Viral Zoonoses: Interactions and Factors Driving Virus Transmission.

The beginning of the 21st century was marked by an increase in the number of emerging/reemerging infectious diseases detected worldwide and by the challenging COVID-19 pandemic [...].

Diverse susceptibilities and responses of human and rodent cells to orthohantavirus infection reveal different levels of cellular restriction.

Orthohantaviruses are rodent-borne emerging viruses that may cause severe diseases in humans but no apparent pathology in their small mammal reservoirs. However, the mechanisms leading to tolerance or pathogenicity in humans and persistence in rodent reservoirs are poorly understood, as is the manner in which they spread within and between organisms. Here, we used a range of cellular and molecular approaches to investigate the interactions of three different orthohantaviruses-Puumala virus (PUUV), responsible for a mild to moderate form of hemorrhagic fever with renal syndrome in humans, Tula virus (TULV) with low pathogenicity, and non-pathogenic Prospect Hill virus (PHV)-with human and rodent host cell lines. Besides the fact that cell susceptibility to virus infection was shown to depend on the cell type and virus strain, the three orthohantaviruses were able to infect Vero E6 and HuH7 human cells, but only the former secreted infectious particles. In cells derived from PUUV reservoir, the bank vole (Myodes glareolus), PUUV achieved a complete viral cycle, while TULV did not enter the cells and PHV infected them but did not produce infectious particles, reflecting differences in host specificity. A search for mature virions by electron microscopy (EM) revealed that TULV assembly occurred in part at the plasma membrane, whereas PHV particles were trapped in autophagic vacuoles in cells of the heterologous rodent host. We described differential interactions of orthohantaviruses with cellular factors, as supported by the cellular distribution of viral nucleocapsid protein with cell compartments, and proteomics identification of cellular partners. Our results also showed that interferon (IFN) dependent gene expression was regulated in a cell and virus species dependent manner. Overall, our study highlighted the complexity of the host-virus relationship and demonstrated that orthohantaviruses are restricted at different levels of the viral cycle. In addition, the study opens new avenues to further investigate how these viruses differ in their interactions with cells to evade innate immunity and how it depends on tissue type and host species.

Inhibition of interferon I induction by non-structural protein NSs of Puumala virus and other vole-associated orthohantaviruses: phenotypic plasticity of the protein and potential functional domains.

The orthohantavirus Puumala virus (PUUV), which is transmitted by bank voles (Clethrionomys glareolus), and other vole-borne hantaviruses contain in their small (S) genome segment two overlapping open reading frames, coding for the nucleocapsid protein and the non-structural protein NSs, a putative type I interferon (IFN-I) antagonist. To investigate the role of NSs of PUUV and other orthohantaviruses, the expression pattern of recombinant NSs constructs and their ability to inhibit human IFN-I promoter activity were investigated. The NSs proteins of PUUV and related cricetid-borne orthohantaviruses showed strong inhibition of IFN-I promoter induction. We identified protein products originating from three and two methionine initiation codons in the NSs ORF of PUUV during transfection and infection, respectively. The three putative start codons are conserved in all PUUV strains analysed. Translation initiation at these start codons influenced the inhibitory activity of the NSs products, with the wild-type (wt) construct expressing two proteins starting at the first and second methionine and showing strong inhibition activity. Analysis of in vitro-generated variants and naturally occurring PUUV NSs proteins indicated that amino acid variation in the NSs protein is well tolerated, suggesting its phenotypic plasticity. The N-terminal 20-amino-acid region of the NSs protein was found to be associated with strong inhibition and to be highly vulnerable to amino acid exchanges and tag fusions. Infection studies using human, bank vole, and Vero E6 cells did not show obvious differences in the replication capacity of PUUV Sotkamo wt and a strain with a truncated NSs protein (NSs21Stop), showing that the lack of a full-length NSs might be compensated by its N-terminal peptide, as seen in transfection experiments. These results contribute to our understanding of virus-host interactions and highlight the importance of future innate immunity studies in reservoir hosts.

Interactions of Viral Proteins from Pathogenic and Low or Non-Pathogenic Orthohantaviruses with Human Type I Interferon Signaling.

