RESUMO
Live-attenuated virus vaccines provide long-lived protection against viral disease but carry inherent risks of residual pathogenicity and genetic reversion. The live-attenuated Candid#1 vaccine was developed to protect Argentines against lethal infection by the Argentine hemorrhagic fever arenavirus, Junín virus. Despite its safety and efficacy in Phase III clinical study, the vaccine is not licensed in the US, in part due to concerns regarding the genetic stability of attenuation. Previous studies had identified a single F427I mutation in the transmembrane domain of the Candid#1 envelope glycoprotein GPC as the key determinant of attenuation, as well as the propensity of this mutation to revert upon passage in cell culture and neonatal mice. To ascertain the consequences of this reversion event, we introduced the I427F mutation into recombinant Candid#1 (I427F rCan) and investigated the effects in two validated small-animal models: in mice expressing the essential virus receptor (human transferrin receptor 1; huTfR1) and in the conventional guinea pig model. We report that I427F rCan displays only modest virulence in huTfR1 mice and appears attenuated in guinea pigs. Reversion at another attenuating locus in Candid#1 GPC (T168A) was also examined, and a similar pattern was observed. By contrast, virus bearing both revertant mutations (A168T+I427F rCan) approached the lethal virulence of the pathogenic Romero strain in huTfR1 mice. Virulence was less extreme in guinea pigs. Our findings suggest that genetic stabilization at both positions is required to minimize the likelihood of reversion to virulence in a second-generation Candid#1 vaccine.IMPORTANCELive-attenuated virus vaccines, such as measles/mumps/rubella and oral poliovirus, provide robust protection against disease but carry with them the risk of genetic reversion to the virulent form. Here, we analyze the genetics of reversion in the live-attenuated Candid#1 vaccine that is used to protect against Argentine hemorrhagic fever, an often-lethal disease caused by the Junín arenavirus. In two validated small-animal models, we find that restoration of virulence in recombinant Candid#1 viruses requires back-mutation at two positions specific to the Candid#1 envelope glycoprotein GPC, at positions 168 and 427. Viruses bearing only a single change showed only modest virulence. We discuss strategies to genetically harden Candid#1 GPC against these two reversion events in order to develop a safer second-generation Candid#1 vaccine virus.
Assuntos
Febre Hemorrágica Americana , Vírus Junin , Vacinas Virais , Animais , Cobaias , Humanos , Camundongos , Glicoproteínas/genética , Febre Hemorrágica Americana/prevenção & controle , Vírus Junin/fisiologia , População da América do Sul , Vacinas Atenuadas/genética , Vacinas Virais/genética , VirulênciaRESUMO
Many enveloped viruses enter host cells by fusing with acidic endosomes. The fusion activity of multiple viral envelope glycoproteins does not generally affect viral membrane permeability. However, fusion induced by the Lassa virus (LASV) glycoprotein complex (GPc) is always preceded by an increase in viral membrane permeability and the ensuing acidification of the virion interior. Here, systematic investigation of this LASV fusion phenotype using single pseudovirus tracking in live cells reveals that the change in membrane barrier function is associated with the fusogenic conformational reorganization of GPc. We show that a small-molecule fusion inhibitor or mutations that impair viral fusion by interfering with GPc refolding into the post-fusion structure prevent the increase in membrane permeability. We find that the increase in virion membrane permeability occurs early during endosomal maturation and is facilitated by virus-cell contact. This increase is observed using diverse arenavirus glycoproteins, whether presented on lentivirus-based pseudoviruses or arenavirus-like particles, and in multiple different cell types. Collectively, these results suggest that conformational changes in GPc triggered by low pH and cell factor binding are responsible for virion membrane permeabilization and acidification of the virion core prior to fusion. We propose that this viroporin-like activity may augment viral fusion and/or post-fusion steps of infection, including ribonucleoprotein release into the cytoplasm.
