α-synuclein dysregulation is a critical aspect of Parkinson's disease pathology. Recent studies have observed that α-synuclein aggregates are cytotoxic to cells in culture and that this toxicity can be spread between cells. However, the molecular mechanisms governing this cytotoxicity and spread are poorly characterized. Recent studies of viruses and bacteria, which achieve their cytoplasmic entry by rupturing intracellular vesicles, have utilized the redistribution of galectin proteins as a tool to measure vesicle rupture by these organisms. Using this approach, we demonstrate that α-synuclein aggregates can induce the rupture of lysosomes following their endocytosis in neuronal cell lines. This rupture can be induced by the addition of α-synuclein aggregates directly into cells as well as by cell-to-cell transfer of α-synuclein. We also observe that lysosomal rupture by α-synuclein induces a cathepsin B dependent increase in reactive oxygen species (ROS) in target cells. Finally, we observe that α-synuclein aggregates can induce inflammasome activation in THP-1 cells. Lysosomal rupture is known to induce mitochondrial dysfunction and inflammation, both of which are well established aspects of Parkinson's disease, thus connecting these aspects of Parkinson's disease to the propagation of α-synuclein pathology in cells.

Cells employ active measures to restrict infection by pathogens, even prior to responses from the innate and humoral immune defenses. In this context selective autophagy is activated upon pathogen induced membrane rupture to sequester and deliver membrane fragments and their pathogen contents for lysosomal degradation. Adenoviruses, which breach the endosome upon entry, escape this fate by penetrating into the cytosol prior to autophagosome sequestration of the ruptured endosome. We show that virus induced membrane damage is recognized through Galectin-8 and sequesters the autophagy receptors NDP52 and p62. We further show that a conserved PPxY motif in the viral membrane lytic protein VI is critical for efficient viral evasion of autophagic sequestration after endosomal lysis. Comparing the wildtype with a PPxY-mutant virus we show that depletion of Galectin-8 or suppression of autophagy in ATG5-/- MEFs rescues infectivity of the PPxY-mutant virus while depletion of the autophagy receptors NDP52, p62 has only minor effects. Furthermore we show that wildtype viruses exploit the autophagic machinery for efficient nuclear genome delivery and control autophagosome formation via the cellular ubiquitin ligase Nedd4.2 resulting in reduced antigenic presentation. Our data thus demonstrate that a short PPxY-peptide motif in the adenoviral capsid permits multi-layered viral control of autophagic processes during entry.

Whether influenza virus replication in macrophages is productive or abortive has been a topic of debate. Utilizing a panel of 28 distinct human, avian, and swine influenza viruses, we found that only a small subset can overcome cellular blocks to productively replicate in murine and primary human macrophages. Murine macrophages have two cellular blocks. The first block is during viral entry, where virions with relatively acid-stable hemagglutinin (HA) proteins are rendered incapable of pH-induced triggering for membrane fusion, resulting in lysosomal degradation. The second block is downstream of viral replication but upstream of late protein synthesis. In contrast, primary human macrophages only have one cellular block that occurs after late protein synthesis. To determine the impact of abortive replication at different stages of the viral life cycle or productive replication on macrophage function, we assessed cytotoxicity, nitric oxide or reactive oxygen species production, and phagocytosis. Intriguingly, productive viral replication decreased phagocytosis of IgG-opsonized bioparticles and Fc receptor CD16 and CD32 surface levels, a function, to our knowledge, never before reported for an RNA virus. These data suggest that replication in macrophages affects cellular function and plays an important role in pathogenesis during infection in vivo.IMPORTANCE Macrophages are a critical first line of defense against respiratory pathogens. Thus, understanding how viruses evade or exploit macrophage function will provide greater insight into viral pathogenicity and antiviral responses. We previously showed that only a subset of highly pathogenic avian (HPAI) H5N1 influenza virus strains could productively replicate in murine macrophages through a hemagglutinin (HA)-mediated mechanism. These studies expand upon this work and demonstrate that productive replication is not specific to unique HPAI H5N1 viruses; an H1N1 strain (A/WSN/33) can also replicate in macrophages. Importantly, we identify two cellular blocks limiting replication that can be overcome by an avian-like pH of activation for nuclear entry and a yet-to-be-identified mechanism(s) to overcome a postnuclear entry block. Overcoming these blocks reduces the cell's ability to phagocytose IgG-opsonized bioparticles by decreasing Fc receptor surface levels, a mechanism previously thought to occur during bacterial and DNA viral infections.KEYWORDS influenza, macrophage, replication M acrophages are one of the first lines of defense against infection. They are poised to secrete large amounts of cytokines, orchestrate the adaptive immune system, and clear infected and dying cells to aid in recovery (1). Further, alveolar macrophages are essential in preventing respiratory failure after infection (2) and for preventing bacterial superinfections during influenza infection (3). However, such responses must be tightly regulated, as excessive cytokine levels contribute to immunopathology and disease severity during infection (4-6).

