Multi-layered control of Galectin-8 mediated autophagy during adenovirus cell entry through a conserved PPxY motif in the viral capsid.

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.

Influenza Virus Overcomes Cellular Blocks To Productively Replicate, Impacting Macrophage Function.

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.

Avian H11 influenza virus isolated from domestic poultry in a Colombian live animal market.

Live animal markets (LAMs) are an essential source of food and trade in Latin American countries; however, they can also serve as 'hotbeds' for the emergence and potential spillover of avian influenza viruses (AIV). Despite extensive knowledge of AIV in Asian LAMs, little is known about the prevalence South American LAMs. To fill this gap in knowledge, active surveillance was carried out at the major LAM in Medellin, Colombia between February and September 2015. During this period, overall prevalence in the market was 2.67% and a North American origin H11N2 AIV most similar to a virus isolated from Chilean shorebirds asymptomatically spread through multiple bird species in the market resulting in 17.0% positivity at peak of infection. Phenotypically, the H11 viruses displayed no known molecular markers associated with increased virulence in birds or mammals, had α2,3-sialic acid binding preference, and caused minimal replication in vitro and little morbidity in vivo. However, the Colombian H11N2 virus replicated and transmitted effectively in chickens explaining the spread throughout the market. Genetic similarity to H11 viruses isolated from North and South American shorebirds suggest that the LAM occurrence may have resulted from a wild bird to domestic poultry spillover event. The ability to spread in domestic poultry as well as potential for human infection by H11 viruses highlight the need for enhanced AIV surveillance in South America in both avian species and humans.

Oral Administration of Astrovirus Capsid Protein Is Sufficient To Induce Acute Diarrhea In Vivo.

The disease mechanisms associated with the onset of astrovirus diarrhea are unknown. Unlike other enteric virus infections, astrovirus infection is not associated with an inflammatory response or cellular damage. In vitro studies in differentiated Caco-2 cells demonstrated that human astrovirus serotype 1 (HAstV-1) capsid protein alone disrupts the actin cytoskeleton and tight junction complex, leading to increased epithelial barrier permeability. In this study, we show that oral administration of purified recombinant turkey astrovirus 2 (TAstV-2) capsid protein results in acute diarrhea in a dose- and time-dependent manner in turkey poults. Similarly to that induced by infectious virus, TAstV-2 capsid-induced diarrhea was independent of inflammation or histological changes but was associated with increased intestinal barrier permeability, as well as redistribution of sodium hydrogen exchanger 3 (NHE3) from the membrane to the cytoplasm of the intestinal epithelium. Unlike other viral enterotoxins that have been identified, astrovirus capsid induces diarrhea after oral administration, reproducing the natural route of infection and demonstrating that ingestion of intact noninfectious capsid protein may be sufficient to provoke acute diarrhea. Based on these data, we hypothesize that the astrovirus capsid acts like an enterotoxin and induces intestinal epithelial barrier dysfunction. IMPORTANCE: Acute gastroenteritis, with its sequela diarrhea, is one of the most important causes of childhood morbidity and mortality worldwide. A variety of infectious agents cause gastroenteritis, and in many cases, an enterotoxin produced by the agent is involved in disease manifestations. Although we commonly think of bacteria as a source of toxins, at least one enteric virus, rotavirus, produces a protein with enterotoxigenic activity during viral replication. In these studies, we demonstrate that oral administration of the turkey astrovirus 2 (TAstV-2) structural (capsid) protein induces acute diarrhea, increases barrier permeability, and causes relocalization of NHE3 in the small intestine, suggesting that rotavirus may not be alone in possessing enterotoxigenic activity.

The Immune Response to Astrovirus Infection.

Astroviruses are one of the leading causes of pediatric gastroenteritis worldwide and are clinically importantly pathogens in the elderly and immunocompromised populations. Although the use of cell culture systems and small animal models have enhanced our understanding of astrovirus infection and pathogenesis, little is known about the immune response to astrovirus infection. Studies from humans and animals suggest that adaptive immunity is important in restricting classic and novel astrovirus infections, while studies from animal models and cell culture systems suggest that an innate immune system plays a role in limiting astrovirus replication. The relative contribution of each arm of the immune system in restricting astrovirus infection remains unknown. This review summarizes our current understanding of the immune response to astrovirus infection and highlights some of the key questions that stem from these studies. A full understanding of the immune response to astrovirus infection is required to be able to treat and control astrovirus-induced gastroenteritis.

Type I Interferon Response Limits Astrovirus Replication and Protects against Increased Barrier Permeability In Vitro and In Vivo.

UNLABELLED: 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-ß]) 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. IMPORTANCE: Human 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.

Spatiotemporal dynamics of adenovirus membrane rupture and endosomal escape.

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.

Emerging roles for ubiquitin in adenovirus cell entry.

Adenovirus relies on numerous interactions between viral and host cell proteins to efficiently enter cells. Undoubtedly, post-translational modifications of host and cellular proteins can impact the efficiency of this cell entry process. Ubiquitylation, once simply thought of as a modification targeting proteins for proteasomal degradation, is now known to regulate protein trafficking within cells, protein-protein interactions and cell signalling pathways. Accumulating evidence suggests that protein ubiquitylation can influence all stages of the life cycle of other viruses such as cell entry, replication and egress. Until recently, the influence of ubiquitylation has only been documented during adenovirus replication. This review highlights the most recent evidence demonstrating direct engagement of host ubiquitylation and SUMOylation machinery by adenovirus during cell entry. Additionally, potential roles for host protein ubiquitylation and the potential for adenovirus regulation of host ubiquitylation machinery during cell entry are discussed.