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1.
J Exp Med ; 218(5)2021 May 03.
Article in English | MEDLINE | ID: mdl-33831141

ABSTRACT

Tick-borne encephalitis virus (TBEV) is an emerging human pathogen that causes potentially fatal disease with no specific treatment. Mouse monoclonal antibodies are protective against TBEV, but little is known about the human antibody response to infection. Here, we report on the human neutralizing antibody response to TBEV in a cohort of infected and vaccinated individuals. Expanded clones of memory B cells expressed closely related anti-envelope domain III (EDIII) antibodies in both groups of volunteers. However, the most potent neutralizing antibodies, with IC50s below 1 ng/ml, were found only in individuals who recovered from natural infection. These antibodies also neutralized other tick-borne flaviviruses, including Langat, louping ill, Omsk hemorrhagic fever, Kyasanur forest disease, and Powassan viruses. Structural analysis revealed a conserved epitope near the lateral ridge of EDIII adjoining the EDI-EDIII hinge region. Prophylactic or early therapeutic antibody administration was effective at low doses in mice that were lethally infected with TBEV.

2.
Cell Host Microbe ; 2021 Mar 29.
Article in English | MEDLINE | ID: mdl-33794184

ABSTRACT

Argonaute (AGO) proteins bind small RNAs to silence complementary RNA transcripts, and they are central to RNA interference (RNAi). RNAi is critical for regulation of gene expression and antiviral defense in Aedes aegypti mosquitoes, which transmit Zika, chikungunya, dengue, and yellow fever viruses. In mosquitoes, AGO1 mediates miRNA interactions, while AGO2 mediates siRNA interactions. We applied AGO-crosslinking immunoprecipitation (AGO-CLIP) for both AGO1 and AGO2, and we developed a universal software package for CLIP analysis (CLIPflexR), identifying 230 small RNAs and 5,447 small RNA targets that comprise a comprehensive RNAi network map in mosquitoes. RNAi network maps predicted expression levels of small RNA targets in specific tissues. Additionally, this resource identified unexpected, context-dependent AGO2 target preferences, including endogenous viral elements and 3'UTRs. Finally, contrary to current thinking, mosquito AGO2 repressed imperfect targets. These findings expand our understanding of small RNA networks and have broad implications for the study of antiviral RNAi.

3.
Proc Natl Acad Sci U S A ; 118(12)2021 Mar 23.
Article in English | MEDLINE | ID: mdl-33723056

ABSTRACT

Human adenosine deaminase acting on RNA 1 (ADAR1) catalyzes adenosine-to-inosine deamination reactions on double-stranded RNA molecules to regulate cellular responses to endogenous and exogenous RNA. Defective ADAR1 editing leads to disorders such as Aicardi-Goutières syndrome, an autoinflammatory disease that manifests in the brain and skin, and dyschromatosis symmetrica hereditaria, a skin pigmentation disorder. Two ADAR1 protein isoforms, p150 (150 kDa) and p110 (110 kDa), are expressed and can edit RNA, but the contribution of each isoform to the editing landscape remains unclear, largely because of the challenges in expressing p150 without p110. In this study, we demonstrate that p110 is coexpressed with p150 from the canonical p150-encoding mRNA due to leaky ribosome scanning downstream of the p150 start codon. The presence of a strong Kozak consensus context surrounding the p110 start codon suggests the p150 mRNA is optimized to leak p110 alongside expression of p150. To reduce leaky scanning and translation initiation at the p110 start codon, we introduced synonymous mutations in the coding region between the p150 and p110 start codons. Cells expressing p150 constructs with these mutations produced significantly reduced levels of p110. Editing analysis of total RNA from ADAR1 knockout cells reconstituted separately with modified p150 and p110 revealed that more than half of the A-to-I edit sites are selectively edited by p150, and the other half are edited by either p150 or p110. This method of isoform-selective editing analysis, making use of the modified p150, has the potential to be adapted for other cellular contexts.

