Protein-driven membrane remodeling: Molecular perspectives from Flaviviridae infections.

The mammalian cell membrane consists of thousands of different lipid species, and this variety is critical for biological function. Alterations to this balance can be dangerous as they can lead to permanent disruption of lipid metabolism, a hallmark in several viral diseases. The Flaviviridae family is made up of positive single-stranded RNA viruses that assemble at or near the location of lipid droplet formation in the endoplasmic reticulum. These viruses are known to interfere with lipid metabolism during the onset of liver disease, albeit to different extents. Pathogenesis of these infections involves specific protein-lipid interactions that alter lipid sorting and metabolism to sustain propagation of the viral infection. Recent experimental studies identify a correlation between viral proteins and lipid content or location in the cell, but these do not assess membrane-embedded interactions. Molecular modeling, specifically molecular dynamics simulations, can provide molecular-level spatial and temporal resolution for characterization of biomolecular interactions. This review focuses on recent advancements and current knowledge gaps in the molecular mechanisms of lipid-mediated liver disease preceded by viral infection. We discuss three viruses from the Flaviviridae family: dengue, zika, and hepatitis C, with a particular focus on lipid interactions with their respective ion channels, known as viroporins.

Infection of Glia by Human Pegivirus Suppresses Peroxisomal and Antiviral Signaling Pathways.

Human pegivirus (HPgV) infects peripheral leukocytes but was recently shown to be a neurotropic virus associated with leukoencephalitis in humans. In the present study, we investigated the neural cell tropism of HPgV as well as its effects on host immune responses. HPgV wild type (WT) and a mutant virus with a deletion in the HPgV NS2 gene (ΔNS2) were able to productively infect human astrocytes and microglia but not neurons or an oligodendrocyte-derived cell line. Of note, the ΔNS2 virus replicated better than WT pegivirus in astrocytes, with both viruses being able to subsequently infect and spread in fresh human astrocyte cultures. Infection of human glia by HPgV WT and ΔNS2 viruses resulted in suppression of peroxisome-associated genes, including PEX11B, ABCD1, PEX7, ABCD3, PEX3, and PEX5L, during peak viral production, which was accompanied by reduced expression of IFNB, IRF3, IRF1, and MAVS, particularly in ΔNS2-infected cells. These data were consistent with analyses of brain tissue from patients infected with HPgV in which we observed suppression of peroxisome and type I interferon gene transcripts, including PEX11B, ABCD3, IRF1, and IRF3, with concurrent loss of PMP70 immunoreactivity in glia. Our data indicate that human astrocytes and microglia are permissive to HPgV infection, resulting in peroxisome injury and suppressed antiviral signaling that is influenced by viral diversity. IMPORTANCE Human pegiviruses are detected in 1 to 5% of the general population, principally infecting leukocytes, although their effects on human health remain uncertain. Here, we show that human pegivirus infects specific neural cell types in culture and human brain and, like other neurotropic flaviviruses, causes suppression of peroxisome and antiviral signaling pathways, which could favor ongoing viral infection and perhaps confer susceptibility to the development of neurological disease.

All genera of Flaviviridae host a conserved Xrn1-resistant RNA motif.

After infection by flaviviruses like Zika and West Nile virus, eukaryotic hosts employ the well-conserved endoribonuclease Xrn1 to degrade the viral genomic RNA. Within the 3' untranslated regions, this enzyme encounters intricate Xrn1-resistant structures. This results in the accumulation of subgenomic flaviviral RNAs, an event that improves viral growth and aggravates viral pathogenicity. Xrn1-resistant RNAs have been established throughout the flaviviral genus, but not yet throughout the entire Flaviviridae family. In this work, we use previously determined characteristics of these structures to identify homologous sequences in many members of the genera pegivirus, hepacivirus and pestivirus. We used structural alignment and mutational analyses to establish that these sequences indeed represent Xrn1-resistant RNA and that they employ the general features of the flaviviral xrRNAs, consisting of a double pseudoknot formed by five base-paired regions stitched together by a crucial triple base interaction. Furthermore, we demonstrate that the pestivirus Bungowannah virus produces subgenomic RNA in vivo. Altogether, these results indicate that viruses make use of a universal Xrn1-resistant RNA throughout the Flaviviridae family.

The First Nonmammalian Pegivirus Demonstrates Efficient In Vitro Replication and High Lymphotropism.