Rodent-borne orthohantaviruses are asymptomatic in their natural reservoir, but they can cause severe diseases in humans. Although an exacerbated immune response relates to hantaviral pathologies, orthohantaviruses have to antagonize the antiviral interferon (IFN) response to successfully propagate in infected cells. We studied interactions of structural and nonstructural (NSs) proteins of pathogenic Puumala (PUUV), low-pathogenic Tula (TULV), and non-pathogenic Prospect Hill (PHV) viruses, with human type I and III IFN (IFN-I and IFN-III) pathways. The NSs proteins of all three viruses inhibited the RIG-I-activated IFNß promoter, while only the glycoprotein precursor (GPC) of PUUV, or its cleavage product Gn/Gc, and the nucleocapsid (N) of TULV inhibited it. Moreover, the GPC of both PUUV and TULV antagonized the promoter of IFN-stimulated responsive elements (ISRE). Different viral proteins could thus contribute to inhibition of IFNß response in a viral context. While PUUV and TULV strains replicated similarly, whether expressing entire or truncated NSs proteins, only PUUV encoding a wild type NSs protein led to late IFN expression and activation of IFN-stimulated genes (ISG). This, together with the identification of particular domains of NSs proteins and different biological processes that are associated with cellular proteins in complex with NSs proteins, suggested that the activation of IFN-I is probably not the only antiviral pathway to be counteracted by orthohantaviruses and that NSs proteins could have multiple inhibitory functions.

Defects in Galactose Metabolism and Glycoconjugate Biosynthesis in a UDP-Glucose Pyrophosphorylase-Deficient Cell Line Are Reversed by Adding Galactose to the Growth Medium.

UDP-glucose (UDP-Glc) is synthesized by UGP2-encoded UDP-Glc pyrophosphorylase (UGP) and is required for glycoconjugate biosynthesis and galactose metabolism because it is a uridyl donor for galactose-1-P (Gal1P) uridyltransferase. Chinese hamster lung fibroblasts harboring a hypomrphic UGP(G116D) variant display reduced UDP-Glc levels and cannot grow if galactose is the sole carbon source. Here, these cells were cultivated with glucose in either the absence or presence of galactose in order to investigate glycoconjugate biosynthesis and galactose metabolism. The UGP-deficient cells display < 5% control levels of UDP-Glc/UDP-Gal and > 100-fold reduction of [6-3H]galactose incorporation into UDP-[6-3H]galactose, as well as multiple deficits in glycoconjugate biosynthesis. Cultivation of these cells in the presence of galactose leads to partial restoration of UDP-Glc levels, galactose metabolism and glycoconjugate biosynthesis. The Vmax for recombinant human UGP(G116D) with Glc1P is 2000-fold less than that of the wild-type protein, and UGP(G116D) displayed a mildly elevated Km for Glc1P, but no activity of the mutant enzyme towards Gal1P was detectable. To conclude, although the mechanism behind UDP-Glc/Gal production in the UGP-deficient cells remains to be determined, the capacity of this cell line to change its glycosylation status as a function of extracellular galactose makes it a useful, reversible model with which to study different aspects of galactose metabolism and glycoconjugate biosynthesis.

Revisiting the genetic diversity of emerging hantaviruses circulating in Europe using a pan-viral resequencing microarray.

Hantaviruses are zoonotic agents transmitted from small mammals, mainly rodents, to humans, where they provoke diseases such as Hemorrhagic fever with Renal Syndrome (HFRS) and its mild form, Nephropathia Epidemica (NE), or Hantavirus Cardio-Pulmonary Syndrome (HCPS). Hantaviruses are spread worldwide and monitoring animal reservoirs is of primary importance to control the zoonotic risk. Here, we describe the development of a pan-viral resequencing microarray (PathogenID v3.0) able to explore the genetic diversity of rodent-borne hantaviruses endemic in Europe. Among about 800 sequences tiled on the microarray, 52 correspond to a tight molecular sieve of hantavirus probes covering a large genetic landscape. RNAs from infected animal tissues or from laboratory strains have been reverse transcribed, amplified, then hybridized to the microarray. A classical BLASTN analysis applied to the sequence delivered through the microarray allows to identify the hantavirus species up to the exact geographical variant present in the tested samples. Geographical variants of the most common European hantaviruses from France, Germany, Slovenia and Finland, such as Puumala virus, Dobrava virus and Tula virus, were genetically discriminated. Furthermore, we precisely characterized geographical variants still unknown when the chip was conceived, such as Seoul virus isolates, recently emerged in France and the United Kingdom.

