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1.
Cell ; 150(6): 1249-63, 2012 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-22980984

RESUMO

A defining feature of vertebrate immunity is the acquisition of immunological memory, which confers enhanced protection against pathogens by mechanisms that are incompletely understood. Here, we compared responses by virus-specific naive T cells (T(N)) and central memory T cells (T(CM)) to viral antigen challenge in lymph nodes (LNs). In steady-state LNs, both T cell subsets localized in the deep T cell area and interacted similarly with antigen-presenting dendritic cells. However, upon entry of lymph-borne virus, only T(CM) relocalized rapidly and efficiently toward the outermost LN regions in the medullary, interfollicular, and subcapsular areas where viral infection was initially confined. This rapid peripheralization was coordinated by a cascade of cytokines and chemokines, particularly ligands for T(CM)-expressed CXCR3. Consequently, in vivo recall responses to viral infection by CXCR3-deficient T(CM) were markedly compromised, indicating that early antigen detection afforded by intranodal chemokine guidance of T(CM) is essential for efficient antiviral memory.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Memória Imunológica , Linfonodos/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Quimiocina CXCL9/imunologia , Células Dendríticas/imunologia , Interferon gama/imunologia , Linfonodos/citologia , Vírus da Coriomeningite Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores CXCR3/imunologia , Células Estromais/imunologia , Vírus da Estomatite Vesicular Indiana
2.
Immunity ; 45(6): 1270-1284, 2016 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-27939671

RESUMO

Infections induce pathogen-specific T cell differentiation into diverse effectors (Teff) that give rise to memory (Tmem) subsets. The cell-fate decisions and lineage relationships that underlie these transitions are poorly understood. Here, we found that the chemokine receptor CX3CR1 identifies three distinct CD8+ Teff and Tmem subsets. Classical central (Tcm) and effector memory (Tem) cells and their corresponding Teff precursors were CX3CR1- and CX3CR1high, respectively. Viral infection also induced a numerically stable CX3CR1int subset that represented ∼15% of blood-borne Tmem cells. CX3CR1int Tmem cells underwent more frequent homeostatic divisions than other Tmem subsets and not only self-renewed, but also contributed to the expanding CX3CR1- Tcm pool. Both Tcm and CX3CR1int cells homed to lymph nodes, but CX3CR1int cells, and not Tem cells, predominantly surveyed peripheral tissues. As CX3CR1int Tmem cells present unique phenotypic, homeostatic, and migratory properties, we designate this subset peripheral memory (tpm) cells and propose that tpm cells are chiefly responsible for the global surveillance of non-lymphoid tissues.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Homeostase/imunologia , Vigilância Imunológica/imunologia , Receptores de Quimiocinas/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Receptor 1 de Quimiocina CX3C , Separação Celular , Citometria de Fluxo , Memória Imunológica/imunologia , Camundongos , Camundongos Endogâmicos C57BL
3.
J Virol ; 97(1): e0138522, 2023 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-36533953

RESUMO

Several mammarenaviruses cause severe hemorrhagic fever (HF) disease in humans and pose important public health problems in their regions of endemicity. There are no United States (US) Food and Drug Administration (FDA)-approved mammarenavirus vaccines, and current anti-mammarenavirus therapy is limited to an off-label use of ribavirin that has limited efficacy. Mammarenaviruses are enveloped viruses with a bi-segmented negative-strand RNA genome. Each genome segment contains two open reading frames (ORF) separated by a noncoding intergenic region (IGR). The large (L) segment encodes the RNA dependent RNA polymerase, L protein, and the Z matrix protein, whereas the small (S) segment encodes the surface glycoprotein precursor (GPC) and nucleoprotein (NP). In the present study, we document the generation of a recombinant form of the prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV) expressing a codon deoptimized (CD) GPC and containing the IGR of the S segment in both the S and L segments (rLCMV/IGR-CD). We show that rLCMV/IGR-CD is fully attenuated in C57BL/6 (B6) mice but able to provide complete protection upon a single administration against a lethal challenge with LCMV. Importantly, rLCMV/IGR-CD exhibited an unbreachable attenuation for its safe implementation as a live-attenuated vaccine (LAV). IMPORTANCE Several mammarenaviruses cause severe disease in humans and pose important public health problems in their regions of endemicity. Currently, no FDA-licensed mammarenavirus vaccines are available, and anti-mammarenaviral therapy is limited to an off-label use of ribavirin whose efficacy is controversial. Here, we describe the generation of recombinant version of the prototypic mammarenavirus lymphocytic choriomeningitis virus (rLCMV) combining the features of a codon deoptimized (CD) GPC and the noncoding intergenic region (IGR) of the S segment in both S and L genome segments, called rLCMV/IGR-CD. We present evidence that rLCMV/IGR-CD has excellent safety and protective efficacy features as live-attenuated vaccine (LAV). Importantly, rLCMV/IGR-CD prevents, in coinfected mice, the generation of LCMV reassortants with increased virulence. Our findings document a well-defined molecular strategy for the generation of mammarenavirus LAV candidates able to trigger long-term protective immunity, upon a single immunization, while exhibiting unique enhanced safety features, including unbreachable attenuation.