4.
Hepatology ; 2021 Mar 13.
Article in English | MEDLINE | ID: mdl-33713356

ABSTRACT

BACKGROUND & AIMS: Equine hepacivirus (EqHV) is phylogenetically the closest relative of hepatitis C virus (HCV) and shares genome organization, hepatotropism, transient or persistent infection outcome, and the ability to cause hepatitis. Thus, EqHV studies are important to understand equine liver disease, and further as an outbred surrogate animal model for HCV pathogenesis and protective immune responses. Here, we aimed to characterize the course of EqHV infection and associated protective immune responses. APPROACH & RESULTS: Seven horses were experimentally inoculated with EqHV, monitored for 6 months, and rechallenged with the same, and subsequently a heterologous EqHV. Clearance was the primary outcome (6 of 7) and was associated with subclinical hepatitis characterized by lymphocytic infiltrate and individual hepatocyte necrosis. Seroconversion was delayed and antibody titers waned slowly. Clearance of primary infection conferred non-sterilizing immunity resulting in shortened duration of viremia after rechallenge. Peripheral blood mononuclear cell responses in horses were minimal, although EqHV specific T cells were identified. Additionally, an interferon stimulated gene signature was detected in the liver during EqHV infection, similar to acute HCV in humans. EqHV, as HCV, is stimulated by direct binding of the liver-specific microRNA, miR-122. Interestingly, we found that EqHV infection sequesters enough miR-122 to functionally affect gene regulation in the liver. This RNA-based mechanism thus could have consequences for pathology. CONCLUSIONS: EqHV infection in horses typically has an acute resolving course, and the protective immune response lasts for at least a year and broadly attenuates subsequent infections. This could have important implications to achieve the primary goal of an HCV vaccine; to prevent chronicity while accepting acute resolving infection after virus exposure.

5.
J Exp Med ; 218(4)2021 04 05.
Article in English | MEDLINE | ID: mdl-33544838

ABSTRACT

Yellow fever virus (YFV) live attenuated vaccine can, in rare cases, cause life-threatening disease, typically in patients with no previous history of severe viral illness. Autosomal recessive (AR) complete IFNAR1 deficiency was reported in one 12-yr-old patient. Here, we studied seven other previously healthy patients aged 13 to 80 yr with unexplained life-threatening YFV vaccine-associated disease. One 13-yr-old patient had AR complete IFNAR2 deficiency. Three other patients vaccinated at the ages of 47, 57, and 64 yr had high titers of circulating auto-Abs against at least 14 of the 17 individual type I IFNs. These antibodies were recently shown to underlie at least 10% of cases of life-threatening COVID-19 pneumonia. The auto-Abs were neutralizing in vitro, blocking the protective effect of IFN-α2 against YFV vaccine strains. AR IFNAR1 or IFNAR2 deficiency and neutralizing auto-Abs against type I IFNs thus accounted for more than half the cases of life-threatening YFV vaccine-associated disease studied here. Previously healthy subjects could be tested for both predispositions before anti-YFV vaccination.


Subject(s)
Antibodies, Neutralizing/immunology , Autoantibodies/immunology , Autoimmune Diseases , Genetic Diseases, Inborn , Interferon-alpha , Receptor, Interferon alpha-beta , Yellow Fever Vaccine , Yellow fever virus , Adolescent , Adult , Aged , Autoimmune Diseases/genetics , Autoimmune Diseases/immunology , /immunology , Female , Genetic Diseases, Inborn/genetics , Genetic Diseases, Inborn/immunology , HEK293 Cells , Humans , Interferon-alpha/genetics , Interferon-alpha/immunology , Male , Middle Aged , Receptor, Interferon alpha-beta/deficiency , Receptor, Interferon alpha-beta/immunology , /immunology , Vaccines, Attenuated/genetics , Vaccines, Attenuated/immunology , Yellow Fever Vaccine/adverse effects , Yellow Fever Vaccine/genetics , Yellow Fever Vaccine/immunology , Yellow fever virus/genetics , Yellow fever virus/immunology
6.
Sci Rep ; 11(1): 736, 2021 Jan 12.
Article in English | MEDLINE | ID: mdl-33436886

ABSTRACT

Aedes (Ae.) aegypti and Ae. albopictus mosquitoes transmit arthropod-borne diseases around the globe, causing ~ 700,000 deaths each year. Genetic mutants are valuable tools to interrogate both fundamental vector biology and mosquito host factors important for viral infection. However, very few genetic mutants have been described in mosquitoes in comparison to model organisms. The relative ease of applying CRISPR/Cas9-based gene editing has transformed genome engineering and has rapidly increased the number of available gene mutants in mosquitoes. Yet, in vivo studies may not be practical for screening large sets of mutants or possible for laboratories that lack insectaries. Thus, it would be useful to adapt CRISPR/Cas9 systems to common mosquito cell lines. In this study, we generated and characterized a mosquito optimized, plasmid-based CRISPR/Cas9 system for use in U4.4 (Ae. albopictus) and Aag2 (Ae. aegypti) cell lines. We demonstrated highly efficient editing of the AGO1 locus and isolated U4.4 and Aag2 cell lines with reduced AGO1 expression. Further, we used homology-directed repair to establish knock-in Aag2 cell lines with a 3xFLAG-tag at the N-terminus of endogenous AGO1. These experimentally verified plasmids are versatile, cost-effective, and efficiently edit immune competent mosquito cell lines that are widely used in arbovirus studies.

