RESUMO
The multi-step process of hepatitis C virus (HCV) entry is facilitated by various host factors, including epidermal growth factor receptor (EGFR) and the tight junction proteins claudin-1 (CLDN1) and occludin (OCLN), which are thought to function at later stages of the HCV entry process. Using single particle imaging of HCV infection of polarized hepatoma spheroids, we observed that EGFR performs multiple functions in HCV entry, both phosphorylation-dependent and -independent. We previously observed, and in this study confirmed, that EGFR is not required for HCV migration to the tight junction. EGFR is required for the recruitment of clathrin to HCV in a phosphorylation-independent manner. EGFR phosphorylation is required for virion internalization at a stage following the recruitment of clathrin. HCV entry activates the RAF-MEK-ERK signaling pathway downstream of EGFR phosphorylation. This signaling pathway regulates the sorting and maturation of internalized HCV into APPL1- and EEA1-associated early endosomes, which form the site of virion uncoating. The tight junction proteins, CLDN1 and OCLN, function at two distinct stages of HCV entry. Despite its appreciated function as a "late receptor" in HCV entry, CLDN1 is required for efficient HCV virion accumulation at the tight junction. Huh-7.5 cells lacking CLDN1 accumulate HCV virions primarily at the initial basolateral surface. OCLN is required for the late stages of virion internalization. This study produced further insight into the unusually complex HCV endocytic process.
Assuntos
Claudina-1 , Hepacivirus , Hepatite C , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Clatrina , Claudina-1/genética , Claudina-1/metabolismo , Receptores ErbB , Hepacivirus/fisiologia , Hepatite C/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Ocludina/metabolismo , Internalização do VírusRESUMO
The signaling pathways and cellular functions regulated by the four Numb-associated kinases are largely unknown. We reported that AAK1 and GAK control intracellular trafficking of RNA viruses and revealed a requirement for BIKE in early and late stages of dengue virus (DENV) infection. However, the downstream targets phosphorylated by BIKE have not yet been identified. Here, to identify BIKE substrates, we conducted a barcode fusion genetics-yeast two-hybrid screen and retrieved publicly available data generated via affinity-purification mass spectrometry. We subsequently validated 19 of 47 putative BIKE interactors using mammalian cell-based protein-protein interaction assays. We found that CLINT1, a cargo-specific adapter implicated in bidirectional Golgi-to-endosome trafficking, emerged as a predominant hit in both screens. Our experiments indicated that BIKE catalyzes phosphorylation of a threonine 294 CLINT1 residue both in vitro and in cell culture. Our findings revealed that CLINT1 phosphorylation mediates its binding to the DENV nonstructural 3 protein and subsequently promotes DENV assembly and egress. Additionally, using live-cell imaging we revealed that CLINT1 cotraffics with DENV particles and is involved in mediating BIKE's role in DENV infection. Finally, our data suggest that additional cellular BIKE interactors implicated in the host immune and stress responses and the ubiquitin proteasome system might also be candidate phosphorylation substrates of BIKE. In conclusion, these findings reveal cellular substrates and pathways regulated by the understudied Numb-associated kinase enzyme BIKE, a mechanism for CLINT1 regulation, and control of DENV infection via BIKE signaling, with potential implications for cell biology, virology, and host-targeted antiviral design.
Assuntos
Vírus da Dengue , Dengue , Animais , Dengue/metabolismo , Vírus da Dengue/metabolismo , Humanos , Fosforilação , Técnicas do Sistema de Duplo-Híbrido , Replicação ViralRESUMO
BACKGROUND: Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) uses full-length angiotensin converting enzyme 2 (ACE2) as a main receptor to enter target cells. The goal of this study was to demonstrate the preclinical efficacy of a novel soluble ACE2 protein with increased duration of action and binding capacity in a lethal mouse model of COVID-19. METHODS: A human soluble ACE2 variant fused with an albumin binding domain (ABD) was linked via a dimerization motif hinge-like 4-cysteine dodecapeptide (DDC) to improve binding capacity to SARS-CoV-2. This novel soluble ACE2 protein (ACE2-1-618-DDC-ABD) was then administered intranasally and intraperitoneally to mice before intranasal inoculation of SARS-CoV-2 and then for two additional days post viral inoculation. RESULTS: Untreated animals became severely ill, and all had to be humanely euthanized by day 6 or 7 and had pulmonary alveolar hemorrhage with mononuclear infiltrates. In contrast, all but one mouse infected with a lethal dose of SARS-CoV-2 that received ACE2-1-618-DDC-ABD survived. In the animals inoculated with SARS-CoV-2 that were untreated, viral titers were high in the lungs and brain, but viral titers were absent in the kidneys. Some untreated animals, however, had variable degrees of kidney proximal tubular injury as shown by attenuation of the proximal tubular brush border and increased NGAL and TUNEL staining. Viral titers in the lung and brain were reduced or nondetectable in mice that received ACE2-1-618-DDC-ABD, and the animals developed only moderate disease as assessed by a near-normal clinical score, minimal weight loss, and improved lung and kidney injury. CONCLUSIONS: This study demonstrates the preclinical efficacy of a novel soluble ACE2 protein, termed ACE2-1-618-DDC-ABD, in a lethal mouse model of SARS-CoV-2 infection that develops severe lung injury and variable degrees of moderate kidney proximal tubular injury.
Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , Enzima de Conversão de Angiotensina 2/uso terapêutico , Animais , COVID-19/terapia , Rim/virologia , Pulmão/virologia , Camundongos , SARS-CoV-2RESUMO
BACKGROUND: There is an urgent need for approaches to prevent and treat SARS-CoV-2 infection. Administration of soluble ACE2 protein acting as a decoy to bind to SARS-CoV-2 should limit viral uptake mediated by binding to membrane-bound full-length ACE2, and further therapeutic benefit should result from ensuring enzymatic ACE2 activity to affected organs in patients with COVID-19. METHODS: A short variant of human soluble ACE2 protein consisting of 618 amino acids (hACE2 1-618) was generated and fused with an albumin binding domain (ABD) using an artificial gene encoding ABDCon, with improved albumin binding affinity. Human kidney organoids were used for infectivity studies of SARS-CoV-2 in a BSL-3 facility to examine the neutralizing effect of these novel ACE2 variants. RESULTS: Whereas plasma ACE2 activity of the naked ACE2 1-618 and ACE2 1-740 lasted about 8 hours, the ACE2 1-618-ABD resulted in substantial activity at 96 hours, and it was still biologically active 3 days after injection. Human kidney organoids express ACE2 and TMPRSS2, and when infected with SARS-CoV-2, our modified long-acting ACE2 variant neutralized infection. CONCLUSIONS: This novel ACE2 1-618-ABD can neutralize SARS-CoV-2 infectivity in human kidney organoids, and its prolonged duration of action should ensure improved efficacy to prevent viral escape and dosing convenience.
RESUMO
Human norovirus is the leading cause of gastroenteritis worldwide, yet basic questions about its life cycle remain unanswered due to an historical lack of robust experimental systems. Recent studies on the closely related murine norovirus (MNV) have identified CD300LF as an indispensable entry factor for MNV. We compared the MNV susceptibilities of cells from different mouse strains and identified polymorphisms in murine CD300LF which are critical for its function as an MNV receptor. Bone marrow-derived macrophages (BMDMs) from I/LnJ mice were resistant to infection from multiple MNV strains which readily infect BMDMs from C57BL/6J mice. The resistance of I/LnJ BMDMs was specific to MNV, since the cells supported infection of other viruses comparably to C57BL/6J BMDMs. Transduction of I/LnJ BMDMs with C57BL/6J CD300LF made the cells permissible to MNV infection, suggesting that the cause of resistance lies in the entry step of MNV infection. In fact, we mapped this phenotype to a 4-amino-acid difference at the CC' loop of CD300LF; swapping of these amino acids between C57BL/6J and I/LnJ CD300LF proteins made the mutant C57BL/6J CD300LF functionally impaired and the corresponding mutant of I/LnJ CD300LF functional as an MNV entry factor. Surprisingly, expression of the I/LnJ CD300LF in other cell types made the cells infectible by MNV, even though the I/LnJ allele did not function as an MNV receptor in macrophage-like cells. Correspondingly, I/LnJ CD300LF bound MNV virions in permissive cells but not in nonpermissive cells. Collectively, our data suggest the existence of a cell type-specific modifier of MNV entry.IMPORTANCE MNV is a prevalent model system for studying human norovirus, which is the leading cause of gastroenteritis worldwide and thus a sizeable public health burden. Elucidating mechanisms underlying susceptibility of host cells to MNV infection can lead to insights on the roles that specific cell types play during norovirus pathogenesis. Here, we show that different alleles of the proteinaceous receptor for MNV, CD300LF, function in a cell type-dependent manner. In contrast to the C57BL/6J allele, which functions as an MNV entry factor in all tested cell types, including human cells, I/LnJ CD300LF does not function as an MNV entry factor in macrophage-like cells but does allow MNV entry in other cell types. Together, these observations indicate the existence of cell type-specific modifiers of CD300LF-dependent MNV entry.