Members of the Pegivirus genus, family Flaviviridae, widely infect humans and other mammals, including nonhuman primates, bats, horses, pigs, and rodents, but are not associated with disease. Here, we report a new, genetically distinct pegivirus in goose (Anser cygnoides), the first identified in a nonmammalian host species. Goose pegivirus (GPgV) can be propagated in goslings, embryonated goose eggs, and primary goose embryo fibroblasts, and is thus the first pegivirus that can be efficiently cultured in vitro Experimental infection of GPgV in goslings via intravenous injection revealed robust replication and high lymphotropism. Analysis of the tissue tropism of GPgV revealed that the spleen and thymus were the organs bearing the highest viral loads. Importantly, GPgV could promote clinical manifestations of goose parvovirus infection, including reduced weight gain and 7% mortality. This finding contrasts with the lack of pathogenicity that is characteristic of previously reported pegiviruses.IMPORTANCE Members of the Pegivirus genus, family Flaviviridae, widely infect humans and other mammals, but are described as causing persistent infection and lacking pathogenicity. The efficiency of in vitro replication systems for pegivirus is poor, thus limiting investigation into viral replication steps. Because of that, the pathogenesis, cellular tropism, route of transmission, biology, and epidemiology of pegiviruses remain largely uncovered. Here, we report a phylogenetically distinct goose pegivirus (GPgV) that should be classified as a new species. GPgV proliferated in cell culture in a species- and cell-type-specific manner. Animal experiments show GPgV lymphotropism and promote goose parvovirus clinical manifestations. This study provides the first cell culture model for pegivirus, opening new possibilities for studies of pegivirus molecular biology. More importantly, our findings stand in contrast to the lack of identified pathogenicity of previously reported pegiviruses, which sheds lights on the pathobiology of pegivirus.

Flaviviridae Viruses and Oxidative Stress: Implications for Viral Pathogenesis.

Oxidative stress is induced once the balance of generation and neutralization of reactive oxygen species (ROS) is broken in the cell, and it plays crucial roles in a variety of natural and diseased processes. Infections of Flaviviridae viruses trigger oxidative stress, which affects both the cellular metabolism and the life cycle of the viruses. Oxidative stress associated with specific viral proteins, experimental culture systems, and patient infections, as well as its correlations with the viral pathogenesis attracts much research attention. In this review, we primarily focus on hepatitis C virus (HCV), dengue virus (DENV), Zika virus (ZIKV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and tick-borne encephalitis virus (TBEV) as representatives of Flaviviridae viruses and we summarize the mechanisms involved in the relevance of oxidative stress for virus-associated pathogenesis. We discuss the current understanding of the pathogenic mechanisms of oxidative stress induced by Flaviviridae viruses and highlight the relevance of autophagy and DNA damage in the life cycle of viruses. Understanding the crosstalk between viral infection and oxidative stress-induced molecular events may offer new avenues for antiviral therapeutics.

In Vivo Dynamics of Reporter Flaviviridae Viruses.

Recombinant viruses possessing reporter proteins have been generated for virus research. In the case of the family Flaviviridae, we recently generated recombinant viruses, including the hepatitis C virus of the genus Hepacivirus, Japanese encephalitis virus (JEV) of the genus Flavivirus, and bovine viral diarrhea virus of the genus Pestivirus; all three viruses possess an 11-amino-acid subunit derived from NanoLuc luciferase (HiBiT). Here, we further developed the recombinant viruses and investigated their utility in vivo Recombinant viruses harboring HiBiT in the E, NS1, or NS3 protein constructed based on the predicted secondary structure, solvent-accessible surface area, and root mean square fluctuation of the proteins exhibited comparable replication to that of the wild-type virus in vitro The recombinant JEV carrying HiBiT in the NS1 protein exhibited propagation in mice comparable to that of the parental virus, and propagation of the recombinant was monitored by the luciferase activity. In addition, the recombinants of classical swine fever virus (CSFV) possessing HiBiT in the Erns or E2 protein also showed propagation comparable to that of the wild-type virus. The recombinant CSFV carrying HiBiT in Erns exhibited similar replication to the parental CSFV in pigs, and detection of viral propagation of this recombinant by luciferase activity was higher than that by quantitative PCR (qPCR). Taken together, these results demonstrated that the reporter Flaviviridae viruses generated herein are powerful tools for elucidating the viral life cycle and pathogeneses and provide a robust platform for the development of novel antivirals.IMPORTANCEIn vivo applications of reporter viruses are necessary to understand viral pathogenesis and provide a robust platform for antiviral development. In developing such applications, determination of an ideal locus to accommodate foreign genes is important, because insertion of foreign genes into irrelevant loci can disrupt the protein functions required for viral replication. Here, we investigated the criteria to determine ideal insertion sites of foreign genes from the protein structure of viral proteins. The recombinant viruses generated by our criteria exhibited propagation comparable to that of parental viruses in vivo Our proteomic approach based on the flexibility profile of viral proteins may provide a useful tool for constructing reporter viruses, including Flaviviridae viruses.