What Do We Know about How Hantaviruses Interact with Their Different Hosts?

Hantaviruses, like other members of the Bunyaviridae family, are emerging viruses that are able to cause hemorrhagic fevers. Occasional transmission to humans is due to inhalation of contaminated aerosolized excreta from infected rodents. Hantaviruses are asymptomatic in their rodent or insectivore natural hosts with which they have co-evolved for millions of years. In contrast, hantaviruses cause different pathologies in humans with varying mortality rates, depending on the hantavirus species and its geographic origin. Cases of hemorrhagic fever with renal syndrome (HFRS) have been reported in Europe and Asia, while hantavirus cardiopulmonary syndromes (HCPS) are observed in the Americas. In some cases, diseases caused by Old World hantaviruses exhibit HCPS-like symptoms. Although the etiologic agents of HFRS were identified in the early 1980s, the way hantaviruses interact with their different hosts still remains elusive. What are the entry receptors? How do hantaviruses propagate in the organism and how do they cope with the immune system? This review summarizes recent data documenting interactions established by pathogenic and nonpathogenic hantaviruses with their natural or human hosts that could highlight their different outcomes.

Discovery of hantavirus circulating among Rattus rattus in French Mayotte island, Indian Ocean.

Hantaviruses are emerging zoonotic viruses that cause human diseases. In this study, sera from 642 mammals from La Réunion and Mayotte islands (Indian Ocean) were screened for the presence of hantaviruses by molecular analysis. None of the mammals from La Réunion island was positive, but hantavirus genomic RNA was discovered in 29/160 (18 %) Rattus rattus from Mayotte island. The nucleoprotein coding region was sequenced from the liver and spleen of all positive individuals allowing epidemiological and intra-strain variability analyses. Phylogenetic analysis based on complete coding genomic sequences showed that this Murinae-associated hantavirus is a new variant of Thailand virus. Further studies are needed to investigate hantaviruses in rodent hosts and in Haemorrhagic Fever with Renal Syndrome (HFRS) human cases.

TNAP, an Essential Player in Membrane Lipid Rafts of Neuronal Cells.

The tissue non-specific alkaline phosphatase (TNAP) is a glycosyl-phosphatidylinositol (GPI) anchored glycoprotein which exists under different forms and is expressed in different tissues. As the other members of the ecto-phosphatase family, TNAP is targeted to membrane lipid rafts. Such micro domains enriched in particular lipids, are involved in cell sorting, are in close contact with the cellular cytoskeleton and play the role of signaling platform. In addition to its location in functional domains, the extracellular orientation of TNAP and the fact this glycoprotein can be shed from plasma membranes, contribute to its different phosphatase activities by acting as a phosphomonoesterase on various soluble substrates (inorganic pyrophosphate -PPi-, pyridoxal phosphate -PLP-, phosphoethanolamine -PEA-), as an ectonucleotidase on nucleotide-phosphate and presumably as a phosphatase able to dephosphorylate phosphoproteins and phospholipids associated to cells or to extra cellular matrix. More and more data accumulate on an involvement of the brain TNAP both in physiological and pathological situations. This review will summarize what is known and expected from the TNAP localization in lipid rafts with a particular emphasis on the role of a neuronal microenvironment on its potential function in the central nervous system.

Role of the cytosolic tails of Rift Valley fever virus envelope glycoproteins in viral morphogenesis.

The correct folding, heterodimerization and trafficking of Gn/Gc envelope glycoproteins of Rift Valley fever virus, RVFV (Bunyaviridae and Phlebovirus genus) are essential for Golgi assembly and budding of viral particles. The Gn and Gc carboxy-terminus contain a Golgi targeting and an ER-retrieval signal, respectively. We generated RVFV-like particles with mutations in the cytosolic tails of Gn or Gc and identified regions important for release of infectious particles. The role of specific amino-acids in these regions was further investigated by creating recombinant mutant viruses by reverse-genetics. Residues outside the suspected Golgi targeting motif, i.e. the di-lysine K29-K30 motif and the N43, R44 and I46 residues of the Gn cytosolic domain, appeared important for Golgi localization and RNP packaging. Concerning the Gc tail, replacement of K2 or K3 in the di-lysine motif, had a drastic impact on Gn trafficking and induced an important organelle redistribution and cell remodeling, greatly affecting particle formation and release.