Assuntos
Infecções por Caliciviridae/virologia , Resistência à Doença/genética , Polimorfismo Genético , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Animais , Sítios de Ligação , Gastroenterite/virologia , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Modelos Moleculares , Norovirus , Conformação Proteica , Receptores Imunológicos/química , Análise de Sequência de Proteína , Internalização do VírusRESUMO
Seventy percent of people infected with hepatitis C virus (HCV) will suffer chronic infection, putting them at risk for liver disease, including hepatocellular carcinoma. The full range of mechanisms that render some people more susceptible to chronic infection and liver disease is still being elucidated. XRN exonucleases can restrict HCV replication and may help to resolve HCV infections. However, it is unknown how 5' triphosphorylated HCV transcripts, primary products of the viral polymerase, become susceptible to attack by 5' monophosphate-specific XRNs. Here, we show that the 5' RNA triphosphatase DUSP11 acts on HCV transcripts, rendering them susceptible to XRN-mediated attack. Cells lacking DUSP11 show substantially enhanced HCV replication, and this effect is diminished when XRN expression is reduced. MicroRNA-122 (miR-122), a target of current phase II anti-HCV drugs, is known to protect HCV transcripts against XRNs. We show that HCV replication is less dependent on miR-122 in cells lacking DUSP11. Combined, these results implicate DUSP11 as an important component of XRN-mediated restriction of HCV.
Assuntos
Hidrolases Anidrido Ácido/metabolismo , Fosfatases de Especificidade Dupla/metabolismo , Exorribonucleases/metabolismo , Hepacivirus/patogenicidade , Interações Hospedeiro-Patógeno/fisiologia , MicroRNAs/metabolismo , Hidrolases Anidrido Ácido/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Fosfatases de Especificidade Dupla/genética , Exorribonucleases/genética , Técnicas de Inativação de Genes , Genoma Viral , Hepacivirus/fisiologia , Hepatite C Crônica/genética , Hepatite C Crônica/virologia , Hepatócitos/virologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virologia , RNA Interferente Pequeno/metabolismo , RNA Viral/metabolismo , Replicação Viral/genéticaRESUMO
All positive-strand RNA viruses reorganize host intracellular membranes to assemble their viral replication complexes (VRCs); however, how these viruses modulate host lipid metabolism to accommodate such membrane proliferation and rearrangements is not well defined. We show that a significantly increased phosphatidylcholine (PC) content is associated with brome mosaic virus (BMV) replication in both natural host barley and alternate host yeast based on a lipidomic analysis. Enhanced PC levels are primarily associated with the perinuclear ER membrane, where BMV replication takes place. More specifically, BMV replication protein 1a interacts with and recruits Cho2p (choline requiring 2), a host enzyme involved in PC synthesis, to the site of viral replication. These results suggest that PC synthesized at the site of VRC assembly, not the transport of existing PC, is responsible for the enhanced accumulation. Blocking PC synthesis by deleting the CHO2 gene resulted in VRCs with wider diameters than those in wild-type cells; however, BMV replication was significantly inhibited, highlighting the critical role of PC in VRC formation and viral replication. We further show that enhanced PC levels also accumulate at the replication sites of hepatitis C virus and poliovirus, revealing a conserved feature among a group of positive-strand RNA viruses. Our work also highlights a potential broad-spectrum antiviral strategy that would disrupt PC synthesis at the sites of viral replication but would not alter cellular processes.