Assuntos
Arenavirus , Arenavirus/genética , Proteínas Viroporinas/metabolismo , Glicoproteínas/metabolismo , Proteínas do Envelope Viral/metabolismo , Vírus Lassa , Internalização do VírusRESUMO
PURPOSE: Over 1 billion doses of COVID-19 vaccines have been already administered across the United States, the United Kingdom and the European Union at the time of writing. Furthermore, 1.82 million booster doses have been administered in the US since 13th August, and similar booster programmes are currently planned or under consideration in the UK and the EU beginning in the autumn of 2021. Early reports showed an association between vaccine administration and the development of ipsilateral axillary and supraclavicular lymphadenopathy, which could interfere with the diagnosis, treatment and follow-up of breast cancer patients. In this paper, we review the available evidence on vaccine-related lymphadenopathy, and we discuss the clinical implications of the same on breast cancer diagnosis and management. METHODS: A literature search was performed - PubMed, Ovid Medline, Scopus, CINHAL, Springer Nature, ScienceDirect, Academic Search Premier and the Directory of Open Access Journals were searched for articles reporting on regional palpable or image-detected lymphadenopathy following COVID-19 vaccination. Separately, we compiled a series of case studies from the University Hospitals of Derby and Burton, United Kingdom and the Mayo Clinic in Minnesota, United States of America, to illustrate the impact that regional lymphadenopathy post-COVID-19 vaccination can have on the diagnosis and management of patients being seen in diagnostic and therapeutic breast clinics. RESULTS: From the literature search, 15 studies met the inclusion criteria (n = 2057 patients, 737 with lymphadenopathy). The incidence of lymphadenopathy ranged between 14.5% and 53% and persisted for >6 weeks in 29% of patients. CONCLUSIONS: Clinicians managing breast cancer patients should be aware that the COVID-19 vaccination may result in regional lymphadenopathy in a significant number of patients, which can result in unnecessary investigations, treatment and increased patient anxiety. An accurate COVID-19 vaccination history should be collected from all patients where regional lymphadenopathy is a clinical and/or an imaging finding and then combined with clinical judgement when managing individual cases.
Assuntos
Neoplasias da Mama/diagnóstico por imagem , Vacinas contra COVID-19/efeitos adversos , Linfadenopatia/diagnóstico por imagem , Mamografia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Vacinação/efeitos adversos , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/terapia , Vacinas contra COVID-19/administração & dosagem , Diagnóstico Diferencial , Erros de Diagnóstico , Feminino , Humanos , Incidência , Linfadenopatia/induzido quimicamente , Linfadenopatia/epidemiologia , Valor Preditivo dos Testes , Prognóstico , Medição de Risco , Fatores de Risco , Fatores de TempoRESUMO
Live-attenuated virus vaccines are highly effective in preventing viral disease but carry intrinsic risks of residual virulence and reversion to pathogenicity. The classically derived Candid#1 virus protects seasonal field workers in Argentina against zoonotic infection by Junín virus (JUNV) but is not approved in the United States, in part due to the potential for reversion at the attenuating locus, a phenylalanine-to-isoleucine substitution at position 427 in the GP2 subunit of the GPC envelope glycoprotein. Previously, we demonstrated facile reversion of recombinant Candid#1 (rCan) in cell culture and identified an epistatic interaction between the attenuating I427 and a secondary K33S mutation in the stable signal peptide (SSP) subunit of GPC that imposes an evolutionary barrier to reversion. The magnitude of this genetic barrier is manifest in our repeated failures to rescue the hypothetical revertant virus. In this study, we show that K33S rCan is safe and attenuated in guinea pigs and capable of eliciting potent virus-neutralizing antibodies. Immunized animals are fully protected against lethal challenge with virulent JUNV. In addition, we employed a more permissive model of infection in neonatal mice to investigate genetic reversion. RNA sequence analysis of the recovered virus identified revertant viruses in pups inoculated with the parental rCan virus and none in mice receiving K33S rCan (P < 0.0001). Taken together, our findings support the further development of K33S rCan as a safe second-generation JUNV vaccine. IMPORTANCE Our most successful vaccines comprise weakened strains of virus that initiate a limited and benign infection in immunized persons. The live-attenuated Candid#1 strain of Junín virus (JUNV) was developed to protect field workers in Argentina from rodent-borne hemorrhagic fever but is not licensed in the United States, in part due to the likelihood of genetic reversion to virulence. A single-amino-acid change in the GPC envelope glycoprotein of the virus is responsible for attenuation, and a single nucleotide change may regenerate the pathogenic virus. Here, we take advantage of a unique genetic interaction between GPC subunits to design a mutant Candid#1 virus that establishes an evolutionary barrier to reversion. The mutant virus (K33S rCan) is fully attenuated and protects immunized guinea pigs against lethal JUNV infection. We find no instances of reversion in mice inoculated with K33S rCan. This work supports the further development of K33S rCan as a second-generation JUNV vaccine.
