AcMNPV P74 is cleaved at R325 and R334 by proteinases of both OB and BBMV to expose a potential fusion peptide for oral infection.

Baculoviruses enter insect midgut epithelial cells via a set of occlusion-derived virion (ODV) envelope proteins called per os infectivity factors (PIFs). P74 of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), which was the first identified PIF, is cleaved by an endogenous proteinase embedded within the occlusion body during per os infection, but the target site(s) and function of the cleavage have not yet been ascertained. Here, based on bioinformatics analyses, we report that cleavage was predicted at an arginine and lysine-rich region in the middle of P74. A series of recombinant viruses with site-directed mutants in this region of P74 were generated. R325 or R334 was identified as primary cleavage site. In addition, we showed that P74 is also cleaved by brush border membrane vesicles (BBMV) of the host insect at R325 or R334, instead of R195, R196, and R199, as previously reported. Simultaneous mutations in R195, R196, and R199 lead to instability of P74 during ODV release. Bioassays showed that mutations at both R325 and R334 significantly affected oral infectivity. Taken together, our data show that both R325 and R334 of AcMNPV P74 are the primary cleavage site for both occlusion body endogenous proteinase and BBMV proteinase during ODV release and are critical for oral infection. IMPORTANCE: Cleavage of viral envelope proteins is usually an important trigger for viral entry into host cells. Baculoviruses are insect-specific viruses that infect host insects via the oral route. P74, a per os infectivity factor of baculoviruses, is cleaved during viral entry. However, the function and precise cleavage sites of P74 remain unknown. In this study, we found that R325 or R334 between the N- and C-conserved domains of P74 was the primary cleavage site by proteinase either from the occlusion body or host midgut. The biological significance of cleavage seems to be the release of the potential fusion peptide at the N-terminus of the cleaved C-terminal P74. Our results shed light on the cleavage model of P74 and imply its role in membrane fusion in baculovirus per os infection.

SARS-CoV-2 replication in airway epithelia requires motile cilia and microvillar reprogramming.

How SARS-CoV-2 penetrates the airway barrier of mucus and periciliary mucins to infect nasal epithelium remains unclear. Using primary nasal epithelial organoid cultures, we found that the virus attaches to motile cilia via the ACE2 receptor. SARS-CoV-2 traverses the mucus layer, using motile cilia as tracks to access the cell body. Depleting cilia blocks infection for SARS-CoV-2 and other respiratory viruses. SARS-CoV-2 progeny attach to airway microvilli 24 h post-infection and trigger formation of apically extended and highly branched microvilli that organize viral egress from the microvilli back into the mucus layer, supporting a model of virus dispersion throughout airway tissue via mucociliary transport. Phosphoproteomics and kinase inhibition reveal that microvillar remodeling is regulated by p21-activated kinases (PAK). Importantly, Omicron variants bind with higher affinity to motile cilia and show accelerated viral entry. Our work suggests that motile cilia, microvilli, and mucociliary-dependent mucus flow are critical for efficient virus replication in nasal epithelia.

Functional analysis of the baculovirus per os infectivity factors 3 and 9 by imaging the interaction between fluorescently labelled virions and isolated midgut cells.

Baculovirus occlusion-derived viruses (ODVs) contain ten known per os infectivity factors (PIFs). These PIFs are crucial for midgut infection of insect larvae and form, with the exception of PIF5, an ODV entry complex. Previously, R18-dequenching assays have shown that PIF3 is dispensable for binding and fusion with midgut epithelial cells. Oral infection nevertheless fails in the absence of PIF3. PIF9 has not been analysed in much depth yet. Here, the biological role of these two PIFs in midgut infection was examined by monitoring the fate of fluorescently labelled ODVs when incubated with isolated midgut cells from Spodoptera exigua larvae. Confocal microscopy showed that in the absence of either PIF3 or PIF9, the ODVs bound to the brush borders, but the nucleocapsids failed to enter the cells. Finally, we discuss how the results obtained for PIF3 with dequenching assays and confocal microscopy can be explained by a two-phase fusion process.

