation d'une nouvelle fonction de la protéine Us11 dans l'échappement à l'autophagie par le virus Herpès Simplex de type 1

L autophagie est un mécanisme vacuolaire de dégradation de matériel cytoplasmique permettant le maintien de l homéostasie cellulaire, mais elle peut être également activée par de nombreux stress, comme l infection virale. Le virus de l Herpès Simplex de type 1 (HSV 1) est capable de contrecarrer ce mécanisme de défense antivirale. HSV-1 possède une protéine ICP34.5 capable d inhiber l autophagie en se liant à Beclin 1, une protéine de la machinerie autophagique. Nous avons mis en évidence une deuxième protéine d HSV-1 capable d inhiber l autophagie, la protéine tardive Us11, qui pourrait avoir un rôle complémentaire à celui d ICP34.5 dans le contrôle de l autophagie par le virus.Nous montrons que l expression ectopique d Us11 permet de bloquer l autophagie induite par différents stimuli, et ce de manière similaire à ICP34.5. De plus, dans un contexte viral, l expression précoce d Us11 dans des cellules infectées par un virus ICP34.5 permet un contrôle de l autophagie comparable à celui d un virus sauvage. Nous avons ensuite recherché le mécanisme d action d Us11. La protéine Us11 a été décrite comme pouvant interagir avec la kinase dépendante de l ARN double brin PKR, empêchant ainsi la phosphorylation de son substrat eIF2 , un facteur d initiation de la traduction. Nous avons observé qu en l absence de PKR, Us11 n est plus capable d inhiber l autophagie. Nous avons pu confirmer qu Us11 a besoin de se lier à PKR pour exercer son activité inhibitrice par la construction de formes tronquées d Us11, permettant de montrer l importance de son domaine d interaction avec PKR dans l inhibition de l autophagie. L étude des formes tronquées d Us11 a soulevé le fait que le domaine N-terminal était également nécessaire. Aucune interaction de ce domaine avec une protéine cellulaire n a été identifiée à ce jour, mais il pourrait permettre l interaction d Us11 avec une autre protéine de la machinerie autophagique. Cependant, nous avons montré qu Us11 n interagissait pas avec Beclin 1 et n avait pas d effet sur la kinase mTOR, une autre voie importante de l autophagie. Enfin, nous avons étudié la modulation de la voie PKR/eIF2 lors de la stimulation de l autophagie par la carence, et nos résultats suggèrent que cette voie joue un rôle sous-estimé dans la réponse à la carence.Le mécanisme d action de la protéine Us11, qui consiste en un blocage de l autophagie en inhibant PKR, n avait jamais été décrit auparavant. Ce travail ouvre de nombreuses perspectives dans l étude de la voie PKR/eIF2 vis à vis de la régulation de l autophagie, ainsi que dans la compréhension de l implication de l autophagie dans la neurovirulence d HSV-1.Autophagy is an evolutionary conserved vacuolar mechanism allowing to degrade cytoplasmic components and to maintaining cellular homeostasis, but it can also be triggered by a variety of stress-related conditions, including viral infection. The herpes simplex virus 1 (HSV-1) is able to counteract this antiviral mechanism. Notably, HSV-1 encodes a protein, IPC34.5, which inhibits autophagy through its interaction with the autophagy machinery protein Beclin 1. In the present work, we uncovered a second anti-autophagic protein from HSV-1, the late protein Us11, which likely plays a complementary role to ICP34.5 regarding the inhibition of autophagy by the virus. We demonstrated that ectopic expression of Us11 inhibited autophagy triggered by different stimuli, as observed for ICP34.5. Moreover, during viral infection, early expression of Us11 was sufficient to block autophagy in cells infected with a ICP34.5 virus, similarly to the wild-type virus. We then explored the mechanism of action of Us11. Us11 has been described as capable of interacting with the dsRNA-dependent kinase PKR, therefore preventing it to phosphorylate its substrate eIF2 , a translation initiation factor. We demonstrated that Us11 was no longer able to inhibit autophagy when expressed in PKR-deficient cells. We confirmed that Us11 binding to PKR was necessary for its function by constructing various truncated forms of Us11 that showed that the PKR-binding domain was crucial. We also unveiled the importance of a domain located within the N-terminal part of Us11. This domain has no cellular molecular partner known, but it can allow Us11 to interact with another protein of the autophagy machinery. However, we further showed that Us11 did not interact with Beclin 1 nor affected the kinase activity of mTOR, another important pathway regulating autophagy. In our work, we also gained insights into regulatory mechanisms of starvation-induced autophagy.The inhibition of autophagy through the specific blockade of PKR by Us11 had never been previously described. This work thus paves the way for studying the involvement of PKR/eIF2 pathway in the regulation of autophagy and for exploring the role of autophagy in HSV-1 neurovirulence.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Gels vaginaux virucides et prévention de l'infection VIH, limites et perspectives