The Rift Valley fever accessory proteins NSm and P78/NSm-GN are distinct determinants of virus propagation in vertebrate and invertebrate hosts.

Rift Valley fever virus (RVFV) is an enzootic virus circulating in Africa that is transmitted to its vertebrate host by a mosquito vector and causes severe clinical manifestations in humans and ruminants. RVFV has a tripartite genome of negative or ambisense polarity. The M segment contains five in-frame AUG codons that are alternatively used for the synthesis of two major structural glycoproteins, GN and GC, and at least two accessory proteins, NSm, a 14-kDa cytosolic protein, and P78/NSm-GN, a 78-kDa glycoprotein. To determine the relative contribution of P78 and NSm to RVFV infectivity, AUG codons were knocked out to generate mutant viruses expressing various sets of the M-encoded proteins. We found that, in the absence of the second AUG codon used to express NSm, a 13-kDa protein corresponding to an N-terminally truncated form of NSm, named NSm', was synthesized from AUG 3. None of the individual accessory proteins had any significant impact on RVFV virulence in mice. However, a mutant virus lacking both NSm and NSm' was strongly attenuated in mice and grew to reduced titers in murine macrophages, a major target cell type of RVFV. In contrast, P78 was not associated with reduced viral virulence in mice, yet it appeared as a major determinant of virus dissemination in mosquitoes. This study demonstrates how related accessory proteins differentially contribute to RVFV propagation in mammalian and arthropod hosts.

Identification of a cellular ligand for the natural cytotoxicity receptor NKp44.

With an array of activating and inhibitory receptors, natural killer (NK) cells are involved in the eradication of infected, transformed, and tumor cells. NKp44 is a member of the natural cytotoxicity receptor family, which is exclusively expressed on activated NK cells. Here, we identify natural cytotoxicity receptor NKp44 (NKp44L), a novel isoform of the mixed-lineage leukemia-5 protein, as a cellular ligand for NKp44. Unlike the other MLL family members, NKp44L is excluded from the nucleus, but expressed at the cell-surface level; its subcellular localization is being associated with the presence of a specific C-terminal motif. Strikingly, NKp44L has not been detected on circulating cells isolated from healthy individuals, but it is expressed on a large panel of the tumor and transformed cells. The sharply decreased NK lysis activity induced by anti-NKp44L antibodies directly demonstrates the role of NKp44L in cytotoxicity. Taken together, these results show that NKp44L could be critical for NK cell-mediated innate immunity. The identification and cellular distribution of NKp44L highlight the role of this self-molecule as a danger signal to alert the NK cell network.

Secreted dengue virus nonstructural protein NS1 is an atypical barrel-shaped high-density lipoprotein.

Dengue virus (DENV) causes the major arboviral disease of the tropics, characterized in its severe forms by signs of hemorrhage and plasma leakage. DENV encodes a nonstructural glycoprotein, NS1, that associates with intracellular membranes and the cell surface. NS1 is eventually secreted as a soluble hexamer from DENV-infected cells and circulates in the bloodstream of infected patients. Extracellular NS1 has been shown to modulate the complement system and to enhance DENV infection, yet its structure and function remain essentially unknown. By combining cryoelectron microscopy analysis with a characterization of NS1 amphipathic properties, we show that the secreted NS1 hexamer forms a lipoprotein particle with an open-barrel protein shell and a prominent central channel rich in lipids. Biochemical and NMR analyses of the NS1 lipid cargo reveal the presence of triglycerides, bound at an equimolar ratio to the NS1 protomer, as well as cholesteryl esters and phospholipids, a composition evocative of the plasma lipoproteins involved in vascular homeostasis. This study suggests that DENV NS1, by mimicking or hijacking lipid metabolic pathways, contributes to endothelium dysfunction, a key feature of severe dengue disease.

Differential expression of the bone and the liver tissue non-specific alkaline phosphatase isoforms in brain tissues.