Investigation of alimentary canal ultrastructure following knockdown of the Dicer-2 gene in planthoppers reveals the potential pathogenicity of southern rice black streaked dwarf virus to its insect vector.

An increasing number of studies are suggesting that plant viruses, including southern rice black-streaked dwarf virus (SRBSDV), can adversely affect biological characteristics of insect vectors by unknown mechanisms. To study the adverse effect of SRBSDV at cellular level on the insect vector, we promoted viral infection by the disruption of the small interfering RNA (siRNA) pathway. The transmission electron microscopy was utilized to describe the ultrastructural changes that occurred in insects when the core component of the siRNA pathway, Dicer-2, was knocked down. The increasing accumulation of SRBSDV in virus-infected vector, the white-backed planthoppers, caused severe cytopathology in the alimentary canal. Similar cytopathology changes in the midgut ultrastructure were characterized in the virus-infected incompetent vector, the small brown planthopper. These results not only add support to the existing evidence suggesting that the siRNA pathway has an antiviral effect, but also reveal the universal and potential ability of SRBSDV to cause damage to the insect tissues of both the vector and non-vector.

In Vitro Evidence Supports Membrane Alanyl Aminopeptidase N as a Receptor for a Plant Virus in the Pea Aphid Vector.

UNLABELLED: Insect-borne plant viruses cause significant agricultural losses and jeopardize sustainable global food production. Although blocking plant virus transmission would allow for crop protection, virus receptors in insect vectors are unknown. Here we identify membrane alanyl aminopeptidase N (APN) as a receptor for pea enation mosaic virus (PEMV) coat protein (CP) in the gut of the pea aphid, Acyrthosiphon pisum, using a far-Western blot method. Pulldown and immunofluorescence binding assays and surface plasmon resonance were used to confirm and characterize CP-APN interaction. PEMV virions and a peptide comprised of PEMV CP fused to a proline-rich hinge (-P-) and green fluorescent protein (CP-P-GFP) specifically bound to APN. Recombinant APN expressed in Sf9 cells resulted in internalization of CP-P-GFP, which was visualized by confocal microscopy; such internalization is an expected hallmark of a functional gut receptor. Finally, in assays with aphid gut-derived brush border membrane vesicles, binding of CP-P-GFP competed with binding of GBP3.1, a peptide previously demonstrated to bind to APN in the aphid gut and to impede PEMV uptake into the hemocoel; this finding supports the hypothesis that GBP3.1 and PEMV bind to and compete for the same APN receptor. These in vitro data combined with previously published in vivo experiments (S. Liu, S. Sivakumar, W. O. Sparks, W. A. Miller, and B. C. Bonning, Virology 401:107-116, 2010, http://dx.doi.org/10.1016/j.virol.2010.02.009) support the identification of APN as the first receptor in a plant virus vector. Knowledge of this receptor will provide for technologies based on PEMV-APN interaction designed to block plant virus transmission and to suppress aphid populations. IMPORTANCE: A significant proportion of global food production is lost to insect pests. Aphids, in addition to weakening plants by feeding on their sap, are responsible for transmitting about half of the plant viruses vectored by insects. Growers rely heavily on the application of chemical insecticides to manage both aphids and aphid-vectored plant viral disease. To increase our understanding of plant virus-aphid vector interaction, we provide in vitro evidence supporting earlier in vivo work for identification of a receptor protein in the aphid gut called aminopeptidase N, which is responsible for entry of the plant virus pea enation mosaic virus into the pea aphid vector. Enrichment of proteins found on the surface of the aphid gut epithelium resulted in identification of this first aphid gut receptor for a plant virus. This discovery is particularly important since the disruption of plant virus binding to such a receptor may enable the development of a nonchemical strategy for controlling aphid-vectored plant viruses to maximize food production.

Influenza virus budding from the tips of cellular microvilli in differentiated human airway epithelial cells.