Assuntos
Bromovirus/fisiologia , Hordeum/metabolismo , Fosfatidilcolinas/biossíntese , Doenças das Plantas/virologia , Replicação Viral/fisiologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Hordeum/genética , Hordeum/virologia , Membranas Intracelulares/metabolismo , Membranas Intracelulares/virologia , Fosfatidilcolinas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Robust dengue virus (DENV) replication requires lipophagy, a selective autophagy that targets lipid droplets. The autophagic mobilization of lipids leads to increased ß-oxidation in DENV-infected cells. The mechanism by which DENV induces lipophagy is unknown. Here, we show that infection with DENV activates the metabolic regulator 5' adenosine-monophosphate activated kinase (AMPK), and that the silencing or pharmacological inhibition of AMPK activity decreases DENV replication and the induction of lipophagy. The activity of the mechanistic target of rapamycin complex 1 (mTORC1) decreases in DENV-infected cells and is inversely correlated with lipophagy induction. Constitutive activation of mTORC1 by depletion of tuberous sclerosis complex 2 (TSC2) inhibits lipophagy induction in DENV-infected cells and decreases viral replication. While AMPK normally stimulates TSC2-dependent inactivation of mTORC1 signaling, mTORC1 inactivation is independent of AMPK activation during DENV infection. Thus, DENV stimulates and requires AMPK signaling as well as AMPK-independent suppression of mTORC1 activity for proviral lipophagy.IMPORTANCE Dengue virus alters host cell lipid metabolism to promote its infection. One mechanism for altered metabolism is the induction of a selective autophagy that targets lipid droplets, termed lipophagy. Lipophagy mobilizes lipid stores, resulting in enhanced ß-oxidation and viral replication. We show here that DENV infection activates and requires the central metabolic regulator AMPK for its replication and the induction of lipophagy. This is required for the induction of lipophagy, but not basal autophagy, in DENV-infected cells.
Assuntos
Adenilato Quinase/metabolismo , Autofagia , Vírus da Dengue/fisiologia , Metabolismo dos Lipídeos , Complexos Multiproteicos/metabolismo , Provírus/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Adenilato Quinase/genética , Replicação do DNA , Regulação Viral da Expressão Gênica , Células HEK293 , Células Hep G2 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Vírion/metabolismo , Replicação ViralRESUMO
Hepatitis C virus (HCV) entry, translation, replication, and assembly occur with defined kinetics in distinct subcellular compartments. It is unclear how HCV spatially and temporally regulates these events within the host cell to coordinate its infection. We have developed a single molecule RNA detection assay that facilitates the simultaneous visualization of HCV (+) and (-) RNA strands at the single cell level using high-resolution confocal microscopy. We detect (+) strand RNAs as early as 2 hours post-infection and (-) strand RNAs as early as 4 hours post-infection. Single cell levels of (+) and (-) RNA vary considerably with an average (+):(-) RNA ratio of 10 and a range from 1-35. We next developed microscopic assays to identify HCV (+) and (-) RNAs associated with actively translating ribosomes, replication, virion assembly and intracellular virions. (+) RNAs display a defined temporal kinetics, with the majority of (+) RNAs associated with actively translating ribosomes at early times of infection, followed by a shift to replication and then virion assembly. (-) RNAs have a strong colocalization with NS5A, but not NS3, at early time points that correlate with replication compartment formation. At later times, only ~30% of the replication complexes appear to be active at a given time, as defined by (-) strand colocalization with either (+) RNA, NS3, or NS5A. While both (+) and (-) RNAs colocalize with the viral proteins NS3 and NS5A, only the plus strand preferentially colocalizes with the viral envelope E2 protein. These results suggest a defined spatiotemporal regulation of HCV infection with highly varied replication efficiencies at the single cell level. This approach can be applicable to all plus strand RNA viruses and enables unprecedented sensitivity for studying early events in the viral life cycle.
Assuntos
Hepacivirus/metabolismo , Hepatite C/metabolismo , RNA Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Vírion/metabolismo , Linhagem Celular , Hepatite C/patologia , HumanosRESUMO
UNLABELLED: To explore mechanisms of hepatitis C viral (HCV) replication we screened a compound library including licensed drugs. Flunarizine, a diphenylmethylpiperazine used to treat migraine, inhibited HCV cell entry in vitro and in vivo in a genotype-dependent fashion. Analysis of mosaic viruses between susceptible and resistant strains revealed that E1 and E2 glycoproteins confer susceptibility to flunarizine. Time of addition experiments and single particle tracking of HCV demonstrated that flunarizine specifically prevents membrane fusion. Related phenothiazines and pimozide also inhibited HCV infection and preferentially targeted HCV genotype 2 viruses. However, phenothiazines and pimozide exhibited improved genotype coverage including the difficult to treat genotype 3. Flunarizine-resistant HCV carried mutations within the alleged fusion peptide and displayed cross-resistance to these compounds, indicating that these drugs have a common mode of action. CONCLUSION: These observations reveal novel details about HCV membrane fusion; moreover, flunarizine and related compounds represent first-in-class HCV fusion inhibitors that merit consideration for repurposing as a cost-effective component of HCV combination therapies.