Près de la moitié des individus vivant avec le VIH sont des femmes qui, dans la gande majorité des cas, ont acquis le virus par exposition sexuelle. En l absence de vaccination, les agents microbicides an application locale vaginale ou rectale font l objet d investigations pour être utilisés comme moyens de prévention contre l infection par le virus. Le développement des microbicides passe par plusieurs étapes, telles que : -le développement préclinique, incluant les essais in vitro, ex vivo, et les essais sur des modèles animaux le développement clinique, incluant les essais cliniques de phase I, II, IIb pour l innocuité, et les essais de phase III pour l efficacité. Les agents microbicides sont répartis en six classes distinctes, en fonction de leur mécanisme d action : les microbicides barrières, les surfactants, les protecteurs du milieu vaginal, les inhibiteurs d entrée du virus, les inhibiteurs de la transcriptase inverse, les inhibiteurs de la fusion. Les essais cliniques de phase I et II ont révélé que la majorité des agents microbicides ont une bonne innocuité et sont bien tolérés, mais à ce jour très peu d études de phase III ont démontré une réelle efficacité contre l infection par le virus. Au fur et à mesure des années et des échecs, les recherches se sont portées vers des formulations de microbicides de plus en plus spécifiques du virus. Ainsi, on part de microbicides barrières agissant comme un préservatif (invisible condom®) jusqu au microbicide spécifique de la transcriptase inverse virale (gel Ténofovir), qui à ce jour cristallise les meilleurs espoirs de développemnt d un composé en application locale.CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocSudocFranceF

Herpesvirus and Autophagy: “All Right, Everybody Be Cool, This Is a Robbery!”

Autophagy is an essential vacuolar process of the cell, leading to lysosomal degradation and recycling of proteins and organelles, which is extremely important in maintaining homeostasis. Multiple roles have been now associated with autophagy, in particular a pro-survival role in nutrient starvation or in stressful environments, a role in life span extension, in development, or in innate and adaptive immunity. This cellular process can also take over microorganisms or viral proteins inside autophagosomes and degrade them directly in autolysosomes and is then called xenophagy and virophagy, respectively. Several Herpesviruses have developed strategies to escape this degradation, by expression of specific anti-autophagic proteins. However, we are increasingly discovering that Herpesviruses hijack autophagy, rather than just fight it. This beneficial effect is obvious since inhibition of autophagy will lead to decreased viral titers for human cytomegalovirus (HCMV), Epstein-Barr virus (EBV) or Varicella-Zoster virus (VZV), for example. Conversely, autophagy stimulation will improve viral multiplication. The autophagic machinery can be used in whole or in part, and can optimize viral propagation or persistence. Some viruses block maturation of autophagosomes to avoid the degradation step, then autophagosomal membranes are used to contribute to the envelopment and/or the egress of viral particles. On the other hand, VZV stimulates the whole process of autophagy to subvert it in order to use vesicles containing ATG (autophagy-related) proteins and resembling amphisomes for their transport in the cytoplasm. During latency, autophagy can also be activated by latent proteins encoded by different oncogenic Herpesviruses to promote cell survival and achieve long term viral persistence in vivo. Finally, reactivation of gammaherpesvirus Murid Herpesvirus 68 (MHV68) in mice appears to be positively modulated by autophagy, in order to control the level of inflammation. Therefore, Herpesviruses appear to behave more like thieves than fugitives

