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.     

Enterovirus 71‐induced autophagy detected in vitro and in vivo promotes viral replication

Enterovirus 71 (EV71) is an important pathogen causing death in children under 5 years old worldwide. However, the underlying pathogenesis remains unclear. This study reveals that EV71 infection in rhabdomyosarcoma (RD) and neuroblastoma (SK‐N‐SH) cells stimulated the autophagic process, which was demonstrated by an increase of punctate GFP‐microtubule‐associated protein 1 light chain 3 (GFP‐LC3), the level of autophagosome‐bound LC3‐II protein and double‐membrane autophagosome formation. EV71‐induced autophagy benefited EV71 replication, which was confirmed by the autophagic inducer rapamycin and the inhibitor 3‐methyladenine. Signaling pathway investigation revealed that the decreased expression of phosphorylated mTOR and phosphorylated p70S6K is involved in EV71‐induced autophagy in a cell‐specific manner. The expression of phosphorylated extracellular signal‐regulated kinase (Erk) was suppressed consistently in EV71‐infected cells. However it did not participate in the autophagic response of the cell. Other signaling pathway molecules, such as Erk, PI3K/Akt, Bcl‐2, BNIP3, and Beclin‐1 were not affected by infection with EV71. Electron microscopy showed co‐localization of autophagosome‐like vesicles with either EV71‐VP1 or LC3 protein in neurons of the cervical spinal cord in ICR mice infected with EV71. In conclusion, EV71 infection triggered autophagic flux and induced autophagosome formation both in vitro and in vivo. Autophagy induced by EV71 is beneficial for viral replication. Understanding the role of autophagy induced by EV71 in vitro and the formation of autophagosome‐like vesicle in vivo provide new insights into the pathogenesis of EV71 infection. J. Med. Virol. 81:1241–1252, 2009. © 2009 Wiley‐Liss, Inc.     

急性离心运动后恢复期大鼠骨骼肌微管形态及自噬体降解特征研究

目的:观察急性离心运动后恢复期大鼠骨骼肌微管形态变化及自噬体降解特征.方法:SD大鼠随机分为安静组、急性离心运动组,于运动后0h、12h、24h、48h、72h取材.分离单根肌纤维检测微管形态,Western Blot检测LC3、p62蛋白表达,腹腔注射秋水仙碱检测恢复前期(12h内)骨骼肌自噬流.结果:①急性离心运动...     

自噬在视网膜光损伤中的作用研究进展

细胞自噬是一种由基因调控的细胞内溶酶体降解途径。自噬几乎存在于所有哺乳动物细胞中,对细胞的生存具有双刃剑作用。一方面,自噬有助于维持细胞内环境稳定;另一方面,过度自噬也可能导致细胞自噬性死亡。本文就视网膜光损伤中细胞自噬作用的相关进展作一综述。     

Autophagic flux without a block differentiates varicella-zoster virus infection from herpes simplex virus infection

Autophagy is a process by which misfolded and damaged proteins are sequestered into autophagosomes, before degradation in and recycling from lysosomes. We have extensively studied the role of autophagy in varicella-zoster virus (VZV) infection, and have observed that vesicular cells are filled with >100 autophagosomes that are easily detectable after immunolabeling for the LC3 protein. To confirm our hypothesis that increased autophagosome formation was not secondary to a block, we examined all conditions of VZV infection as well as carrying out two assessments of autophagic flux. We first investigated autophagy in human skin xenografts in the severe combined immunodeficiency (SCID) mouse model of VZV pathogenesis, and observed that autophagosomes were abundant in infected human skin tissues. We next investigated autophagy following infection with sonically prepared cell-free virus in cultured cells. Under these conditions, autophagy was detected in a majority of infected cells, but was much less than that seen after an infected-cell inoculum. In other words, inoculation with lower-titered cell-free virus did not reflect the level of stress to the VZV-infected cell that was seen after inoculation of human skin in the SCID mouse model or monolayers with higher-titered infected cells. Finally, we investigated VZV-induced autophagic flux by two different methods (radiolabeling proteins and a dual-colored LC3 plasmid); both showed no evidence of a block in autophagy. Overall, therefore, autophagy within a VZV-infected cell was remarkably different from autophagy within an HSV-infected cell, whose genome contains two modifiers of autophagy, ICP34.5 and US11, not present in VZV.VZV induces macroautophagy (hereafter referred to as autophagy) in skin cells within the typical exanthem associated with either primary VZV infection (varicella or chickenpox) or VZV reactivation (herpes zoster or shingles). During prior studies, the extent of autophagy was gauged by enumeration of autophagosomes by both 2D and 3D microscopy (1, 2). The usual number of autophagosomes seen by 3D animation was 100 per infected cell, but sometimes approached 200 per cell. In contrast, a typical nonstressed cell usually has fewer than 4 autophagosomes (3, 4). When monolayers were inoculated with VZV-infected cells, the traditional method for VZV infection, autophagy was again easily seen after enumeration of autophagosomes and immunoblotting for the LC3-phosphatidylethanolamine conjugate (LC3-II). Again these results suggested that autophagic flux was present during VZV infection in cultured cells.As part of a more extensive assessment of autophagy after VZV-induced cellular stress, we have now investigated autophagy in infected human skin xenografts from the SCID mouse model of VZV infection. This model is the most accurate representation of the skin manifestation of varicella in the human host (5, 6). Finally, we addressed an important point about the nature of VZV-induced autophagy. Because the number of autophagosomes seen in the human vesicle cells from varicella and herpes zoster patients is so high, the question has arisen whether there is a late block in the maturation of autophagosomes to autolysosomes. In this report, we demonstrate that (i) autophagy induced by VZV infection is related to the overall stress to the cell, namely, a higher inoculum leads to greater autophagy; and that (ii) autophagosomes induced during VZV infection mature into autolysosomes without an obvious block before final maturation. The autophagic flux assay results confirm that VZV infection induces autophagy that proceeds to completion, possibly allowing the cell to alleviate the cellular stress caused by the viral infection (7). In previous work, we showed that inhibition of autophagy led to a significant decrease in VZV titer. Overall, therefore, autophagy within a VZV-infected cell is remarkably different from autophagy within an HSV-infected cell, an alphaherpesvirus whose genome contains two modifiers of autophagy, ICP34.5 and US11 (8–13). In contrast, autophagy appears to be proviral in the life cycle of VZV.     

