Endosomophagy clears disrupted early endosomes but not virus particles during virus entry into cells

Enveloped viruses fuse with host membranes without affecting cell integrity. Nonenveloped viruses and bacteria penetrate by rupturing endosomal membranes and thus expose complex-type carbohydrates from the endosome lumen to cytosolic proteins. Here we report on the dynamics and initial marker analyses of Galectin-3 (Gal3)-positive membranes triggered by incoming adenovirus species B/C in HeLa cells. Using mCherry-Gal3 reporter constructs, immunolabeling, confocal and electron microscopy, we detected robust signals from Gal3-containing, early endosomal antigen 1-positive membranes 1 h post-infection (pi). Adenoviruses penetrate from non-acidic endosomes with high efficiency, 15 min pi, and largely outnumbered the Gal3-positive membranes, suggesting that Gal3 recruitment to broken membranes is transient, or Gal3-positive membranes are rapidly turned-over. In support of rapid turn-over, Gal3 was foundwithin single-membrane vesicles and degradative autophagosomes. The Gal3 membranes contained ubiquitin and the poly-ubiquitin binding protein p62/sequestosome-1, but only low amounts of virus, or membrane-lytic protein VI exposed from virions. Remarkably, the Gal3-positive membranes were cleared 3 h pi, slower than protein VI, whichwas cleared 30min pi. The data show that broken early endosomes, but not virus particles, are rapidly removed by a process involving autophagy, which we term ‘endosomophagy’. We speculate that endosomophagy is pro-viral and attenuates innate immunity. Introduction All viruses and many bacteria that enter eukaryotic cells activate a genetic program for replication and immune evasion [2] [3] [4]. Invariably, host defense against the pathogens or host cell damage antagonizes the intruders [5] [6] [7]. Damage to host cells occurs during invasion of bacteria and non-enveloped viruses, including Salmonella, Shigella, Listeria or Adenovirus, which disrupt phagocytic vacuoles and endosomes [8] [9]. Disrupted vacuoles recruit beta-galactoside binding lectins, such as galectin-3 (Gal3) and Gal8, both widely expressed in epithelial cells [10] [11] [12] [13]. Cytosolic galectins can also be subject to export by leader peptide-independent mechanisms [14] [15] [16]. They can bind to enveloped viruses and bacteria, and act as pattern recognition receptors [17]. Intracellular Gal8 coordinates the destruction of disrupted late endosomes and associated bacteria by autophagy, whereas Gal3 is involved in membrane sorting by clustering glycoproteins and glycolipids, and modulation of cell-cell contacts during organogenesis [18] [19] [20]. Recently, Gal3 was reported to be recruited to endosomes disrupted by incoming human adenovirus type 5 (HAdV-C5) [13] [21]. Adenoviruses are non-enveloped human pathogens and widely used vectors in clinical gene therapy and vaccination [22] [23] [24] [25]. HAdV-C2 and C5 enter epithelial cells by receptor-mediated endocytosis and a stepwise uncoating program initiated at the plasma membrane by the differential movements of two receptors, integrin and coxsackievirus adenovirus receptor (CAR) [26] [27] [28]. Virus penetration into the cytosol occurs from early endosomes in a pHindependent manner [29] [30]. It requires the membrane-lytic viral protein VI [31] [32] [33], lysosomal secretion, the sphingolipid ceramide [34] [35], and gates the pathway for viral DNA genome separation from the capsid and nuclear delivery of the viral genome [36] [37]. Here, we describe a novel observation, the recruitment of Gal3, ubiquitin and the poly-ubiquitin binding protein p62/SQSTM1 to ruptured early endosomes, followed by clearance of disrupted membranes without clearance of virus particles from the inEndosomophagy clears disrupted early endosomes but not virus particles during virus entry into cells DOI: 10.19185/matters.201606000013 Matters (ISSN: 2297-8240) | 2 fected cell. p62/SQSTM1 is an adaptor linking poly-ubiquitination to macroautophagy [38] [39]. We speculate that this mode of clearance is pro-viral, in contrast to the recruitment of Gal8 to ruptured phagosomal vacuoles in Salmonella infected cells, which presents a mode of pathogen restriction [12]. Objective Ruptured host membranes are danger signals, and enhance innate immunity against infections. The objective here was to analyze the composition and dynamics of ruptured endosomes induced upon virus entry into cells. Endosomophagy clears disrupted early endosomes but not virus particles during virus entry into cells DOI: 10.19185/matters.201606000013 Matters (ISSN: 2297-8240) | 3 a