7.
Cell Syst ; 12(3): 210-219.e3, 2021 Mar 17.
Article in English | MEDLINE | ID: mdl-33515490

ABSTRACT

While decades of research have elucidated many steps of the alphavirus lifecycle, the earliest replication dynamics have remained unclear. This missing time window has obscured early replicase strand-synthesis behavior and prevented elucidation of how the first events of infection might influence subsequent viral competition. Using quantitative live-cell and single-molecule imaging, we observed the initial replicase activity and its strand preferences in situ and measured the trajectory of replication over time. Under this quantitative framework, we investigated viral competition, where one alphavirus is able to exclude superinfection by a second homologous virus. We show that this appears as an indirect phenotypic consequence of a bidirectional competition between the two species, coupled with the rapid onset of viral replication and a limited total cellular carrying capacity. Together, these results emphasize the utility of analyzing viral kinetics within single cells.

8.
Proc Natl Acad Sci U S A ; 118(1)2021 01 05.
Article in English | MEDLINE | ID: mdl-33443154

ABSTRACT

The journey from plasma membrane to nuclear pore is a critical step in the lifecycle of DNA viruses, many of which must successfully deposit their genomes into the nucleus for replication. Viral capsids navigate this vast distance through the coordinated hijacking of a number of cellular host factors, many of which remain unknown. We performed a gene-trap screen in haploid cells to identify host factors for adenovirus (AdV), a DNA virus that can cause severe respiratory illness in immune-compromised individuals. This work identified Mindbomb 1 (MIB1), an E3 ubiquitin ligase involved in neurodevelopment, as critical for AdV infectivity. In the absence of MIB1, we observed that viral capsids successfully traffic to the proximity of the nucleus but ultimately fail to deposit their genomes within. The capacity of MIB1 to promote AdV infection was dependent on its ubiquitination activity, suggesting that MIB1 may mediate proteasomal degradation of one or more negative regulators of AdV infection. Employing complementary proteomic approaches to characterize proteins proximal to MIB1 upon AdV infection and differentially ubiquitinated in the presence or absence of MIB1, we observed an intersection between MIB1 and ribonucleoproteins (RNPs) largely unexplored in mammalian cells. This work uncovers yet another way that viruses utilize host cell machinery for their own replication, highlighting a potential target for therapeutic interventions that counter AdV infection.

9.
Cell ; 184(1): 120-132.e14, 2021 01 07.
Article in English | MEDLINE | ID: mdl-33382968

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic has claimed the lives of over one million people worldwide. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a member of the Coronaviridae family of viruses that can cause respiratory infections of varying severity. The cellular host factors and pathways co-opted during SARS-CoV-2 and related coronavirus life cycles remain ill defined. To address this gap, we performed genome-scale CRISPR knockout screens during infection by SARS-CoV-2 and three seasonal coronaviruses (HCoV-OC43, HCoV-NL63, and HCoV-229E). These screens uncovered host factors and pathways with pan-coronavirus and virus-specific functional roles, including major dependency on glycosaminoglycan biosynthesis, sterol regulatory element-binding protein (SREBP) signaling, bone morphogenetic protein (BMP) signaling, and glycosylphosphatidylinositol biosynthesis, as well as a requirement for several poorly characterized proteins. We identified an absolute requirement for the VMP1, TMEM41, and TMEM64 (VTT) domain-containing protein transmembrane protein 41B (TMEM41B) for infection by SARS-CoV-2 and three seasonal coronaviruses. This human coronavirus host factor compendium represents a rich resource to develop new therapeutic strategies for acute COVID-19 and potential future coronavirus pandemics.


Subject(s)
Coronavirus Infections/genetics , Genome-Wide Association Study , /physiology , A549 Cells , Cell Line , Clustered Regularly Interspaced Short Palindromic Repeats , Coronavirus 229E, Human/physiology , Coronavirus Infections/virology , Coronavirus NL63, Human/physiology , Coronavirus OC43, Human/physiology , Gene Knockout Techniques , HEK293 Cells , Host-Pathogen Interactions/drug effects , Humans , Membrane Proteins/metabolism , Metabolic Networks and Pathways/drug effects , Protein Interaction Mapping
10.
Cell ; 184(1): 133-148.e20, 2021 01 07.
Article in English | MEDLINE | ID: mdl-33338421

ABSTRACT

Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection, we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results, we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms present at nearly 20% in East Asian populations reduce flavivirus infection. Based on our mechanistic studies, we propose that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication.