Assuntos
Febre Hemorrágica Americana/prevenção & controle , Vírus Junin/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Cobaias , Febre Hemorrágica Americana/imunologia , Imunogenicidade da Vacina , Vírus Junin/genética , Vírus Junin/patogenicidade , Masculino , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Células Vero , Vacinas Virais/genética , VirulênciaAssuntos
COVID-19 , Linfadenopatia , Vacinas contra COVID-19 , Humanos , Atenção Primária à Saúde , SARS-CoV-2 , VacinaçãoRESUMO
BACKGROUND: The primary aim of this prospective, multicentre feasibility study was to determine whether the biopsied axillary node can be marked using black carbon dye and successfully identified at the time of surgery. METHODS: We included breast cancer patients undergoing needle biopsy of the axillary node. The biopsied node was tattooed at the time of needle biopsy (fine needle aspiration or core biopsy) or at a separate visit with black carbon dye (Spot® or Black Eye™). Participants underwent primary surgery or neoadjuvant chemotherapy (NACT) and axillary surgery (SNB or ALND) as per routine care. RESULTS: 110 patients were included. Median age of the women was 59 (range 31-88) years. 48 (44%) underwent SNB and 62 (56%) ALND. Median volume of dye injected was 2.0 ml (range 0.2-4.2). Tattooed node was identified intra-operatively in 90 (82%) patients. The identification rate was higher (76 of 88, 86%) in the primary surgery group compared with NACT (14 of 22, 64%) (p = 0.03). Of those undergoing NACT, the identification rate was better in the patients undergoing SNB (3 of 4, 75%) compared with ALND (11 of 18, 61%) (p > 0.99). The tattooed node was the sentinel node in 78% (28 of 36) patients in the primary surgery group and 100% (3 of 3) in the NACT group. There was no learning curve for surgeons or radiologists. The identification rate did not vary with timing between dye injection and surgery (p = 0.56), body mass index (p = 0.62) or volume of dye injected (p = 0.25). CONCLUSION: It is feasible to mark the axillary node with carbon dye and identify it intra-operatively. ClinicalTrials.gov: NCT03640819.
Assuntos
Neoplasias da Mama , Biópsia de Linfonodo Sentinela , Tatuagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Axila/patologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Carbono , Estudos de Viabilidade , Feminino , Humanos , Excisão de Linfonodo , Linfonodos/patologia , Linfonodos/cirurgia , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Estudos ProspectivosRESUMO
Old World (Africa) and New World (South America) arenaviruses are associated with human hemorrhagic fevers. Efforts to develop small molecule therapeutics have yielded several chemical series including the 4-acyl-1,6-dialkylpiperazin-2-ones. Herein, we describe an extensive exploration of this chemotype. In initial Phase I studies, R1 and R4 scanning libraries were assayed to identify potent substituents against Old World (Lassa) virus. In subsequent Phase II studies, R6 substituents and iterative R1, R4 and R6 substituent combinations were evaluated to obtain compounds with improved Lassa and New World (Machupo, Junin, and Tacaribe) arenavirus inhibitory activity, in vitro human liver microsome metabolic stability and aqueous solubility.
Assuntos
Antivirais/farmacologia , Arenavirus/efeitos dos fármacos , Piperazinas/farmacologia , Proteínas do Envelope Viral/antagonistas & inibidores , Antivirais/síntese química , Antivirais/química , Arenavirus/metabolismo , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Piperazinas/síntese química , Piperazinas/química , Relação Estrutura-Atividade , Proteínas do Envelope Viral/metabolismoRESUMO
For many viruses that enter their target cells through pH-dependent fusion of the viral and endosomal membranes, cell-cell fusion assays can provide an experimental platform for investigating the structure-function relationships that promote envelope glycoprotein membrane-fusion activity. Typically, these assays employ effector cells expressing the recombinant envelope glycoprotein on the cell surface and target cells engineered to quantitatively report fusion with the effector cell. In the protocol described here, Vero cells are transfected with a plasmid encoding the arenavirus envelope glycoprotein complex GPC and infected with the vTF7-3 vaccinia virus expressing the bacteriophage T7 RNA polymerase. These effector cells are mixed with target cells infected with the vCB21R-lacZ vaccinia virus encoding a ß-galactosidase reporter under the control of the T7 promoter. Cell-cell fusion is induced upon exposure to low-pH medium (pH 5.0), and the resultant expression of the ß-galactosidase reporter is quantitated using a chemiluminescent substrate. We have utilized this robust microplate cell-cell fusion assay extensively to study arenavirus entry and its inhibition by small-molecule fusion inhibitors.