La Résistance naturelle au VIH-1 (mécanismes de résistance et perspectives thérapeutiques)

Dans la très grande majorité des cas, l'infection par le VIH-1 se traduit par une période asymptomatique très longue pendant laquelle le virus se multiplie activement pendant que le taux de lymphocytes TCD4+ baisse progressivement. Quand ce taux atteint un certain seuil (200 à 400/ml), apparaissent de nombreuses infections opportunistes et cancers, la mort survenant généralement au bout de 8 à 10 ans. Cependant, un petit nombre de sujets présentent une résistance naturelle au VIH-1, se traduisant soit par l'absence d'infection malgré des expositions répétées (sujets exposés non infectés : ENI), soit par un contrôle spontané de l'infection caractérisé par une charge virale indétectable ou très faible en l'absence de tout traitement antirétroviral (elite controllers). L'étude des mécanismes de la résistance naturelle au VIH-1 a révélé l'existence de différents facteurs génétiques ou immunitaires. La connaissance de ces facteurs a permis d'entrevoir de nouvelles pistes thérapeutiques par modifications de certains de ces facteurs génétiques et la mise au point de vaccins permettant la production d'anticorps neutralisants polyvalents.CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocSudocFranceF

Herpes Simplex Virus 1 Induces Cytoplasmic Accumulation of TIA-1/TIAR and both Synthesis and Cytoplasmic Accumulation of Tristetraprolin, Two Cellular Proteins That Bind and Destabilize AU-Rich RNAs

Herpes simplex virus 1 causes a shutoff of cellular protein synthesis through the degradation of RNA that is mediated by the virion host shutoff (Vhs) protein encoded by the U(L)41 gene. We reported elsewhere that the Vhs-dependent degradation of RNA is selective, and we identified RNAs containing AU-rich elements (AREs) that were upregulated after infection but degraded by deadenylation and progressive 3′-to-5′ degradation. We also identified upregulated RNAs that were not subject to Vhs-dependent degradation (A. Esclatine, B. Taddeo, L. Evans, and B. Roizman, Proc. Natl. Acad. Sci. USA 101:3603-3608, 2004). Among the latter was the RNA encoding tristetraprolin, a protein that binds AREs and is known to be associated with the degradation of RNAs containing AREs. Prompted by this observation, we examined the status of the ARE binding proteins tristetraprolin and TIA-1/TIAR in infected cells. We report that tristetraprolin was made and accumulated in the cytoplasm of wild-type virus-infected human foreskin fibroblasts as early as 2 h and in HEp-2 cells as early as 6 h after infection. The amounts of tristetraprolin that accumulated in the cytoplasm of cells infected with a mutant virus lacking U(L)41 were significantly lower than those in wild-type virus-infected cells. The localization of tristetraprolin was not modified in cells infected with a mutant lacking the gene encoding infected cell protein 4 (ICP4). TIA-1 and TIAR are two other proteins that are associated with the regulation of ARE-containing RNAs and that normally reside in nuclei. In infected cells, they started to accumulate in the cytoplasm after 6 h of infection. In cells infected with the mutant virus lacking U(L)41, TIA-1/TIAR accumulated in the cytoplasm in granular structures reminiscent of stress granules in a significant percentage of the cells. In addition, an antibody to tristetraprolin coprecipitated the Vhs protein from lysates of cells late in infection. The results indicate that the Vhs-dependent degradation of ARE-containing RNAs correlates with the transactivation, cytoplasmic accumulation, and persistence of tristetraprolin in infected cells