GABARAPs regulate PI4P-dependent autophagosome:lysosome fusion

The Atg8 autophagy proteins are essential for autophagosome biogenesis and maturation. The γ-aminobutyric acid receptor-associated protein (GABARAP) Atg8 family is much less understood than the LC3 Atg8 family, and the relationship between the GABARAPs’ previously identified roles as modulators of transmembrane protein trafficking and autophagy is not known. Here we report that GABARAPs recruit palmitoylated PI4KIIα, a lipid kinase that generates phosphatidylinositol 4-phosphate (PI4P) and binds GABARAPs, from the perinuclear Golgi region to autophagosomes to generate PI4P in situ. Depletion of either GABARAP or PI4KIIα, or overexpression of a dominant-negative kinase-dead PI4KIIα mutant, decreases autophagy flux by blocking autophagsome:lysosome fusion, resulting in the accumulation of abnormally large autophagosomes. The autophagosome defects are rescued by overexpressing PI4KIIα or by restoring intracellular PI4P through “PI4P shuttling.” Importantly, PI4KIIα’s role in autophagy is distinct from that of PI4KIIIβ and is independent of subsequent phosphatidylinositol 4,5 biphosphate (PIP₂) generation. Thus, GABARAPs recruit PI4KIIα to autophagosomes, and PI4P generation on autophagosomes is critically important for fusion with lysosomes. Our results establish that PI4KIIα and PI4P are essential effectors of the GABARAP interactome’s fusion machinery.Macroautophagy (autophagy) is orchestrated by multiple autophagy-related (Atg) proteins (1). Among these, the Atg8 proteins are essential for autophagosome biogenesis and maturation. Mammals have at least six Atg8 orthologs that can be broadly classified into two large subfamilies: LC3s (light-chain 3) and GABARAPs (γ-aminobutyric acid receptor-associated proteins)/GATE-16s (Golgi-associated ATPase enhancer of 16 kDa), hereafter referred to collectively as GABARAPs. GABARAPs were initially identified as trafficking modulators for transmembrane receptors from the Golgi to the plasma membrane (2), and subsequently as Atg8s (1). Functional studies in mammalian cells have placed GABARAPs downstream of LC3 during autophagy (3).The complexity of the mammalian Atg8 protein network was highlighted by a recent screen that revealed a cohort of at least 67 binding partners, a third of which are unique to either the LC3 or GABARAP families (4). Phosphatidylinositol 4-kinase IIα (PI4KIIα), which generates phosphatidylinositol 4-phosphate (PI4P) from phosphatidylinositol, was identified as a binding partner for GABARAPs, but not for LC3 (4). PI4KIIα is one of four vertebrate PI4Ks (5), and it has not been previously implicated in autophagy. Here we establish for the first time, to our knowledge, that GABARAPs govern the fusion of autophagosomes with lysosomes (A:L fusion) through PI4KIIα-mediated in situ PI4P generation on autophagosomes. We propose a working model that integrates GABARAPs’ critical roles as trafficking modulators and autophagy effectors through PI4KIIα.