Audrey Esclatine

Overview of macroautophagy regulation in mammalian cells

Macroautophagy is a multistep, vacuolar, degradation pathway terminating in the lysosomal compartment, and it is of fundamental importance in tissue homeostasis. In this review, we consider macroautophagy in the light of recent advances in our understanding of the formation of autophagosomes, which are double-membrane-bound vacuoles that sequester cytoplasmic cargos and deliver them to lysosomes. In most cases, this final step is preceded by a maturation step during which autophagosomes interact with the endocytic pathway. The discovery of AuTophaGy-related genes has greatly increased our knowledge about the mechanism responsible for autophagosome formation, and there has also been progress in the understanding of molecular aspects of autophagosome maturation. Finally, the regulation of autophagy is now better understood because of the discovery that the activity of Atg complexes is targeted by protein kinases, and owing to the importance of nuclear regulation via transcription factors in regulating the expression of autophagy genes.

Autophagy in health and disease. 1. Regulation and significance of autophagy: an overview

Macroautophagy is a vacuolar degradation pathway that terminates in the lysosomal compartment after formation of a cytoplasmic vacuole or autophagosome that engulfs macromolecules and organelles. The identification of ATG (autophagy-related) genes that are involved in the formation of autophagosomes has greatly increased our knowledge of the molecular basis of macroautophagy, and its roles in cell function, which extend far beyond degradation and quality control of the cytoplasm. Macroautophagy, which plays a major role in tissue homeostasis, is now recognized as contributing to innate and adaptive immune responses. Recently, several mediators of apoptosis have been shown to control macroautophagy. Deciphering the cross talk between macroautophagy and apoptosis probably should help increase understanding of the role of macroautophagy in human disease and is likely to be of therapeutic importance.

The Human Cytomegalovirus Protein TRS1 Inhibits Autophagy via Its Interaction with Beclin 1

ABSTRACT Human cytomegalovirus modulates macroautophagy in two opposite directions. First, HCMV stimulates autophagy during the early stages of infection, as evident by an increase in the number of autophagosomes and a rise in the autophagic flux. This stimulation occurs independently of de novo viral protein synthesis since UV-inactivated HCMV recapitulates the stimulatory effect on macroautophagy. At later time points of infection, HCMV blocks autophagy (M. Chaumorcel, S. Souquere, G. Pierron, P. Codogno, and A. Esclatine, Autophagy 4:1–8, 2008) by a mechanism that requires de novo viral protein expression. Exploration of the mechanisms used by HCMV to block autophagy unveiled a robust increase of the cellular form of Bcl-2 expression. Although this protein has an anti-autophagy effect via its interaction with Beclin 1, it is not responsible for the inhibition induced by HCMV, probably because of its phosphorylation by c-Jun N-terminal kinase. Here we showed that the HCMV TRS1 protein blocks autophagosome biogenesis and that a TRS1 deletion mutant is defective in autophagy inhibition. TRS1 has previously been shown to neutralize the PKR antiviral effector molecule. Although phosphorylation of eIF2α by PKR has been described as a stimulatory signal to induce autophagy, the PKR-binding domain of TRS1 is dispensable to its inhibitory effect. Our results show that TRS1 interacts with Beclin 1 to inhibit autophagy. We mapped the interaction with Beclin 1 to the N-terminal region of TRS1, and we demonstrated that the Beclin 1-binding domain of TRS1 is essential to inhibit autophagy.

Human cytomegalovirus controls a new autophagy-dependent cellular antiviral defense mechanism