A key step in adenovirus cell entry is viral penetration of cellular membranes to gain access to the cytoplasm and deliver the genome to the nucleus. Yet little is known about this important event in the adenoviral life cycle. Using the cytosolic protein galectin-3 (gal3) as a marker of membrane rupture with both live-and fixed-cell imaging, we demonstrate that in the majority of instances, exposure of pVI and recruitment of gal3 to ruptured membranes occur early at or near the cell surface and occur minimally in EEA-1-positive (EEA-1 ؉ ) early endosomes or LAMP-1 ؉ late endosomes/lysosomes. Live-cell imaging of Ad5 egress from gal3؉ endosomes occurs most frequently from perinuclear locations. While the Ad5 capsid is observed escaping from gal3 ؉ endosomes, pVI appears to remain associated with the gal3 ؉ ruptured endosomes. Thus, Ad5 membrane rupture and endosomal escape appear to be both spatially and temporally distinct events.

Little is known about intrinsic epithelial cell responses against astrovirus infection. Here we show that human astrovirus type 1 (HAstV-1) infection induces type I interferon (beta interferon [IFN-␤]) production in differentiated Caco2 cells, which not only inhibits viral replication by blocking positive-strand viral RNA and capsid protein synthesis but also protects against HAstV-1-increased barrier permeability. Excitingly, we found similar results in vivo using a murine astrovirus (MuAstV) model, providing new evidence that virus-induced type I IFNs may protect against astrovirus replication and pathogenesis in vivo. IMPORTANCEHuman astroviruses are a major cause of pediatric diarrhea, yet little is known about the immune response. Here we show that type I interferon limits astrovirus infection and preserves barrier permeability both in vitro and in vivo. Importantly, we characterized a new mouse model for studying astrovirus replication and pathogenesis. The successful replication and spread of many enteric viruses depend upon modulating immune factors produced by intestinal epithelial cells (IECs) including interferons (IFNs) (1, 2). For instance, enteric adenoviruses are sensitive to IEC-produced type I IFNs, unlike respiratory adenoviruses (3), while rotavirus exploits type I IFN signaling in IECs to promote early viral replication (4). However, nothing is known about the impact of IFN on astrovirus infection.Astroviruses are small, nonenveloped, RNA viruses that are one of the most important causes of pediatric acute gastroenteritis worldwide (5-8). Infection begins by binding to an unidentified receptor(s) on epithelial cells after fecal-oral transmission followed by entry via endosomes (9). After viral uncoating, the positive-sense, single-stranded RNA genome is translated into a polyprotein precursor that is subsequently cleaved into proteins required for replication and the assembly of progeny virions. The genome contains three open reading frames: ORF1a, ORF1b, and ORF2. ORF1a and ORF1b encode nonstructural proteins involved in transcription and replication of the virus, while ORF2 encodes the capsid protein (10, 11). Negative-strand RNA is produced from the positive genomic strand, which can be detected 6 to 12 h postinfection (hpi) (12). Transcription of the negativestrand genome yields the genomic and subgenomic RNA. Human astrovirus (HAstV) proteins have been associated with membranes in infected cells likely serving as the site for replication and assembly (13-15). After assembly, the progeny virions egress from the cell, a process promoted by caspase activation (16).Recently, Guix et al. found that HAstV type 4 (HAstV-4) replication induces type I IFN production and that pretreatment of Caco2 cells with beta interferon (IFN-␤) reduced HAstV-4 capsid protein synthesis and progeny virion production (17). However, whether the IFN-␤ produced during astrovirus infection is sufficient to limit astrovirus replication, and at what step in the viral life cycle IFN-␤ affects astrovirus, remains...