The enzyme tissue non-specific alkaline phosphatase (TNAP) belongs to the ectophosphatase family. It is present in large amounts in bone in which it plays a role in mineralization but little is known about its function in other tissues. Arguments are accumulating for its involvement in the brain, in particular in view of the neurological symptoms accompanying human TNAP deficiencies. We have previously shown, by histochemistry, alkaline phosphatase (AP) activity in monkey brain vessels and parenchyma in which AP exhibits specific patterns. Here, we clearly attribute this activity to TNAP expression rather than to other APs in primates (human and marmoset) and in rodents (rat and mouse). We have not found any brain-specific transcripts but our data demonstrate that neuronal and endothelial cells exclusively express the bone TNAP transcript in all species tested, except in mouse neurons in which liver TNAP transcripts have also been detected. Moreover, we highlight the developmental regulation of TNAP expression; this also acts during neuronal differentiation. Our study should help to characterize the regulation of the expression of this ectophosphatase in various cell types of the central nervous system.

The cellular prion protein interacts with the tissue non-specific alkaline phosphatase in membrane microdomains of bioaminergic neuronal cells.

BACKGROUND: The cellular prion protein, PrP(C), is GPI anchored and abundant in lipid rafts. The absolute requirement of PrP(C) in neurodegeneration associated to prion diseases is well established. However, the function of this ubiquitous protein is still puzzling. Our previous work using the 1C11 neuronal model, provided evidence that PrP(C) acts as a cell surface receptor. Besides a ubiquitous signaling function of PrP(C), we have described a neuronal specificity pointing to a role of PrP(C) in neuronal homeostasis. 1C11 cells, upon appropriate induction, engage into neuronal differentiation programs, giving rise either to serotonergic (1C11(5-HT)) or noradrenergic (1C11(NE)) derivatives. METHODOLOGY/PRINCIPAL FINDINGS: The neuronal specificity of PrP(C) signaling prompted us to search for PrP(C) partners in 1C11-derived bioaminergic neuronal cells. We show here by immunoprecipitation an association of PrP(C) with an 80 kDa protein identified by mass spectrometry as the tissue non-specific alkaline phosphatase (TNAP). This interaction occurs in lipid rafts and is restricted to 1C11-derived neuronal progenies. Our data indicate that TNAP is implemented during the differentiation programs of 1C11(5-HT) and 1C11(NE) cells and is active at their cell surface. Noteworthy, TNAP may contribute to the regulation of serotonin or catecholamine synthesis in 1C11(5-HT) and 1C11(NE) bioaminergic cells by controlling pyridoxal phosphate levels. Finally, TNAP activity is shown to modulate the phosphorylation status of laminin and thereby its interaction with PrP. CONCLUSION/SIGNIFICANCE: The identification of a novel PrP(C) partner in lipid rafts of neuronal cells favors the idea of a role of PrP in multiple functions. Because PrP(C) and laminin functionally interact to support neuronal differentiation and memory consolidation, our findings introduce TNAP as a functional protagonist in the PrP(C)-laminin interplay. The partnership between TNAP and PrP(C) in neuronal cells may provide new clues as to the neurospecificity of PrP(C) function.

The effects of microcarrier culture on recombinant CHO cells under biphasic hypothermic culture conditions.

A Chinese hamster ovary (CHO) cell line, producing recombinant secreted human placental alkaline phosphatase (SEAP) was investigated under three different culture conditions (suspension cells, cells attached to Cytodex 3 and Cytopore 1 microcarriers) in a biphasic culture mode using a temperature shift to mild hypothermic conditions (33 degrees C) in a fed-batch bioreactor. The cell viability in both the suspension and the Cytodex 3 cultures was maintained for significantly longer periods under hypothermic conditions than in the single-temperature cultures, leading to higher integrated viable cell densities. For all culture conditions, the specific productivity of SEAP increased after the temperature reduction; the specific productivities of the microcarrier cultures increased approximately threefold while the specific productivity of the suspension culture increased nearly eightfold. The glucose and glutamine consumption rates and lactate and ammonia production rates were significantly lowered after the temperature reduction, as were the yields of lactate from glucose. However, the yield of ammonia from glutamine increased in response to the temperature shift.

Glycosylation-related genes are variably expressed depending on the differentiation state of a bioaminergic neuronal cell line: implication for the cellular prion protein.