The epithelium of conducting airways represents the main target for influenza virus in mammals. However, the peculiarities of virus interactions with differentiated airway epithelial cells remain largely unknown. Here, influenza virus budding was studied in differentiated cultures of human tracheobronchial epithelial cells using transmission electron microscopy. Budding of spherical and filamentous virions was observed on the apical surfaces of cells with no association with cilia and secretory granules. Quantitative analysis of the distribution of viral buds on the cell surface indicated that the tips of the microvilli represented a prominent site of influenza virus budding in the human airway epithelium. As the microvilli of differentiated cells are involved in many fundamental cell functions, these data will prompt further studies on the biological significance of microvilli-associated budding for virus replication, transmission and pathogenicity.

Identification of sugar residues involved in the binding of TGEV to porcine brush border membranes.

Coronaviruses most often infect the respiratory or intestinal tract. Transmissible gastroenteritis virus (TGEV), a group 1 coronavirus, infects the porcine small intestine. Piglets up to the age of 3 weeks die from diarrhea caused by the viral gastroenteritis unless they are protected by antibodies. In addition to the cellular receptor, porcine aminopeptidase N, the TGEV spike protein binds to sialic acid residues. We have shown that the sialic acid binding activity mediates the binding of TGEV to a mucin-like glycoprotein present in porcine brush border membranes. This was shown by performing a virus overlay binding assay with proteins obtained from brush border membranes by lectin precipitation. Because of the reactivity with specific lectins we assume that the recognized glycoprotein has the characteristics of a mucin.

Trypsin cleavage of the baculovirus occlusion-derived virus attachment protein P74 is prerequisite in per os infection.

Baculovirus occlusion-derived virions (ODVs) contain a number of infectivity factors essential for the initiation of infection in larval midgut cells. Deletion of any of these factors neutralizes infectivity by the per os route. We have observed that P74 of the group I alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is N-terminally cleaved when a soluble form of the protein was incubated with insect midgut tissues under alkaline conditions and that cleavage was prevented by soybean trypsin inhibitor (SBTI). Presently, biological assays were carried out that suggest SBTI inhibits and trypsin enhances baculovirus per os infectivity. We developed a method to rescue per os infectivity of a P74 null virus involving co-transfection of viral DNA with a plasmid that transiently expresses p74. We used this plasmid rescue method to functionally characterize P74. A series of site-directed mutants were generated at the N terminus to evaluate if trypsin cleavage sites were necessary for function. Mutagenesis of R195, R196 and R199 compromised per os infectivity and rendered P74 resistant to midgut trypsin.

[Physiopathology of Rotavirus diarrhea]. / Physiopathologie de la diarrhée à Rotavirus.

The rotavirus is the major cause of infantile gastroenteritis. The virus infects the mature enterocytes of the villus tip of the small intestine and induces a watery diarrhea. Diarrhea can occur in the absence of histological changes in the intestine, and, conversely, the histological changes can be asymptomatic. Rotavirus decreases the activities of digestive enzymes at the apical brush border membrane and inhibits Na+ -solute cotransport systems. Accumulation of carbohydrates in the intestinal lumen as well as malabsorption of nutrients and a concomitant inhibition of water absorption can lead to a malabsorptive component of diarrhea. Since the discovery of the NSP4 enterotoxin, several hypotheses have been proposed in favour of an additional secretion component in the pathogenesis of diarrhea. Rotavirus induces a moderate net chloride secretion at the onset of the diarrhea. The mechanisms appear to different from those used by bacterial enterotoxin that cause pure secretory diarrhea. Rotavirus stimulated C1- reabsorption in villi, and failed to stimulate C1- secretion in crypt. Intestinal villi could secrete chloride as a result of rotavirus infection. The chloride secretory response is regulated by a dependant calcium signalling pathway induced by NSP4. The overall response is weak, suggesting that NSP4 may exert both secretory and subsequent antisecretory actions, hence limiting C1- secretion.