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that remains the major infectious cause of birth defects, as well as being an important opportunistic pathogen. Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved process responsible for the degradation of cytoplasmic macromolecules, and the elimination of damaged organelles via a lysosomal pathway. This process is also triggered in organisms by stressful conditions and by certain diseases. Previous observations have suggested that autophagy (also known as xenophagy in this case) may contribute to innate immunity against viral infections. Recent studies on HSV-1, another herpesvirus, have shown that HSV-1 is able to avoid this cellular defense by means of a viral protein, ICP34.5, which antagonizes the host autophagy response. However, it was not known whether HCMV was also able to counteract autophagy. Here, we show that HCMV infection drastically inhibits autophagosome formation in primary human fibroblasts. Autophagy was assessed by GFP–LC3 redistribution, LC3-II accumulation and electron microscopy. Inhibition of autophagy occurred early in the infection by a mechanism involving viral protein(s). Indeed, only infected cells expressing viral proteins displayed a striking decrease of autophagy; whereas bystander, non-infected cells displayed a level of autophagy similar to that of control cells. HCMV activated the mTOR signaling pathway, and rendered infected cells resistant to rapamycin-induced autophagy. Moreover, infected cells also became resistant to the stimulation of autophagy by lithium chloride, an mTOR-independent inducer of autophagy. These findings suggest that HCMV has developed efficient strategies for blocking the induction of autophagy during infection.

The patterns of accumulation of cellular RNAs in cells infected with a wild-type and a mutant herpes simplex virus 1 lacking the virion host shutoff gene

Cellular RNA extracted from quiescent human foreskin fibroblasts harvested at 1, 3, 7, or 12 h after infection was profiled on Affymetrix HG-U95Av2 arrays designed to detect 12,626 unique human transcripts. We also profiled RNA extracted from cells harvested at 1 and 7 h after infection with a mutant lacking the gene (ΔUL41) encoding a protein (vhs) brought into cells by the virus and responsible for nonselective degradation of RNA early in infection. We report the following: (i) of the 12 tested genes, up-regulated at least 3-fold relative to the values of mock infected cells, 9 were confirmed by real-time PCR. The microchip assays analyses indicate that there were 475 genes up-regulated ≥3-fold. The up-regulated genes were clustered into 15 groups with respect to temporal pattern of transcript accumulation, and classified into 20 groups on the basis of their function. The preponderance of cellular genes up-regulated early in infection play a predominant role in transcription, whereas those up-regulated at later times respond to intracellular stress or concern themselves with the cell cycle and apoptosis. (ii) The number of genes up-regulated early in infection was higher in cells infected with the ΔUL41 mutant. Conversely, more genes were down-regulated late in infection with wild-type virus than with mutant viruses. Both observations are compatible with the known function of the UL41 gene product early in infection and with degradation of cellular RNAs in the absence of replenishment by de novo transcription of cellular genes.

The Herpes Simplex Virus 1 Us11 Protein Inhibits Autophagy through Its Interaction with the Protein Kinase PKR

ABSTRACT Autophagy is now known to be an essential component of host innate and adaptive immunity. Several herpesviruses have developed various strategies to evade this antiviral host defense. Herpes simplex virus 1 (HSV-1) blocks autophagy in fibroblasts and in neurons, and the ICP34.5 protein is important for the resistance of HSV-1 to autophagy because of its interaction with the autophagy machinery protein Beclin 1. ICP34.5 also counteracts the shutoff of protein synthesis mediated by the double-stranded RNA (dsRNA)-dependent protein kinase PKR by inhibiting phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α) in the PKR/eIF2α signaling pathway. Us11 is a late gene product of HSV-1, which is also able to preclude the host shutoff by direct inhibition of PKR. In the present study, we unveil a previously uncharacterized function of Us11 by demonstrating its antiautophagic activity. We show that the expression of Us11 is able to block autophagy and autophagosome formation in both HeLa cells and fibroblasts. Furthermore, immediate-early expression of Us11 by an ICP34.5 deletion mutant virus is sufficient to render the cells resistant to PKR-induced and virus-induced autophagy. PKR expression and the PKR binding domain of Us11 are required for the antiautophagic activity of Us11. However, unlike ICP34.5, Us11 did not interact with Beclin 1. We suggest that the inhibition of autophagy observed in cells infected with HSV-1 results from the activity of not only ICP34.5 on Beclin 1 but also Us11 by direct interaction with PKR.