Subject(s)
Flavivirus Infections/genetics , Flavivirus/physiology , Membrane Proteins/metabolism , Animals , Asian Continental Ancestry Group/genetics , Autophagy , /metabolism , CRISPR-Cas Systems , Cell Line , Flavivirus Infections/immunology , Flavivirus Infections/metabolism , Flavivirus Infections/virology , Gene Knockout Techniques , Genome-Wide Association Study , Host-Pathogen Interactions , Humans , Immunity, Innate , Membrane Proteins/genetics , Polymorphism, Single Nucleotide , Virus Replication , Yellow fever virus/physiology , Zika Virus/physiology
11.
Cell Host Microbe ; 29(2): 267-280.e5, 2021 02 10.
Article in English | MEDLINE | ID: mdl-33357464

ABSTRACT

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has devastated the global economy and claimed more than 1.7 million lives, presenting an urgent global health crisis. To identify host factors required for infection by SARS-CoV-2 and seasonal coronaviruses, we designed a focused high-coverage CRISPR-Cas9 library targeting 332 members of a recently published SARS-CoV-2 protein interactome. We leveraged the compact nature of this library to systematically screen SARS-CoV-2 at two physiologically relevant temperatures along with three related coronaviruses (human coronavirus 229E [HCoV-229E], HCoV-NL63, and HCoV-OC43), allowing us to probe this interactome at a much higher resolution than genome-scale studies. This approach yielded several insights, including potential virus-specific differences in Rab GTPase requirements and glycosylphosphatidylinositol (GPI) anchor biosynthesis, as well as identification of multiple pan-coronavirus factors involved in cholesterol homeostasis. This coronavirus essentiality catalog could inform ongoing drug development efforts aimed at intercepting and treating coronavirus disease 2019 (COVID-19) and help prepare for future coronavirus outbreaks.


Subject(s)
/virology , /metabolism , CRISPR-Cas Systems , Coronavirus 229E, Human/genetics , Coronavirus 229E, Human/metabolism , Coronavirus NL63, Human/genetics , Coronavirus NL63, Human/metabolism , Coronavirus OC43, Human , Genes, Viral , Host-Pathogen Interactions , Humans , Viral Proteins/genetics , Viral Proteins/metabolism
12.
Sci Transl Med ; 2020 Dec 07.
Article in English | MEDLINE | ID: mdl-33288661

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19), primarily infects cells at mucosal surfaces. Serum neutralizing antibody responses are variable and generally low in individuals that suffer mild forms of COVID-19. Although potent IgG antibodies can neutralize the virus, less is known about secretory antibodies such as IgA that might impact the initial viral spread and transmissibility from the mucosa. Here we characterize the IgA response to SARS-CoV-2 in a cohort of 149 convalescent individuals following diagnosis with COVID-19. IgA responses in plasma generally correlated with IgG responses. Further, clones of IgM-, IgG-, and IgA-producing B cells were derived from common progenitor cells. Plasma IgA monomers specific to SARS-CoV-2 proteins were demonstrated to be two-fold less potent than IgG equivalents. However, IgA dimers, the primary form of antibody in the nasopharynx, were on average fifteen times more potent than IgA monomers against the same target. Thus, dimeric IgA responses may be particularly valuable for protection against SARS-CoV-2 and for vaccine efficacy.

13.
Sci Adv ; 6(45)2020 Nov.
Article in English | MEDLINE | ID: mdl-33148654

ABSTRACT

Hepatitis C virus (HCV) has no animal reservoir, infecting only humans. To investigate species barrier determinants limiting infection of rodents, murine liver complementary DNA library screening was performed, identifying transmembrane proteins Cd302 and Cr1l as potent restrictors of HCV propagation. Combined ectopic expression in human hepatoma cells impeded HCV uptake and cooperatively mediated transcriptional dysregulation of a noncanonical program of immunity genes. Murine hepatocyte expression of both factors was constitutive and not interferon inducible, while differences in liver expression and the ability to restrict HCV were observed between the murine orthologs and their human counterparts. Genetic ablation of endogenous Cd302 expression in human HCV entry factor transgenic mice increased hepatocyte permissiveness for an adapted HCV strain and dysregulated expression of metabolic process and host defense genes. These findings highlight human-mouse differences in liver-intrinsic antiviral immunity and facilitate the development of next-generation murine models for preclinical testing of HCV vaccine candidates.