Evidence for proteolytic cleavage of the baculovirus occlusion-derived virion envelope protein P74.

Baculovirus occlusion-derived virions (ODVs) are released from occlusion bodies by the alkaline environment of the insect midgut. The ODV envelope protein P74 is required for oral infectivity. A soluble form of the Autographa californica multiple nucleopolyhedrovirus P74 protein, P74sol, was engineered as part of a chimeric protein with jellyfish green fluorescent protein (GFP). P74sol-GFP was overproduced by the baculovirus expression system and purified away from the wild-type P74. Brush border membrane vesicles (BBMVs) were prepared from the midguts of third-instar Helicoverpa zea larvae. When P74sol-GFP was incubated under alkaline conditions with BBMVs, a P74sol-GFP product with a smaller molecular mass was produced. Immunoblots indicated that the smaller product was generated by N-terminal cleavage of P74. This cleavage was prevented by soybean trypsin inhibitor. Analysis of the peptide sequences of P74 homologues identified a conserved trypsin cleavage site that could generate the observed P74sol-GFP BBMV-specific cleavage product.

The function of envelope protein P74 from Autographa californica multiple nucleopolyhedrovirus in primary infection to host.

This research investigated the function of envelope protein P74 of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) in primary infection to host. A p74-inactivation recombinant baculovirus, rAc-gfp(Delta) p74, was constructed by inserting gfp driven by AcMNPV polyhedrin promoter into the p74 locus of AcMNPV genome. Bioassays showed that the P74-null occlusion bodies (OBs) failed to infect its natural host larvae, Spodoptera exigua, per os, while the p74-null budded virus (BVs) could infect host larvae by injection. However, its inability for oral infectivity was rescued by a mixed infection with wild-type OBs or with the purified P74 protein expressed in Spodoptera frugiperda Sf-9 cells, and the P74 protein rescue was in a dosage-dependent manner. The 50% lethal dosage (LD50) value of a P74 overexpression recombinant virus, rAc-p74(++)-polh+, which contained two copies of p74 gene, was not significantly different from that of wild-type virus. One-step growth curve assays of viruses suggested that BV production from cells infected with p74-null virus was similar to that from cells infected with wild-type virus or the P74 overexpression virus. ELISA analysis indicated that P74 protein could bind its host brush border membrane vesicles (BBMV) efficiently with saturation, but it could only bind its sensitive midgut BBMV specifically. In vitro pull-down assay showed that a protein of approximately 35 kDa in the BBMV was involved in the specific binding. These results demonstrated that the P74 protein is essential for oral infectivity of occlusion-derived virus (ODV) and plays a role in midgut attachment and fusion.

Binding of transmissible gastroenteritis coronavirus to brush border membrane sialoglycoproteins.

Transmissible gastroenteritis coronavirus (TGEV) is a porcine pathogen causing enteric infections that are lethal for suckling piglets. The enterotropism of TGEV is connected with the sialic acid binding activity of the viral surface protein S. Here we show that, among porcine intestinal brush border membrane proteins, TGEV recognizes a mucin-type glycoprotein designated MGP in a sialic acid-dependent fashion. Virus binding assays with cryosections of the small intestine from a suckling piglet revealed the binding of TGEV to mucin-producing goblet cells. A nonenteropathogenic mutant virus that lacked a sialic acid binding activity was unable to bind to MGP and to attach to goblet cells. Our results suggest a role of MGP in the enteropathogenicity of TGEV.

Human cytomegalovirus-caused damage to placental trophoblasts mediated by immediate-early gene-induced tumor necrosis factor-alpha.