Assuntos
Arenavirus/metabolismo , Bioensaio/métodos , Endossomos/metabolismo , Glicoproteínas/metabolismo , Animais , Chlorocebus aethiops , Concentração de Íons de Hidrogênio , Fusão de Membrana/fisiologia , Células Vero , Proteínas do Envelope Viral/metabolismo , beta-Galactosidase/metabolismoRESUMO
The Candid#1 strain of Junín virus was developed using a conventional attenuation strategy of serial passage in nonhost animals and cultured cells. The live-attenuated Candid#1 vaccine is used in Argentina to protect at-risk individuals against Argentine hemorrhagic fever, but it has not been licensed in the United States. Recent studies have revealed that Candid#1 attenuation is entirely dependent on a phenylalanine-to-isoleucine substitution at position 427 in the fusion subunit (GP2) of the viral envelope glycoprotein complex (GPC), thereby raising concerns regarding the potential for reversion to virulence. In this study, we report the identification and characterization of an intragenic epistatic interaction between the attenuating F427I mutation in GP2 and a lysine-to-serine mutation at position 33 in the stable signal peptide (SSP) subunit of GPC, and we demonstrate the utility of this interaction in creating an evolutionary barrier against reversion to the pathogenic genotype. In the presence of the wild-type F427 residue, the K33S mutation abrogates the ability of ectopically expressed GPC to mediate membrane fusion at endosomal pH. This defect is rescued by the attenuating F427I mutation. We show that the recombinant Candid#1 (rCan) virus bearing K33S GPC is viable and retains its attenuated genotype under cell culture conditions that readily select for reversion in the parental rCan virus. If back-mutation to F427 offers an accessible pathway to increase fitness in rCan, reversion in K33S-GPC rCan is likely to be lethal. The epistatic interaction between K33S and F427I thus may minimize the likelihood of reversion and enhance safety in a second-generation Candid#1 vaccine.IMPORTANCE The live-attenuated Candid#1 vaccine strain of Junín virus is used to protect against Argentine hemorrhagic fever. Recent findings that a single missense mutation in the viral envelope glycoprotein complex (GPC) is responsible for attenuation raise the prospect of facile reversion to pathogenicity. Here, we characterize a genetic interaction between GPC subunits that evolutionarily forces retention of the attenuating mutation. By incorporating this secondary mutation into Candid#1 GPC, we hope to minimize the likelihood of reversion and enhance safety in a second-generation Candid#1 vaccine. A similar approach may guide the design of live-attenuated vaccines against Lassa and other arenaviral hemorrhagic fevers.
Assuntos
Epistasia Genética , Glicoproteínas/genética , Vírus Junin/genética , Vírus Junin/imunologia , Proteínas do Envelope Viral/genética , Vacinas Virais/genética , Animais , Chlorocebus aethiops , Evolução Molecular , Genótipo , Febre Hemorrágica Americana/prevenção & controle , Humanos , Vírus Junin/metabolismo , Vírus Junin/patogenicidade , Fusão de Membrana , Mutação , Vacinas Atenuadas/genética , Células Vero , Virulência , Internalização do VírusRESUMO
UNLABELLED: Arenaviruses are responsible for severe and often fatal hemorrhagic disease. In the absence of effective antiviral therapies and vaccines, these viruses pose serious threats to public health and biodefense. Arenaviruses enter the host cell by fusion of the viral and endosomal membranes, a process mediated by the virus envelope glycoprotein GPC. Unlike other class I viral fusion proteins, GPC retains its stable signal peptide (SSP) as an essential third subunit in the mature complex. SSP spans the membrane twice and is myristoylated at its cytoplasmic N terminus. Mutations that abolish SSP myristoylation have been shown to reduce pH-induced cell-cell fusion activity of ectopically expressed GPC to â¼20% of wild-type levels. In order to examine the role of SSP myristoylation in the context of the intact virus, we used reverse genetics to generate Junín viruses (Candid #1 isolate) in which the critical glycine-2 residue in SSP was either replaced by alanine (G2A) or deleted (ΔG2). These mutant viruses produced smaller foci of infection in Vero cells and showed an â¼5-fold reduction in specific infectivity, commensurate with the defect in cell-cell fusion. However, virus assembly and GPC incorporation into budded virions were unaffected. Our findings suggest that the myristate moiety is cryptically disposed in the prefusion GPC complex and may function late in the fusion process to promote merging of the viral and cellular membranes. IMPORTANCE: Hemorrhagic fever arenaviruses pose significant threats to public health and biodefense. Arenavirus entry into the host cell is promoted by the virus envelope glycoprotein GPC. Unlike other viral envelope glycoproteins, GPC contains a myristoylated stable signal peptide (SSP) as an essential third subunit. Myristoylation has been shown to be important for the membrane fusion activity of recombinantly expressed GPC. Here, we use reverse genetics to study the role of SSP myristoylation in the context of the intact virion. We find that nonmyristoylated GPC mutants of the Candid #1 strain of Junín virus display a commensurate deficiency in their infectivity, albeit without additional defects in virion assembly and budding. These results suggest that SSP myristoylation may function late in the fusion process to facilitate merging of the viral and cellular membranes. Antiviral agents that target this novel aspect of GPC membrane fusion may be useful in the treatment of arenavirus hemorrhagic fevers.