The UL41 protein of herpes simplex virus mediates selective stabilization or degradation of cellular mRNAs

The UL41 protein of herpes simplex virus 1 has been reported to mediate the degradation of both viral and cellular mRNAs. Extensive studies on β-actin and some viral mRNAs were consonant with this conclusion. In earlier studies, we reported that the UL41-dependent degradation of cellular mRNAs up-regulated after infection was selective. One class of the up-regulated mRNAs, exemplified by the stress-inducible immediate-early 1 mRNA, is deadenylated, 3′ to 5′ degraded and is not translated. Another class of up-regulated mRNAs, exemplified by GADD45β, does not undergo this pattern of degradation and is translated. A puzzling feature of the earlier results is that the amounts of up-regulated mRNAs accumulating in the cytoplasm of ΔUL41 mutant virus-infected cells was lower than in WT virus-infected cells, a contradiction, inasmuch as if the rates of accumulation were identical and degradation of the mRNAs were higher in WT virus-infected cells, the steady-state levels should have been higher in ΔUL41 mutant virus-infected cells. In this report, we show that in ΔUL41 mutant virus-infected cells, the rates of degradation of the stress-inducible immediate-early response gene 1 and other up-regulated mRNAs are approximately the same as those observed in mock-infected cells and are faster than in WT virus-infected cells. This is contrary to the observed UL41-dependent degradation of β-actin and other mRNAs. The UL41 protein thus mediates two functions, i.e., it mediates rapid degradation of some mRNAs exemplified by β-actin and stabilizes or delays the degradation of other mRNAs exemplified by GADD45β, tristetraprolin, etc. A model unifying both activities of the UL41 protein is presented.

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.

ABSTRACT 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 UL41 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 UL41 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 UL41, 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.

Macroautophagy Signaling and Regulation

Macroautophagy is a vacuolar degradation pathway that terminates in the lysosomal compartment. Macroautophagy is a multistep process involving: (1) signaling events that occur upstream of the molecular machinery of autophagy; (2) molecular machinery involved in the formation of the autophagosome, the initial multimembrane-bound compartment formed in the autophagic pathway; and (3) maturation of autophagosomes, which acquire acidic and degradative capacities. In this chapter we summarize what is known about the regulation of the different steps involved in autophagy, and we also discuss how macroautophagy can be manipulated using drugs or genetic approaches that affect macroautophagy signaling, and the subsequent formation and maturation of the autophagosomes. Modulating autophagy offers a promising new therapeutic approach to human diseases that involve macroautophagy.

The Stress-Inducible Immediate-Early Responsive Gene IEX-1 Is Activated in Cells Infected with Herpes Simplex Virus 1, but Several Viral Mechanisms, Including 3′ Degradation of Its RNA, Preclude Expression of the Gene

ABSTRACT The accumulation of cellular transcripts from cells infected with herpes simplex virus 1 (HSV-1) as measured with the aid of Affymetrix microchips has been reported elsewhere. Among these transcripts were genes that respond to stress and that could have a noxious effect on viral replication. We have selected the stress-inducible cellular gene encoding the immediate-early response protein IEX-1 to verify and determine the significance of the accumulation of these transcripts in infected cells. We report that we verified the increase in accumulation of IEX-1 transcripts after infection by Northern analyses and real-time PCR. These transcripts reach peak levels between 3 and 7 h after infection and decrease thereafter. However, IEX-1 protein was detected in cells 1 h after infection but not at later intervals. Studies designed to elucidate the failure of IEX-1 protein to be synthesized revealed the following points. (i) IEX-1 RNA transported to the cytoplasm after 1 h of infection consisted of at least two populations, a partially degraded population and a population consisting of unspliced IEX-1 RNA. Neither of these RNAs could translate the authentic IEX-1 protein. (ii) The partially degraded IEX-1 RNA was not detected in the cytoplasm of cells infected with a mutant virus lacking the UL41 gene encoding the virion host shutoff protein (vhs). Although degradation of RNA mediated by vhs was reported to be 5′ to 3′, the partially degraded IEX-1 RNA lacked the 3′ sequences rather than the 5′ sequences. (iii) The unspliced pre-RNA form containing the IEX-1 intron sequences was detected in the cytoplasm of cell infected with wild-type virus but not in those infected with a mutant lacking the α27 gene encoding the infected cell protein No. 27. (iv) Overexpression of IEX-1 protein by transduction of the gene prior to infection with 1 PFU of HSV-1 per cell had no effect on the accumulation of late genes and virus yield. We conclude that the failure of IEX-1 to express its protein reflects the numerous mechanisms by which the virus thwarts the cells from expressing its genes after infection.