Infection of the fetal epithelium (trophoblast) lining the villous placenta by human cytomegalovirus (HCMV) accompanies placental inflammations and fetal intrauterine growth restriction. However, the consequences of infection on the villous trophoblast have not been explored. We show that HCMV infection of primary immature (cytotrophoblast-like) or mature (syncytiotrophoblast-like) cultures results in loss of half of the cells within 24 hours of virus challenge. Two-color immunofluorescence of HCMV immediate early (IE) gene expression and apoptosis (terminal dUTP nick-end labeling) revealed apoptosis only in uninfected cells. Antibody to tumor necrosis factor (TNF)-alpha completely inhibited infection-induced trophoblast apoptosis and cell loss, as did co-incubation with epidermal growth factor, known to inhibit trophoblast apoptosis. Transfection with HCMV immediate early- (IE)1-72 and IE2-86, but not IE2-55, expression plasmids induced paracrine trophoblast apoptosis inhibitable by epidermal growth factor or antibody to TNF-alpha. These results show that HCMV infection of villous trophoblasts leads to rapid loss of neighboring cells mediated by viral IE protein-induced TNF-alpha secretion. We propose that HCMV infection damages the placental trophoblast barrier by accelerating trophoblast turnover and decreasing its capacity for renewal.

Ionic strength- and temperature-induced K(Ca) shifts in the uncoating reaction of rotavirus strains RF and SA11: correlation with membrane permeabilization.

The hydrodynamic diameters of native rotavirus particles, bovine RF and simian SA11 strains, were determined by quasielastic light scattering. By using this method and agarose gel electrophoresis, the Ca(2+) dissociation constant, K(Ca), governing the transition from triple-layer particles (TLPs) to double-layer particles (DLPs), was shown to increase, at constant pH, as the temperature and/or the ionic strength of the incubation medium increased. We report the novel observation that, under physiological conditions, K(Ca) values for both RF and SA11 rotaviruses were well above the intracytoplasmic Ca(2+) concentrations of various cells, which may explain why TLP uncoating takes place within vesicles (possibly endosomes) during the entry process. A correlation between TLP uncoating and cell membrane permeabilization was found, as shown by the release of carboxyfluorescein (CF) from CF-loaded intestinal brush-border membrane vesicles. Conditions stabilizing the virion in the TLP form inhibited CF release, whereas conditions favoring the TLP-to-DLP transformation activated this process. We conclude that membrane permeabilization must be preceded by the loss of the outer-capsid proteins from trypsinized TLP and that physiological ionic strength is required for permeabilization to take place. Finally, the paper develops an alternative explanation for the mechanism of rotavirus entry, compatible with the Ca(2+)-dependent endocytic pathway. We propose that there must be an iterative process involving tight coupling in time between the lowering of endosomal Ca(2+) concentration, virion decapsidation, and membrane permeabilization, which would cause the transcriptionally active DLPs to enter the cytoplasm of cells.

Avian-to-mammal transmission of an avian rotavirus: analysis of its pathogenicity in a heterologous mouse model.

It has been suggested that group A avian rotaviruses can be transmitted to mammals, but there is no direct evidence that such viruses induce disease in mammals. Suckling mice were orally inoculated with two avian rotaviruses. A pigeon rotavirus, PO-13, was found to induce diarrhea, but a turkey rotavirus, Ty-3, did not. The diarrhea induced by PO-13 was dependent on the age of the mouse. In histopathological examinations, antigens of PO-13 were sporadically detected in absorptive cells in the ileum, and lesions were observed as ballooning degenerations of absorptive cells in a region from the duodenum to the ileum. However, the rotavirus antigen was not detected in the majority of these degenerative cells. These results indicated that PO-13 could infect and induce diarrhea in suckling mice. This is the first evidence of an avian rotavirus being experimentally transmissible to a mammal.

Pathogenesis of rotavirus gastroenteritis.