Assuntos
Vírus Junin/fisiologia , Glicoproteínas de Membrana/metabolismo , Ácido Mirístico/metabolismo , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas , Proteínas do Envelope Viral/metabolismo , Montagem de Vírus , Internalização do Vírus , Substituição de Aminoácidos , Animais , Chlorocebus aethiops , Glicoproteínas/genética , Glicoproteínas/metabolismo , Vírus Junin/genética , Glicoproteínas de Membrana/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Genética Reversa , Deleção de Sequência , Células Vero , Proteínas do Envelope Viral/genética , Vírion/metabolismoRESUMO
UNLABELLED: Arenavirus species are responsible for severe life-threatening hemorrhagic fevers in western Africa and South America. Without effective antiviral therapies or vaccines, these viruses pose serious public health and biodefense concerns. Chemically distinct small-molecule inhibitors of arenavirus entry have recently been identified and shown to act on the arenavirus envelope glycoprotein (GPC) to prevent membrane fusion. In the tripartite GPC complex, pH-dependent membrane fusion is triggered through a poorly understood interaction between the stable signal peptide (SSP) and the transmembrane fusion subunit GP2, and our genetic studies have suggested that these small-molecule inhibitors act at this interface to antagonize fusion activation. Here, we have designed and synthesized photoaffinity derivatives of the 4-acyl-1,6-dialkylpiperazin-2-one class of fusion inhibitors and demonstrate specific labeling of both the SSP and GP2 subunits in a native-like Lassa virus (LASV) GPC trimer expressed in insect cells. Photoaddition is competed by the parental inhibitor and other chemically distinct compounds active against LASV, but not those specific to New World arenaviruses. These studies provide direct physical evidence that these inhibitors bind at the SSP-GP2 interface. We also find that GPC containing the uncleaved GP1-GP2 precursor is not susceptible to photo-cross-linking, suggesting that proteolytic maturation is accompanied by conformational changes at this site. Detailed mapping of residues modified by the photoaffinity adducts may provide insight to guide the further development of these promising lead compounds as potential therapeutic agents to treat Lassa hemorrhagic fever. IMPORTANCE: Hemorrhagic fever arenaviruses cause lethal infections in humans and, in the absence of licensed vaccines or specific antiviral therapies, are recognized to pose significant threats to public health and biodefense. Lead small-molecule inhibitors that target the arenavirus envelope glycoprotein (GPC) have recently been identified and shown to block GPC-mediated fusion of the viral and cellular endosomal membranes, thereby preventing virus entry into the host cell. Genetic studies suggest that these inhibitors act through a unique pH-sensing intersubunit interface in GPC, but atomic-level structural information is unavailable. In this report, we utilize novel photoreactive fusion inhibitors and photoaffinity labeling to obtain direct physical evidence for inhibitor binding at this critical interface in Lassa virus GPC. Future identification of modified residues at the inhibitor-binding site will help elucidate the molecular basis for fusion activation and its inhibition and guide the development of effective therapies to treat arenaviral hemorrhagic fevers.
Assuntos
Antivirais/farmacologia , Fusão de Membrana/efeitos dos fármacos , Sinais Direcionadores de Proteínas , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus/efeitos dos fármacos , Animais , Chlorocebus aethiops , Humanos , Concentração de Íons de Hidrogênio , Vírus Lassa , Subunidades Proteicas , Células Vero , Proteínas do Envelope Viral/químicaRESUMO
The membrane-anchored proteins of enveloped viruses form labile spikes on the virion surface, primed to undergo large-scale conformational changes culminating in virus-cell membrane fusion and viral entry. The prefusion form of these envelope glycoproteins thus represents an important molecular target for antiviral intervention. A critical roadblock to this endeavor has been our inability to produce the prefusion envelope glycoprotein trimer for biochemical and structural analysis. Through our studies of the GPC envelope glycoprotein of the hemorrhagic fever arenaviruses, we have shown that GPC is unique among class I viral fusion proteins in that the mature complex retains a stable signal peptide (SSP) in addition to the conventional receptor-binding and transmembrane fusion subunits. In this report we show that the recombinant GPC precursor can be produced as a discrete native-like trimer and that its proteolytic cleavage generates the mature glycoprotein. Proteoliposomes containing the cleaved GPC mediate pH-dependent membrane fusion, a characteristic feature of arenavirus entry. This reaction is inhibited by arenavirus-specific monoclonal antibodies and small-molecule fusion inhibitors. The in vitro reconstitution of GPC-mediated membrane-fusion activity offers unprecedented opportunities for biochemical and structural studies of arenavirus entry and its inhibition. To our knowledge, this report is the first to demonstrate functional reconstitution of membrane fusion by a viral envelope glycoprotein.