Assuntos
Flunarizina/farmacologia , Hepacivirus/efeitos dos fármacos , Proteínas Virais de Fusão/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Células Cultivadas , Genótipo , Hepacivirus/genética , Humanos , Proteínas Virais de Fusão/genéticaRESUMO
The hepatitis C virus (HCV) genome contains numerous RNA elements that are required for its replication. Most of the identified RNA structures are located within the 5' and 3' untranslated regions (UTRs). One prominent RNA structure, termed the cis-acting replication element (CRE), is located within the NS5B coding region. Mutation of part of the CRE, the 5BSL3.2 stem-loop, impairs HCV RNA replication. This loop has been implicated in a kissing interaction with a complementary stem-loop structure in the 3' UTR. Although it is clear that this interaction is required for viral replication, the function of the interaction, and its regulation are unknown. In order to gain insight into the CRE function, we isolated cellular proteins that preferentially bind the CRE and identified them using mass spectrometry. This approach identified EWSR1 as a CRE-binding protein. Silencing EWSR1 expression impairs HCV replication and infectious virus production but not translation. While EWRS1 is a shuttling protein that is extensively nuclear in hepatocytes, substantial amounts of EWSR1 localize to the cytosol in HCV-infected cells and colocalize with sites of HCV replication. A subset of EWRS1 translocates into detergent-resistant membrane fractions, which contain the viral replicase proteins, in cells with replicating HCV. EWSR1 directly binds the CRE, and this is dependent on the intact CRE structure. Finally, EWSR1 preferentially interacts with the CRE in the absence of the kissing interaction. This study implicates EWSR1 as a novel modulator of CRE function in HCV replication.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Elementos Facilitadores Genéticos/fisiologia , Regulação Viral da Expressão Gênica , Hepacivirus/fisiologia , Proteínas de Ligação a RNA/metabolismo , Proteínas não Estruturais Virais/genética , Replicação Viral/genética , Proteínas de Ligação a Calmodulina/genética , Linhagem Celular Tumoral , Células HEK293 , Hepacivirus/genética , Hepacivirus/metabolismo , Humanos , RNA Viral/genética , RNA Viral/metabolismo , Proteína EWS de Ligação a RNA , Proteínas de Ligação a RNA/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
The current model of hepatitis C virus (HCV) production involves the assembly of virions on or near the surface of lipid droplets, envelopment at the ER in association with components of VLDL synthesis, and egress via the secretory pathway. However, the cellular requirements for and a mechanistic understanding of HCV secretion are incomplete at best. We combined an RNA interference (RNAi) analysis of host factors for infectious HCV secretion with the development of live cell imaging of HCV core trafficking to gain a detailed understanding of HCV egress. RNAi studies identified multiple components of the secretory pathway, including ER to Golgi trafficking, lipid and protein kinases that regulate budding from the trans-Golgi network (TGN), VAMP1 vesicles and adaptor proteins, and the recycling endosome. Our results support a model wherein HCV is infectious upon envelopment at the ER and exits the cell via the secretory pathway. We next constructed infectious HCV with a tetracysteine (TC) tag insertion in core (TC-core) to monitor the dynamics of HCV core trafficking in association with its cellular cofactors. In order to isolate core protein movements associated with infectious HCV secretion, only trafficking events that required the essential HCV assembly factor NS2 were quantified. TC-core traffics to the cell periphery along microtubules and this movement can be inhibited by nocodazole. Sub-populations of TC-core localize to the Golgi and co-traffic with components of the recycling endosome. Silencing of the recycling endosome component Rab11a results in the accumulation of HCV core at the Golgi. The majority of dynamic core traffics in association with apolipoprotein E (ApoE) and VAMP1 vesicles. This study identifies many new host cofactors of HCV egress, while presenting dynamic studies of HCV core trafficking in infected cells.