[Human Genetic Predisposition to Diseases Caused by Viruses from Flaviviridae Family].

The identification of human predisposition genes to severe forms of infectious diseases is important for understanding the mechanisms of pathogenesis, as well as for the detection of the risk groups. This will allow one to carry out targeted vaccination and preventive therapy. The most common approaches to the genetic risk estimation include conducting association studies, in which the groups of patients and control individuals are compared using both preliminarily selected candidate genes and using genome-wide analysis. To search for genetic variants predisposed to severe forms of infectious diseases, it is expedient to form a control that consists of patients with clinically proven infections with asymptomatic or mild forms of the disease. The examples of the use of these approaches to identify genetic factors that predispose one to severe forms of infections caused by viruses from the Flaviviridae family are considered in the review. At present, a number of genetic markers associated with predisposition to tick-borne encephalitis, West Nile fever, and Dengue fever have already been detected. These associations must be confirmed in independent samples. Genetic variants, for which the association with spontaneous recovery during infection with hepatitis C virus, patient's reaction on antiviral drugs, and the development of liver fibrosis was established, were also detected. The gene variants with more pronounced phenotypic effects will probably be found during further studies; they can be used in clinical practice as prognostic markers of the course and outcomes of infection with the Flaviviridae, as well as of the response to treatment.

Modulation of the Translational Landscape During Herpesvirus Infection.

Herpesviral mRNAs are produced and translated by cellular machinery, rendering them susceptible to the network of regulatory events that impact translation. In response, these viruses have evolved to infiltrate and hijack translational control pathways as well as to integrate specialized host translation strategies into their own repertoire. They are robust systems to dissect mechanisms of mammalian translational regulation and continue to offer insight into cis-acting mRNA features that impact assembly and activity of the translation apparatus. Here, I discuss recent advances revealing the extent to which the three herpesvirus subfamilies regulate both host and viral translation, thereby dramatically impacting the landscape of protein synthesis in infected cells.

NS2 proteins of GB virus B and hepatitis C virus share common protease activities and membrane topologies.

UNLABELLED: GB virus B (GBV-B), which is hepatotropic in experimentally infected small New World primates, is a member of the Hepacivirus genus but phylogenetically relatively distant from hepatitis C virus (HCV). To gain insights into the role and specificity of hepaciviral nonstructural protein 2 (NS2), which is required for HCV polyprotein processing and particle morphogenesis, we investigated whether NS2 structural and functional features are conserved between HCV and GBV-B. We found that GBV-B NS2, like HCV NS2, has cysteine protease activity responsible for cleavage at the NS2/NS3 junction, and we experimentally confirmed the location of this junction within the viral polyprotein. A model for GBV-B NS2 membrane topology was experimentally established by determining the membrane association properties of NS2 segments fused to green fluorescent protein (GFP) and their nuclear magnetic resonance structures using synthetic peptides as well as by applying an N-glycosylation scanning approach. Similar glycosylation studies confirmed the HCV NS2 organization. Together, our data show that despite limited amino acid sequence similarity, GBV-B and HCV NS2 proteins share a membrane topology with 3 N-terminal transmembrane segments, which is also predicted to apply to other recently discovered hepaciviruses. Based on these data and using trans-complementation systems, we found that intragenotypic hybrid NS2 proteins with heterologous N-terminal membrane segments were able to efficiently trans-complement an assembly-deficient HCV mutant with a point mutation in the NS2 C-terminal domain, while GBV-B/HCV or intergenotypic NS2 chimeras were not. These studies indicate that virus- and genotype-specific intramolecular interactions between N- and C-terminal domains of NS2 are critically involved in HCV morphogenesis. IMPORTANCE: Nonstructural protein 2 (NS2) of hepatitis C virus (HCV) is a multifunctional protein critically involved in polyprotein processing and virion morphogenesis. To gain insights into NS2 mechanisms of action, we investigated whether NS2 structural and functional features are conserved between HCV and GB virus B (GBV-B), a phylogenetically relatively distant primate hepacivirus. We showed that GBV-B NS2, like HCV NS2, carries cysteine protease activity. We experimentally established a model for GBV-B NS2 membrane topology and demonstrated that despite limited sequence similarity, GBV-B and HCV NS2 share an organization with three N-terminal transmembrane segments. We found that the role of HCV NS2 in particle assembly is genotype specific and relies on critical interactions between its N- and C-terminal domains. This first comparative analysis of NS2 proteins from two hepaciviruses and our structural predictions of NS2 from other newly identified mammal hepaciviruses highlight conserved key features of the hepaciviral life cycle.