Assuntos
Glicoproteínas/metabolismo , Vírus Junin/metabolismo , Fusão de Membrana , Proteínas do Envelope Viral/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Chlorocebus aethiops , Furina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Lipídeos/química , Proteínas Mutantes/metabolismo , Testes de Neutralização , Conformação Proteica , Proteolipídeos/metabolismo , Proteólise , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície , Células Vero , Eliminação de Partículas ViraisRESUMO
Arenaviruses are responsible for acute hemorrhagic fevers with high mortality and pose significant threats to public health and biodefense. These enveloped negative-sense RNA viruses replicate in the cell cytoplasm and express four proteins. To better understand how these proteins insinuate themselves into cellular processes to orchestrate productive viral replication, we have identified and characterized novel cytosolic structures involved in arenavirus replication and transcription. In cells infected with the nonpathogenic Tacaribe virus or the attenuated Candid#1 strain of Junín virus, we find that newly synthesized viral RNAs localize to cytosolic puncta containing the nucleoprotein (N) of the virus. Density gradient centrifugation studies reveal that these replication-transcription complexes (RTCs) are associated with cellular membranes and contain full-length genomic- and antigenomic-sense RNAs. Viral mRNAs segregate at a higher buoyant density and are likewise scant in immunopurified RTCs, consistent with their translation on bulk cellular ribosomes. In addition, confocal microscopy analysis reveals that RTCs contain the lipid phosphatidylinositol-4-phosphate and proteins involved in cellular mRNA metabolism, including the large and small ribosomal subunit proteins L10a and S6, the stress granule protein G3BP1, and a subset of translation initiation factors. Elucidating the structure and function of RTCs will enhance our understanding of virus-cell interactions that promote arenavirus replication and mitigate against host cell immunity. This knowledge may lead to novel intervention strategies to limit viral virulence and pathogenesis.
Assuntos
Infecções por Arenaviridae/virologia , Arenavirus do Novo Mundo/fisiologia , Citosol/virologia , Vírus Junin/fisiologia , RNA Viral/biossíntese , Replicação Viral , Animais , Arenavirus do Novo Mundo/patogenicidade , Membrana Celular/virologia , Centrifugação com Gradiente de Concentração , Chlorocebus aethiops , Citosol/ultraestrutura , Vírus Junin/patogenicidade , Nucleoproteínas , Fosfatos de Fosfatidilinositol/análise , RNA Viral/genética , Transcrição Gênica , Células VeroRESUMO
The arenavirus envelope glycoprotein (GPC) retains a stable signal peptide (SSP) as an essential subunit in the mature complex. The 58-amino-acid residue SSP comprises two membrane-spanning hydrophobic regions separated by a short ectodomain loop that interacts with the G2 fusion subunit to promote pH-dependent membrane fusion. Small-molecule compounds that target this unique SSP-G2 interaction prevent arenavirus entry and infection. The interaction between SSP and G2 is sensitive to the phylogenetic distance between New World (Junín) and Old World (Lassa) arenaviruses. For example, heterotypic GPC complexes are unable to support virion entry. In this report, we demonstrate that the hybrid GPC complexes are properly assembled, proteolytically cleaved, and transported to the cell surface but are specifically defective in their membrane fusion activity. Chimeric SSP constructs reveal that this incompatibility is localized to the first transmembrane segment of SSP (TM1). Genetic changes in TM1 also affect sensitivity to small-molecule fusion inhibitors, generating resistance in some cases and inhibitor dependence in others. Our studies suggest that interactions of SSP TM1 with the transmembrane domain of G2 may be important for GPC-mediated membrane fusion and its inhibition.
Assuntos
Arenavirus/fisiologia , Glicoproteínas/metabolismo , Sinais Direcionadores de Proteínas , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Sequência de Aminoácidos , Animais , Arenavirus/genética , Chlorocebus aethiops , Glicoproteínas/genética , Modelos Moleculares , Dados de Sequência Molecular , Células Vero , Proteínas do Envelope Viral/genéticaRESUMO
Arenaviruses comprise a diverse family of enveloped negative-strand RNA viruses that are endemic to specific rodent hosts worldwide. Several arenaviruses cause severe hemorrhagic fevers in humans, including Junín and Machupo viruses in South America and Lassa fever virus in western Africa. Arenavirus entry into the host cell is mediated by the envelope glycoprotein complex, GPC. The virion is endocytosed on binding to a cell-surface receptor, and membrane fusion is initiated in response to physiological acidification of the endosome. As with other class I virus fusion proteins, GPC-mediated membrane fusion is promoted through a regulated sequence of conformational changes leading to formation of the classical postfusion trimer-of-hairpins structure. GPC is, however, unique among the class I fusion proteins in that the mature complex retains a stable signal peptide (SSP) as a third subunit, in addition to the canonical receptor-binding and fusion proteins. We will review the curious properties of the tripartite GPC complex and describe evidence that SSP interacts with the fusion subunit to modulate pH-induced activation of membrane fusion. This unusual solution to maintaining the metastable prefusion state of GPC on the virion and activating the class I fusion cascade at acidic pH provides novel targets for antiviral intervention.