Assuntos
Apolipoproteínas E , Retículo Endoplasmático , Complexo de Golgi , Hepacivirus/fisiologia , Vesículas Secretórias , Proteína 1 Associada à Membrana da Vesícula , Liberação de Vírus/fisiologia , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Transporte Biológico Ativo/genética , Linhagem Celular , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Complexo de Golgi/virologia , Humanos , Vesículas Secretórias/genética , Vesículas Secretórias/metabolismo , Vesículas Secretórias/virologia , Proteína 1 Associada à Membrana da Vesícula/genética , Proteína 1 Associada à Membrana da Vesícula/metabolismo , Montagem de Vírus/fisiologiaRESUMO
Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) utilizes angiotensin-converting enzyme 2 (ACE2) as its main receptor for cell entry. We bioengineered a soluble ACE2 protein termed ACE2 618-DDC-ABD that has increased binding to SARS-CoV-2 and prolonged duration of action. Here, we investigated the protective effect of this protein when administered intranasally to k18-hACE2 mice infected with the aggressive SARS-CoV-2 Delta variant. k18-hACE2 mice were infected with the SARS-CoV-2 Delta variant by inoculation of a lethal dose (2 × 104 PFU). ACE2 618-DDC-ABD (10 mg/kg) or PBS was administered intranasally six hours prior and 24 and 48 h post-viral inoculation. All animals in the PBS control group succumbed to the disease on day seven post-infection (0% survival), whereas, in contrast, there was only one casualty in the group that received ACE2 618-DDC-ABD (90% survival). Mice in the ACE2 618-DDC-ABD group had minimal disease as assessed using a clinical score and stable weight, and both brain and lung viral titers were markedly reduced. These findings demonstrate the efficacy of a bioengineered soluble ACE2 decoy with an extended duration of action in protecting against the aggressive Delta SARS-CoV-2 variant. Together with previous work, these findings underline the universal protective potential against current and future emerging SARS-CoV-2 variants.
Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , Melfalan , gama-Globulinas , Humanos , Camundongos , Animais , Peptidil Dipeptidase A/metabolismo , SARS-CoV-2/metabolismoRESUMO
Therapeutic antibodies have become one of the most influential therapeutics in modern medicine to fight against infectious pathogens, cancer, and many other diseases. However, experimental screening for highly efficacious targeting antibodies is labor-intensive and of high cost, which is exacerbated by evolving antigen targets under selective pressure such as fast-mutating viral variants. As a proof-of-concept, we developed a machine learning-assisted antibody generation pipeline AbGen that greatly accelerates the screening and re-design of immunoglobulins G (IgGs) against a broad spectrum of SARS-CoV-2 coronavirus variant strains. Our AbGen centers around a novel antibody language model (AbLM) that is pretrained on 12 million generic protein domain sequences and fine-tuned on 4,000+ paired VH-VL sequences, with IgG-specific CDR-masking and VH-VL cross-attention. AbLM provides a latent space of IgG sequence embeddings for AbGen, including (a) landscapes of IgGs' activities in neutralizing the wild-type virus are analyzed through structure prediction for IgG and IgG-antigen (viral protein spike's receptor binding domain, RBD) interactions; and (b) landscapes of IgGs' susceptibility in neutralizing variant viruses are predicted through Gaussian process regression, despite that as few as 14 clinical antibodies' responses to variants of concern are available. The AbGen pipeline was applied to over 1300 IgG sequences we collected from RBD-binding B cells of convalescent patients. With experimental validations, AbGen efficiently prioritized IgG candidates against a broad spectrum of viral variants (wildtype, Delta, and Omicron), preventing the infection of host cells in vitro and hACE2 transgenic mice in vivo. Compared to other existing protein language models that require 10-100 times more model parameters, AbLM improved the precision from around 50% to 75% to predict IgGs with low variant susceptibility. Furthermore, AbGen enables structure-based computational protein redesign for selected IgG clones with single amino acid substitutions at the RBD-binding interface that doubled the IgG blockade efficacy for one of the severe, therapy-resistant strains - Delta (B.1.617). Our work expedites applications of artificial intelligence in antibody screen and re-design combining data-driven protein language models and Kriging for antibody sequence analysis and activity prediction, in synergy with physics-driven protein docking and design for antibody-antigen interface analyses and functional optimization.