The outcome of intestinal infection with rotaviruses is more complex than initially appreciated, and it is affected by a complex interplay of host and viral factors. Rotaviruses infect intestinal enterocytes, and the early events in infection are mediated by virus-epithelial cell interactions. Diarrhoea may be caused by several mechanisms including (i) malabsorption that occurs secondary to the destruction of enterocytes, (ii) villus ischaemia and activation of the enteric nervous system that may be evoked by release of a vasoactive agent from infected epithelial cells in the absence of significant pathologic lesions or enterocyte damage, and (iii) intestinal secretion stimulated by the intracellular or extracellular action of the rotavirus non-structural protein, NSP4, a novel enterotoxin and secretory agonist with pleiotropic properties. New studies of rotavirus infection of polarized intestinal epithelial cells show that rotaviruses infect cells differently depending on whether or not they require sialic acid for initial binding, and infection alters epithelial cell functions. NSP4 also affects epithelial cell function and interactions. NSP4 (i) induces an age- and dose-dependent diarrhoeal response in young rodents that is similar to virus-induced disease, (ii) stimulates a Ca(2+)-dependent cell permeability where the secretory response is age-dependent, and (iii) alters epithelial cell integrity. Antibody to NSP4 protects mouse pups from diarrhoea induced by homotypic and heterotypic viruses. These data support a new mechanism of rotavirus-induced diarrhoea whereby a viral enterotoxin triggers a signal transduction pathway that alters epithelial cell permeability and chloride secretion. This new information about how a gastrointestinal virus causes disease demonstrates common pathogenic mechanisms for viral and bacterial pathogens not previously appreciated. These results also suggest new approaches to prevent or treat rotavirus-induced diarrhoea.

Rotavirus infection impairs intestinal brush-border membrane Na(+)-solute cotransport activities in young rabbits.

The mechanism of rotavirus diarrhea was investigated by infecting young, specific pathogen-free, New Zealand rabbits with a lapine rotavirus, strain La/RR510. With 4-wk-old animals, virus shedding into the intestinal lumen peaked at 72 h postinfection (hpi), and a mild, watery diarrhea appeared at 124 hpi. No intestinal lesions were seen up to 144 hpi, indicating that diarrhea does not follow mucosal damage but can precede it, as if cell dysfunction were the cause, not the consequence, of the histological lesions. Kinetic analyses with brush-border membrane vesicles isolated from infected rabbits revealed strong inhibition of both Na(+)-D-glucose (SGLT1) and Na(+)-L-leucine symport activities. For both symporters, only maximum velocity decreased with time. The density of phlorizin-binding sites and SGLT1 protein antigen in the membrane remained unaffected, indicating that the virus effect on this symporter is direct. Because SGLT1 supports water reabsorption under physiological conditions, the mechanism of rotavirus diarrhea may involve a generalized inhibition of Na(+)-solute symport systems, hence, of water reabsorption. Massive water loss through the intestine may eventually overwhelm the capacity of the organ for water reabsorption, thereby helping the diarrhea to get established.

Differences in virus receptor for type I and type II feline infectious peritonitis virus.

Feline infectious peritonitis viruses (FIPVs) are classified into type I and type II serogroups. Here, we report that feline aminopeptidase N (APN), a cell-surface metalloprotease on the intestinal, lung and kidney epithelial cells, is a receptor for type II FIPV but not for type I FIPV. A monoclonal antibody (MAb) R-G-4, which blocks infection of Felis catus whole fetus (fcwf-4) cells by type II FIPV, was obtained by immunizing mice with fcwf-4 cells which are highly susceptible to FIPV. This MAb also blocked infection of fcwf-4 cells by type II feline enteric coronavirus (FECV), canine coronavirus (CCV), and transmissible gastroenteritis virus (TGEV). On the other hand, it did not block infection by type I FIPVs. MAb R-G-4 recognized a polypeptide of relative molecular mass 120-130 kDa in feline intestinal brush-border membrane (BBM) proteins. The polypeptide possessed aminopeptidase activity, and the first 15 N-terminal amino acid sequence was identical to that of the feline APN. Feline intestinal BBM proteins and the polypeptide reacted with MAb R-G-4 (feline APN) inhibited the infectivity of type II FIPV, type II FECV, CCV and TGEV to fcwf-4 cells, but did not inhibit the infectivity of type I FIPVs.