Assuntos
Arenaviridae/fisiologia , Internalização do Vírus , Sequência de Aminoácidos , Animais , Antígenos CD/fisiologia , Antivirais/farmacologia , Arenaviridae/classificação , Infecções por Arenaviridae/virologia , Sequência Conservada , Reservatórios de Doenças , Endossomos/virologia , Previsões , Humanos , Fusão de Membrana/efeitos dos fármacos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/fisiologia , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Conformação Proteica , Sinais Direcionadores de Proteínas/fisiologia , Receptores da Transferrina/fisiologia , Receptores Virais/fisiologia , Roedores/virologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/fisiologia , Internalização do Vírus/efeitos dos fármacosRESUMO
Arenaviruses are responsible for acute hemorrhagic fevers worldwide and are recognized to pose significant threats to public health and biodefense. Small molecule compounds have recently been discovered that inhibit arenavirus entry and protect against lethal infection in animal models. These chemically distinct inhibitors act on the tripartite envelope glycoprotein (GPC) through its unusual stable signal peptide subunit to stabilize the complex against pH-induced activation of membrane fusion in the endosome. Here, we report the production and characterization of the intact transmembrane GPC complex of Junín arenavirus and its interaction with these inhibitors. The solubilized GPC is antigenically indistinguishable from the native protein and forms a homogeneous trimer in solution. When reconstituted into a lipid bilayer, the purified complex interacts specifically with its cell-surface receptor transferrin receptor-1. We show that small molecule entry inhibitors specific to New World or Old World arenaviruses bind to the membrane-associated GPC complex in accordance with their respective species selectivities and with dissociation constants comparable with concentrations that inhibit GPC-mediated membrane fusion. Furthermore, competitive binding studies reveal that these chemically distinct inhibitors share a common binding pocket on GPC. In conjunction with previous genetic studies, these findings identify the pH-sensing interface of GPC as a highly vulnerable target for antiviral intervention. This work expands our mechanistic understanding of arenavirus entry and provides a foundation to guide the development of small molecule compounds for the treatment of arenavirus hemorrhagic fevers.
Assuntos
Antígenos CD/metabolismo , Antivirais/farmacologia , Infecções por Arenaviridae/tratamento farmacológico , Vírus Junin/metabolismo , Receptores da Transferrina/metabolismo , Proteínas do Envelope Viral/antagonistas & inibidores , Proteínas do Envelope Viral/metabolismo , Animais , Antígenos CD/genética , Antivirais/química , Infecções por Arenaviridae/genética , Infecções por Arenaviridae/metabolismo , Chlorocebus aethiops , Humanos , Vírus Junin/genética , Receptores da Transferrina/genética , Células Vero , Proteínas do Envelope Viral/genética , Internalização do Vírus/efeitos dos fármacosRESUMO
Arenaviruses cause acute hemorrhagic fevers with high mortality. Entry of the virus into the host cell is mediated by the viral envelope glycoprotein, GPC. In contrast to other class I viral envelope glycoproteins, the mature GPC complex contains a cleaved stable signal peptide (SSP) in addition to the canonical receptor-binding (G1) and transmembrane fusion (G2) subunits. SSP is critical for intracellular transport of the GPC complex to the cell surface and for its membrane-fusion activity. Previous studies have suggested that SSP is retained in GPC through interaction with a zinc-binding domain (ZBD) in the cytoplasmic tail of G2. Here we used NMR spectroscopy to determine the structure of Junín virus (JUNV) ZBD (G2 residues 445-485) and investigate its interaction with a conserved Cys residue (Cys-57) in SSP. We show that JUNV ZBD displays a novel fold containing two zinc ions. One zinc ion is coordinated by His-447, His-449, Cys-455, and His-485. The second zinc ion is coordinated by His-459, Cys-467, and Cys-469 and readily accepts Cys-57 from SSP as the fourth ligand. Our studies describe the structural basis for retention of the unique SSP subunit and suggest a mechanism whereby SSP is positioned in the GPC complex to modulate pH-dependent membrane fusion.
Assuntos
Vírus Junin/química , Glicoproteínas de Membrana/química , Proteínas do Envelope Viral/química , Internalização do Vírus , Zinco/química , Sequência de Aminoácidos , Sequência Conservada , Concentração de Íons de Hidrogênio , Vírus Junin/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/fisiologia , Sinais Direcionadores de Proteínas/fisiologia , Estrutura Terciária de Proteína , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Zinco/metabolismo , Dedos de Zinco/fisiologiaRESUMO
The arenavirus envelope glycoprotein (GPC) initiates infection in the host cell through pH-induced fusion of the viral and endosomal membranes. As in other class I viral fusion proteins, this process proceeds through a structural reorganization in GPC in which the ectodomain of the transmembrane fusion subunit (G2) engages the host cell membrane and subsequently refolds to form a highly stable six-helix bundle structure that brings the two membranes into apposition for fusion. Here, we describe a G2-directed monoclonal antibody, F100G5, that prevents membrane fusion by binding to an intermediate form of the protein on the fusion pathway. Inhibition of syncytium formation requires that F100G5 be present concomitant with exposure of GPC to acidic pH. We show that F100G5 recognizes neither the six-helix bundle nor the larger trimer-of-hairpins structure in the postfusion form of G2. Rather, Western blot analysis using recombinant proteins and a panel of alanine-scanning GPC mutants revealed that F100G5 binding is dependent on an invariant lysine residue (K283) near the N terminus of G2, in the so-called fusion peptide that inserts into the host cell membrane during the fusion process. The F100G5 epitope is located in the internal segment of the bipartite GPC fusion peptide, which also contains four conserved cysteine residues, raising the possibility that this fusion peptide may be highly structured. Collectively, our studies indicate that F100G5 identifies an on-path intermediate form of GPC. Binding to the transiently exposed fusion peptide may interfere with G2 insertion into the host cell membrane. Strategies to effectively target fusion peptide function in the endosome may lead to novel classes of antiviral agents.