RESUMO
Dengue virus (DENV), an emerging mosquito-transmitted pathogen capable of causing severe disease in humans, interacts with host cell factors to create a more favorable environment for replication. However, few interactions between DENV and human proteins have been reported to date. To identify DENV-human protein interactions, we used high-throughput yeast two-hybrid assays to screen the 10 DENV proteins against a human liver activation domain library. From 45 DNA-binding domain clones containing either full-length viral genes or partially overlapping gene fragments, we identified 139 interactions between DENV and human proteins, the vast majority of which are novel. These interactions involved 105 human proteins, including six previously implicated in DENV infection and 45 linked to the replication of other viruses. Human proteins with functions related to the complement and coagulation cascade, the centrosome, and the cytoskeleton were enriched among the DENV interaction partners. To determine if the cellular proteins were required for DENV infection, we used small interfering RNAs to inhibit their expression. Six of 12 proteins targeted (CALR, DDX3X, ERC1, GOLGA2, TRIP11, and UBE2I) caused a significant decrease in the replication of a DENV replicon. We further showed that calreticulin colocalized with viral dsRNA and with the viral NS3 and NS5 proteins in DENV-infected cells, consistent with a direct role for calreticulin in DENV replication. Human proteins that interacted with DENV had significantly higher average degree and betweenness than expected by chance, which provides additional support for the hypothesis that viruses preferentially target cellular proteins that occupy central position in the human protein interaction network. This study provides a valuable starting point for additional investigations into the roles of human proteins in DENV infection.
Assuntos
Calreticulina/metabolismo , Vírus da Dengue/fisiologia , Interações Hospedeiro-Patógeno , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Calreticulina/genética , Linhagem Celular Tumoral , Proteínas do Citoesqueleto , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA Viral/metabolismo , Dengue/virologia , Técnicas de Silenciamento de Genes , Genes Reporter , Humanos , Luciferases/biossíntese , Luciferases/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Mapas de Interação de Proteínas , Transporte Proteico , RNA Helicases/metabolismo , Interferência de RNA , Serina Endopeptidases/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação ViralRESUMO
Dengue virus (DENV) modifies cellular membranes to establish its sites of replication. Although the 3D architecture of these structures has recently been described, little is known about the cellular pathways required for their formation and expansion. In this report, we examine the host requirements for DENV replication using a focused RNAi analysis combined with validation studies using pharmacological inhibitors. This approach identified three cellular pathways required for DENV replication: autophagy, actin polymerization, and fatty acid biosynthesis. Further characterization of the viral modulation of fatty acid biosynthesis revealed that a key enzyme in this pathway, fatty acid synthase (FASN), is relocalized to sites of DENV replication. DENV nonstructural protein 3 (NS3) is responsible for FASN recruitment, inasmuch as (i) NS3 expressed in the absence of other viral proteins colocalizes with FASN and (ii) NS3 interacts with FASN in a two-hybrid assay. There is an associated increase in the rate of fatty acid biosynthesis in DENV-infected cells, and de novo synthesized lipids preferentially cofractionate with DENV RNA. Finally, purified recombinant NS3 stimulates the activity of FASN in vitro. Taken together, these experiments suggest that DENV co-opts the fatty acid biosynthetic pathway to establish its replication complexes. This study provides mechanistic insight into DENV membrane remodeling and highlights the potential for the development of therapeutics that inhibit DENV replication by targeting the fatty acid biosynthetic pathway.
Assuntos
Vírus da Dengue/fisiologia , Ácido Graxo Sintases/metabolismo , Ácidos Graxos/biossíntese , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/fisiologia , Animais , Linhagem Celular , Vírus da Dengue/patogenicidade , Ácido Graxo Sintases/genética , Humanos , Interferência de RNA , Técnicas do Sistema de Duplo-Híbrido , Proteínas não Estruturais Virais/genéticaRESUMO
Replication complexes (RCs), formed by positive-strand (+) RNA viruses through rearrangements of host endomembranes, protect their replicating RNA from host innate immune defenses. We have shown that two evolutionarily conserved defense systems, autophagy and interferon (IFN), target viral RCs and inhibit viral replication collaboratively. However, the mechanism by which autophagy proteins target viral RCs and the role of IFN-inducible GTPases in the disruption of RCs remains poorly understood. Here, using murine norovirus (MNV) as a model (+) RNA virus, we show that the guanylate binding protein 1 (GBP1) is the human GTPase responsible for inhibiting RCs. Furthermore, we found that ATG16L1 mediates the LC3 targeting of MNV RC by binding to WIPI2B and CAPRIN1, and that IFN gamma-mediated control of MNV replication was dependent on CAPRIN1. Collectively, this study identifies a novel mechanism for the autophagy machinery-mediated recognition and inhibition of viral RCs, a hallmark of (+) RNA virus replication. IMPORTANCE Replication complexes provide a microenvironment important for (+) RNA virus replication and shield it from host immune response. Previously we have shown that interferon gamma (IFNG) disrupts the RC of MNV via evolutionarily conserved autophagy proteins and IFN-inducible GTPases. Elucidating the mechanism of targeting of viral RC by ATG16L1 and IFN-induced GTPase will pave the way for development of therapeutics targeting the viral replication complexes. Here, we have identified GBP1 as the sole GBP targeting viral RC and uncovered the novel role of CAPRIN1 in recruiting ATG16L1 to the viral RC.