14.
bioRxiv ; 2020 Oct 02.
Article in English | MEDLINE | ID: mdl-33024965

ABSTRACT

T cell-mediated immunity may play a critical role in controlling and establishing protective immunity against SARS-CoV-2 infection; yet the repertoire of viral epitopes responsible for T cell response activation remains mostly unknown. Identification of viral peptides presented on class I human leukocyte antigen (HLA-I) can reveal epitopes for recognition by cytotoxic T cells and potential incorporation into vaccines. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two human cell lines at different times post-infection using mass spectrometry. We found HLA-I peptides derived not only from canonical ORFs, but also from internal out-of-frame ORFs in Spike and Nucleoprotein not captured by current vaccines. Proteomics analyses of infected cells revealed that SARS-CoV-2 may interfere with antigen processing and immune signaling pathways. Based on the endogenously processed and presented viral peptides that we identified, we estimate that a pool of 24 peptides would provide one or more peptides for presentation by at least one HLA allele in 99% of the human population. These biological insights and the list of naturally presented SARS-CoV-2 peptides will facilitate data-driven selection of peptides for immune monitoring and vaccine development.

15.
bioRxiv ; 2020 Oct 08.
Article in English | MEDLINE | ID: mdl-33052332

ABSTRACT

The COVID-19 pandemic has claimed the lives of more than one million people worldwide. The causative agent, SARS-CoV-2, is a member of the Coronaviridae family, which are viruses that cause respiratory infections of varying severity. The cellular host factors and pathways co-opted by SARS-CoV-2 and other coronaviruses in the execution of their life cycles remain ill-defined. To develop an extensive compendium of host factors required for infection by SARS-CoV-2 and three seasonal coronaviruses (HCoV-OC43, HCoV-NL63, and HCoV-229E), we performed parallel genome-scale CRISPR knockout screens. These screens uncovered multiple host factors and pathways with pan-coronavirus and virus-specific functional roles, including major dependency on glycosaminoglycan biosynthesis, SREBP signaling, and glycosylphosphatidylinositol biosynthesis, as well as an unexpected requirement for several poorly characterized proteins. We identified an absolute requirement for the VTT-domain containing protein TMEM41B for infection by SARS-CoV-2 and all other coronaviruses. This human Coronaviridae host factor compendium represents a rich resource to develop new therapeutic strategies for acute COVID-19 and potential future coronavirus spillover events. HIGHLIGHTS: Genome-wide CRISPR screens for SARS-CoV-2, HCoV-OC43, HCoV-NL63, and HCoV-229E coronavirus host factors.Parallel genome-wide CRISPR screening uncovered host factors and pathways with pan-coronavirus and virus-specific functional roles.Coronaviruses co-opt multiple biological pathways, including glycosaminoglycan biosynthesis, SREBP signaling, and glycosylphosphatidylinositol biosynthesis and anchoring, among others.TMEM41B - a poorly understood factor with roles in autophagy and lipid mobilization - is a critical pan-coronavirus host factor.

16.
bioRxiv ; 2020 Oct 11.
Article in English | MEDLINE | ID: mdl-33052348

ABSTRACT

Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms (SNPs) present at nearly twenty percent in East Asian populations reduce flavivirus infection. Based on our mechanistic studies we hypothesize that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication. HIGHLIGHTS: TMEM41B and VMP1 are required for both autophagy and flavivirus infection, however, autophagy is not required for flavivirus infection.TMEM41B associates with viral proteins and likely facilitates membrane remodeling to establish viral RNA replication complexes.TMEM41B single nucleotide polymorphisms (SNPs) present at nearly twenty percent in East Asian populations reduce flavivirus infection.TMEM41B-deficient cells display an exaggerated innate immune response upon high multiplicity flavivirus infection.