Assuntos
Anticorpos/farmacologia , Glicoproteínas/imunologia , Vírus Junin/fisiologia , Fusão de Membrana/efeitos dos fármacos , Proteínas Virais de Fusão/imunologia , Sequência de Aminoácidos , Animais , Infecções por Arenaviridae/imunologia , Infecções por Arenaviridae/virologia , Chlorocebus aethiops , Glicoproteínas/química , Glicoproteínas/genética , Concentração de Íons de Hidrogênio , Vírus Junin/química , Vírus Junin/efeitos dos fármacos , Vírus Junin/genética , Dados de Sequência Molecular , Alinhamento de Sequência , Células Vero , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genéticaRESUMO
The mature arenavirus envelope glycoprotein GPC is a tripartite complex comprising a stable signal peptide (SSP) in addition to the receptor-binding (G1) and transmembrane fusion (G2) subunits. We have shown previously that SSP is a key element in GPC-mediated membrane fusion, and that GPC sensitivity to acidic pH is modulated in part through the lysine residue at position 33 in the ectodomain loop of SSP (J. York and J. H. Nunberg, J. Virol. 80:7775-7780, 2006). A glutamine substitution at this position stabilizes the native GPC complex and thereby prevents the induction of pH-dependent membrane fusion. In efforts to identify the intersubunit interactions of K33, we performed alanine-scanning mutagenesis at charged residues in the membrane-proximal ectodomain of G2 and determined the ability of these mutations to rescue the fusion deficiency in K33Q GPC. Four second-site mutations that specifically complement K33Q were identified (D400A, E410A, R414A, and K417A). Moreover, complementation was also observed at three hydrophobic positions in the membrane-spanning domain of G2 (F427, W428, and F438). Interestingly, all of the complementing mutations restored wild-type pH sensitivity to the K33Q mutant, while none themselves affected the pH of membrane fusion. Our studies demonstrate a specific interaction between SSP and G2 that is involved in priming the native GPC complex for pH-induced membrane fusion. Importantly, this pH-dependent interaction has been shown to be vulnerable to small-molecule compounds that stabilize the native complex and prevent the activation of membrane fusion. A detailed mechanistic understanding of the control of GPC-mediated membrane fusion will be important in guiding the development of effective therapeutics against arenaviral hemorrhagic fever.
Assuntos
Glicoproteínas/metabolismo , Vírus Junin/metabolismo , Fusão de Membrana , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Sequência de Aminoácidos , Animais , Chlorocebus aethiops , Glicoproteínas/química , Glicoproteínas/genética , Concentração de Íons de Hidrogênio , Vírus Junin/genética , Dados de Sequência Molecular , Mutação/genética , Peptídeos/genética , Peptídeos/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Alinhamento de Sequência , Células Vero , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
The arenavirus envelope glycoprotein (GPC) mediates viral entry through pH-induced membrane fusion in the endosome. This crucial process in the viral life cycle can be specifically inhibited in the New World arenaviruses by the small-molecule compound ST-294. Here, we show that ST-294 interferes with GPC-mediated membrane fusion by targeting the interaction of the G2 fusion subunit with the stable signal peptide (SSP). We demonstrate that amino acid substitutions at lysine-33 of the Junín virus SSP confer resistance to ST-294 and engender de novo sensitivity to ST-161, a chemically distinct inhibitor of the Old World Lassa fever virus. These compounds, as well as a broadly active inhibitor, ST-193, likely share a molecular target at the SSP-G2 interface. We also show that both ST-294 and ST-193 inhibit pH-induced dissociation of the G1 receptor-binding subunit from GPC, a process concomitant with fusion activation. Interestingly, the inhibitory activity of these molecules can in some cases be overcome by further lowering the pH used for activation. Our results suggest that these small molecules act to stabilize the prefusion GPC complex against acidic pH. The pH-sensitive interaction between SSP and G2 in GPC represents a robust molecular target for the development of antiviral compounds for the treatment of arenavirus hemorrhagic fevers.