GB virus type C infection polarizes T-cell cytokine gene expression toward a Th1 cytokine profile via NS5A protein expression.

Human immunodeficiency virus (HIV) disease progression is associated with a helper T cell 1 (Th1) to helper T cell 2 (Th2) cytokine profile switch. Persistent GB virus type C (GBV-C) infection is associated with survival and a serum Th1 cytokine profile in HIV-infected individuals. We found that GBV-C infection increased gene expression of Th1 cytokines and decreased Th2 cytokine expression in peripheral blood mononuclear cells. Furthermore, expression of GBV-C NS5A protein in a CD4(+) cell line resulted in upregulation of Th1 cytokines (tumor necrosis factor α) and downregulation of Th2 cytokines (interleukin 4, interleukin 5, interleukin 10, interleukin 13). GBV-C-induced modulation in T-cell cytokines may contribute to the beneficial effect of GBV-C in HIV-infected individuals.

An experimental model of meningoencephalomyelitis by Rocio flavivirus in BALB/c mice: inflammatory response, cytokine production, and histopathology.

Rocio virus (ROCV) is a flavivirus, probably transmitted by Culex mosquitoes and maintained in nature as a zoonosis of wild birds. Rocio virus caused a human epidemic of severe encephalitis that lasted from 1973 to 1980 in the Ribeira valley, in the southeastern coast of Brazil. After this outbreak, serologic evidence of ROCV circulation has been reported and public health authorities are concerned about a return of ROCV outbreaks in Brazil. We show here a study on the pathogenesis and the physiopathology of ROCV disease in the central nervous system of a Balb/C young adult mice experimental model. The animals were intraperitoneally infected by ROCV and followed from 0 to 9 days after infection, when all of them died. Nervous tissue samples were collected from infected animals for immunohistochemistry and molecular biology analysis. We observed the virus in the central nervous system, the inflammatory changes induced by Th1 and Th2 cytokines, and the final irreversible damage of nervous tissues by neuronal degeneration and apoptosis. These findings can help to better understand the pathogenesis and physiopathology of the human meningoencephalomyelitis by ROCV and other flaviviruses.

Down-regulation of intra-hepatic T-cell signaling associated with GB virus C in a HCV/HIV co-infected group with reduced liver disease.

BACKGROUND & AIMS: Studies have shown that GB virus C (GBV-C) infection leads to reduced liver disease in hepatitis C virus (HCV)/human immunodeficiency virus (HIV) co-infection. Considering that the underlying mechanism(s) are unknown, we aim to identify differential gene and protein expression associated with GBV-C in HCV/HIV co-infection that may be responsible for reduced liver disease. METHODS: Liver, peripheral blood mononuclear cells (PBMCs), and plasma samples were collected from 43 HCV/HIV patients. Plasma was tested for GBV-C RNA by RT-PCR with NS5B gene primers. A microarray was performed on the liver and RT-qPCRs on the liver/PBMC samples. Hepatic protein expression was measured by immunohistochemistry. RESULTS: Sixteen out of 43 patients had GBV-C RNA. GBV-C was associated with reduced hepatic fibrosis (p=0.005) and inflammation (p=0.007). The microarray analysis of the liver samples (n=10) showed down-regulation of genes critical to intra-hepatic T-cell signaling associated with GBV-C. Quantitative RT-PCR of the liver samples (n=13) confirmed the down-regulation of lymphocyte-specific protein tyrosine kinase (LCK) (p=0.02) and docking protein 2 (DOK2) (p=0.04). No differences in the expression levels of these genes were observed in PBMCs (n=22) according to the GBV-C status. The hepatic expression of the LCK protein, measured by immunohistochemistry (n=36), was decreased in CD3-positive T-cells within portal tracts associated with GBV-C (p=0.003). This remained significant in multivariate analysis controlling for hepatic fibrosis and inflammation (p=0.027). No differences were observed in plasma cytokine concentrations (n=25) or ex-vivo peripheral T-cell responses (n=13) versus GBV-C status. CONCLUSIONS: GBV-C infection is associated with down-regulation of critical genes involved in intra-hepatic T-cell signaling in HCV/HIV co-infection. This may be relevant to the pathogenesis of reduced HCV-related liver disease in HIV co-infection.