A striking feature of the cellular prion protein (PrP(C)) is the heterogeneity of its glycoforms, whose contribution to PrP(C) function has yet to be defined. Using the 1C11 neuronal bioaminergic differentiation model and a glycomics approach, we show here a correlation between differential PrP(C) N-glycosylations in 1C11(5-HT) serotonergic and 1C11(NE) noradrenergic cells compared to their 1C11 precursor cells and a variation of the glycogenome expression status in these cells. In particular, expression of genes involved in N-glycan synthesis or in the modeling of chondroitin and heparan sulfate proteoglycans appeared to be modulated. Our results highlight that, the expression of glycosylation-related genes is regulated during bioaminergic neuronal differentiation, consistent with a participation of glycoconjugates in neuronal development and plasticity. A neuronal regulation of glycosylation processes may have direct implications on some neurospecific functions of PrP(C) and may participate in specific brain targeting of prion strains.

The effects of culture conditions on the glycosylation of secreted human placental alkaline phosphatase produced in Chinese hamster ovary cells.

The effects of different culture conditions, suspension and microcarrier culture and temperature reduction on the structures of N-linked glycans attached to secreted human placental alkaline phosphatase (SEAP) were investigated for CHO cells grown in a controlled bioreactor. Both mass spectrometry and anion-exchange chromatography were used to probe the N-linked glycan structures and distribution. Complex-type glycans were the dominant structures with small amounts of high mannose glycans observed in suspension and reduced temperature cultures. Biantennary glycans were the most common structures detected by mass spectrometry, but triantennary and tetraantennary forms were also detected. The amount of sialic acid present was relatively low, approximately 0.4 mol sialic acid/mol SEAP for suspension cultures. Microcarrier cultures exhibited a decrease in productivity compared with suspension culture due to a decrease in both maximum viable cell density (15-20%) and specific productivity (30-50%). In contrast, a biphasic suspension culture in which the temperature was reduced at the beginning of the stationary phase from 37 to 33 degrees C, showed a 7% increase in maximum viable cell density, a 62% increase in integrated viable cell density, and a 133% increase in specific productivity, leading to greater than threefold increase in total productivity. Both microcarrier and reduced temperature cultures showed increased sialylation and decreased fucosylation when compared to suspension culture. Our results highlight the importance of glycoform analysis after process modification as even subtle changes (e.g., changing from one microcarrier to another) may affect glycan distributions.

Involvement of the Gab2 scaffolding adapter in type I interferon signalling.

Interferons (IFNs) are pleiotropic cytokines involved in the regulation of physiological and pathological processes. Upon interaction with their specific receptors, IFNs activate the Jak/STAT signalling pathway. Numerous studies suggest, however, that the classical Jak/STAT pathway cannot alone account for the wide range of IFN's biological effects. To better understand the role of alternative signalling pathways in the type I IFNs response, we analyzed novel tyrosine-phosphorylated proteins following IFN-alpha2 stimulation. We showed for the first time that the Grb2-associated binder 2 (Gab2) protein is differentially phosphorylated upon the IFN subtype employed and the cells stimulated. We demonstrated that IFNAR1 physically interacts with Gab2. Moreover, the cellular content of Gab2 varies as a function of IFN receptor chain expression levels, and in particular of the ratio of IFNAR1 to IFNAR2, suggesting that Gab2 and IFNAR2 compete for interaction with IFNAR1. Analysis of Gab2 deletion mutants indicates that IFNAR1 might interact with a Gab2 region containing p85-PI3'kinase binding sites. Our results shed new light on recent data involving both Gab2 and type I IFNs in osteoclastogenesis and oncogenesis.

Cell attachment to microcarriers affects growth, metabolic activity, and culture productivity in bioreactor culture.

It is not well understood how changes from suspension to microcarrier cultures affect cell growth, metabolism, and yield of recombinant proteins. To investigate the effects of culture conditions on cell characteristics, fed-batch bioreactor cultures were performed under different culture conditions (suspension cultures, cultures attached to Cytodex 3 and Cytopore 1 microcarriers) using two different Chinese hamster ovary cell lines producing either secreted human placental alkaline phosphatase (TR2-255) or tissue plasminogen activator (CHO 1-15-500). In controlled, agitated bioreactors, suspension cultures reached cell densities and product titers higher than those in microcarrier cultures, in contrast to the results in static flask cultures. Growth and metabolic activities showed similar trends in suspension and microcarrier culture regardless of cell line. However, the responses of the specific productivities to the different culture conditions differed significantly between the cell lines.