17.
Elife ; 92020 10 28.
Article in English | MEDLINE | ID: mdl-33112236

ABSTRACT

Neutralizing antibodies elicited by prior infection or vaccination are likely to be key for future protection of individuals and populations against SARS-CoV-2. Moreover, passively administered antibodies are among the most promising therapeutic and prophylactic anti-SARS-CoV-2 agents. However, the degree to which SARS-CoV-2 will adapt to evade neutralizing antibodies is unclear. Using a recombinant chimeric VSV/SARS-CoV-2 reporter virus, we show that functional SARS-CoV-2 S protein variants with mutations in the receptor-binding domain (RBD) and N-terminal domain that confer resistance to monoclonal antibodies or convalescent plasma can be readily selected. Notably, SARS-CoV-2 S variants that resist commonly elicited neutralizing antibodies are now present at low frequencies in circulating SARS-CoV-2 populations. Finally, the emergence of antibody-resistant SARS-CoV-2 variants that might limit the therapeutic usefulness of monoclonal antibodies can be mitigated by the use of antibody combinations that target distinct neutralizing epitopes.

18.
J Infect ; 81(5): 766-775, 2020 Nov.
Article in English | MEDLINE | ID: mdl-32987099

ABSTRACT

OBJECTIVES: Screening for genes differentially expressed in placental tissues, aiming to identify transcriptional signatures that may be involved in ZIKV congenital pathogenesis. METHODS: Transcriptome data from placental tissues of pregnant women naturally infected with Zika virus during the third trimester were compared to those from women who tested negative for Zika infection. The findings were validated using both a cell culture model and an immunohistochemistry/morphological analysis of naturally infected placental tissues. RESULTS: Transcriptome analysis revealed that Zika virus infection induces downregulation of insulin-like growth factor II (IGF2) gene, an essential factor for fetal development. The Caco-2 cell culture model that constitutively expresses IGF2 was used for the transcriptome validation. Asiatic and African Zika virus strains infection caused downregulated IGF2 gene expression in Caco-2 cells, whereas other flaviviruses, such as dengue serotype 1, West Nile and wild-type yellow fever viruses, had no effect on this gene expression. Immunohistochemical assays on decidual tissues corroborated our transcriptome analysis, showing that IGF2 is reduced in the decidua of Zika virus-infected women. CONCLUSIONS: Our results draw attention to IGF2 modulation in uterine tissues, and this finding is expected to support future studies on strategies to ameliorate the harmful effects of Zika virus infection during pregnancy.

19.
Antiviral Res ; 183: 104939, 2020 11.
Article in English | MEDLINE | ID: mdl-32980446

ABSTRACT

Yellow fever virus (YFV), a member of the Flaviviridae family, is an arthropod-borne virus that can cause severe disease in humans with a lethality rate of up to 60%. Since 2017, increases in YFV activity in areas of South America and Africa have been described. Although a vaccine is available, named strain 17D (Theiler and Smith, 1937), it is contraindicated for use in the elderly, expectant mothers, immunocompromised people, among others. To this day there is no antiviral treatment against YFV to reduce the severity of viral infection. Here, we used a circular polymerase extension reaction (CPER)-based reverse genetics approach to generate a full-length reporter virus (YFVhb) by introducing a small HiBit tag in the NS1 protein. The reporter virus replicates at a similar rate to the parental YFV in HuH-7 cells. Using YFVhb, we designed a high throughput antiviral screening luciferase-based assay to identify inhibitors that target any step of the viral replication cycle. We validated our assay by using a range of inhibitors including drugs, immune sera and neutralizing single chain variable fragments (scFv). In light of the recent upsurge in YFV and a potential spread of the virus, this assay is a further tool in the development of antiviral therapy against YFV.

20.
PLoS Pathog ; 16(9): e1008927, 2020 09.
Article in English | MEDLINE | ID: mdl-32997711

ABSTRACT

Viruses cleave cellular proteins to remodel the host proteome. The study of these cleavages has revealed mechanisms of immune evasion, resource exploitation, and pathogenesis. However, the full extent of virus-induced proteolysis in infected cells is unknown, mainly because until recently the technology for a global view of proteolysis within cells was lacking. Here, we report the first comprehensive catalog of proteins cleaved upon enterovirus infection and identify the sites within proteins where the cleavages occur. We employed multiple strategies to confirm protein cleavages and assigned them to one of the two enteroviral proteases. Detailed characterization of one substrate, LSM14A, a p body protein with a role in antiviral immunity, showed that cleavage of this protein disrupts its antiviral function. This study yields a new depth of information about the host interface with a group of viruses that are both important biological tools and significant agents of disease.


Subject(s)
Cysteine Endopeptidases/metabolism , Enterovirus Infections/virology , Enterovirus/pathogenicity , Virus Replication/physiology , Antiviral Agents/metabolism , Enterovirus/metabolism , Host-Pathogen Interactions/physiology , Humans , Proteolysis , Viral Proteins/metabolism
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