Assuntos
Interferon gama , Interferons , Humanos , Animais , Camundongos , GTP Fosfo-Hidrolases/metabolismo , Replicação Viral , RNA , Proteínas de Ciclo CelularRESUMO
A soluble ACE2 protein bioengineered for long duration of action and high affinity to SARS-CoV-2 was administered either intranasally (IN) or intraperitoneally (IP) to SARS-CoV-2-inoculated k18hACE2 mice. This decoy protein (ACE2 618-DDC-ABD) was given either IN or IP, pre- and post-inoculation, or IN, IP, or IN + IP but only post-inoculation. Survival by day 5 was 0% in untreated mice, 40% in the IP-pre, and 90% in the IN-pre group. In the IN-pre group, brain histopathology was essentially normal and lung histopathology significantly improved. Consistent with this, brain SARS-CoV-2 titers were undetectable and lung titers reduced in the IN-pre group. When ACE2 618-DDC-ABD was administered only post-inoculation, survival was 30% in the IN + IP, 20% in the IN, and 20% in the IP group. We conclude that ACE2 618-DDC-ABD results in markedly improved survival and provides organ protection when given intranasally as compared with when given either systemically or after viral inoculation, and that lowering brain titers is a critical determinant of survival and organ protection.
Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , Animais , Camundongos , SARS-CoV-2 , EncéfaloRESUMO
Studying viral-host protein-protein interactions can facilitate the discovery of therapies for viral infection. We use high-throughput yeast two-hybrid experiments and mass spectrometry to generate a comprehensive SARS-CoV-2-human protein-protein interactome network consisting of 739 high-confidence binary and co-complex interactions, validating 218 known SARS-CoV-2 host factors and revealing 361 novel ones. Our results show the highest overlap of interaction partners between published datasets and of genes differentially expressed in samples from COVID-19 patients. We identify an interaction between the viral protein ORF3a and the human transcription factor ZNF579, illustrating a direct viral impact on host transcription. We perform network-based screens of >2,900 FDA-approved or investigational drugs and identify 23 with significant network proximity to SARS-CoV-2 host factors. One of these drugs, carvedilol, shows clinical benefits for COVID-19 patients in an electronic health records analysis and antiviral properties in a human lung cell line infected with SARS-CoV-2. Our study demonstrates the value of network systems biology to understand human-virus interactions and provides hits for further research on COVID-19 therapeutics.
Assuntos
COVID-19 , Mapeamento de Interação de Proteínas , Humanos , Linhagem Celular , Regulação da Expressão Gênica , SARS-CoV-2/genética , Proteínas Virais/metabolismoRESUMO
Phosphatidylinositol 4-kinase III alpha (PI4KA) is an essential cofactor of hepatitis C virus (HCV) replication. We initiated this study to determine whether HCV directly engages PI4KA to establish its replication. PI4KA kinase activity was found to be absolutely required for HCV replication using a small interfering RNA transcomplementation assay. Moreover, HCV infection or subgenomic HCV replicons produced a dramatic increase in phosphatidylinositol 4-phosphate (PI4P) accumulation throughout the cytoplasm, which partially colocalized with the endoplasmic reticulum. In contrast, the majority of PI4P accumulated at the Golgi bodies in uninfected cells. The increase in PI4P was not observed after infection with UV-inactivated HCV and did not reflect changes in PI4KA protein or RNA abundance. In an analysis of U2OS cell lines with inducible expression of the HCV polyprotein or individual viral proteins, viral polyprotein expression resulted in enhanced cytoplasmic PI4P production. Increased PI4P accumulation following HCV protein expression was precluded by silencing the expression of PI4KA, but not the related PI4KB. Silencing PI4KA also resulted in aberrant agglomeration of viral replicase proteins, including NS5A, NS5B, and NS3. NS5A alone, but not other viral proteins, stimulated PI4P production in vivo and enhanced PI4KA kinase activity in vitro. Lastly, PI4KA coimmunoprecipitated with NS5A from infected Huh-7.5 cells and from dually transfected 293T cells. In sum, these results suggest that HCV NS5A modulation of PI4KA-dependent PI4P production influences replication complex formation.