Assuntos
Engenharia Genética , Vírus da Coriomeningite Linfocítica , Vacinas Virais , Animais , Humanos , Camundongos , Códon/genética , DNA Intergênico/genética , Vírus da Coriomeningite Linfocítica/genética , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos Endogâmicos C57BL , Vacinas Atenuadas/imunologia , Desenvolvimento de Vacinas
4.
J Virol ; 97(2): e0168822, 2023 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-36656012

RESUMO

The eukaryotic chaperonin containing tailless complex polypeptide 1 ring complex (CCT, also known as TCP-1 Ring Complex, TRiC/CCT) participates in the folding of 5% to 10% of the cellular proteome and has been involved in the life cycle of several viruses, including dengue, Zika, and influenza viruses, but the mechanisms by which the TRiC/CCT complex contributes to virus multiplication remain poorly understood. Here, we document that the nucleoprotein (NP) of the mammarenavirus lymphocytic choriomeningitis virus (LCMV) is a substrate of the human TRiC/CCT complex, and that pharmacological inhibition of TRiC/CCT complex function, or RNAi-mediated knockdown of TRiC/CCT complex subunits, inhibited LCMV multiplication in human cells. We obtained evidence that the TRiC/CCT complex is required for the production of NP-containing virus-like particles (VLPs), and the activity of the virus ribonucleoprotein (vRNP) responsible for directing replication and transcription of the viral genome. Pharmacological inhibition of the TRIC/CCT complex also restricted multiplication of the live-attenuated vaccine candidates Candid#1 and ML29 of the hemorrhagic fever causing Junin (JUNV) and Lassa (LASV) mammarenaviruses, respectively. Our findings indicate that the TRiC/CCT complex is required for mammarenavirus multiplication and is an attractive candidate for the development of host directed antivirals against human-pathogenic mammarenaviruses. IMPORTANCE Host-directed antivirals have gained great interest as an antiviral strategy to counteract the rapid emergence of drug-resistant viruses. The chaperonin TRiC/CCT complex has been involved in the life cycle of several viruses, including dengue, Zika, and influenza viruses. Here, we have provided evidence that the chaperonin TRiC/CCT complex participates in mammarenavirus infection via its interaction with the viral NP. Importantly, pharmacological inhibition of TRiC/CCT function significantly inhibited multiplication of LCMV and the distantly related mammarenavirus JUNV in human cells. Our findings support that the TRiC/CCT complex is required for multiplication of mammarenaviruses and that the TRiC/CCT complex is an attractive host target for the development of antivirals against human-pathogenic mammarenaviruses.


Assuntos
Chaperonina com TCP-1 , Vírus da Coriomeningite Linfocítica , Nucleoproteínas , Humanos , Antivirais , Chaperonina com TCP-1/metabolismo , Replicação Viral
5.
Curr Top Microbiol Immunol ; 439: 265-303, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36592249

RESUMO

Members of the family Arenaviridae are classified into four genera: Antennavirus, Hartmanivirus, Mammarenavirus, and Reptarenavirus. Reptarenaviruses and hartmaniviruses infect (captive) snakes and have been shown to cause boid inclusion body disease (BIBD). Antennaviruses have genomes consisting of 3, rather than 2, segments, and were discovered in actinopterygian fish by next-generation sequencing but no biological isolate has been reported yet. The hosts of mammarenaviruses are mainly rodents and infections are generally asymptomatic. Current knowledge about the biology of reptarenaviruses, hartmaniviruses, and antennaviruses is very limited and their zoonotic potential is unknown. In contrast, some mammarenaviruses are associated with zoonotic events that pose a threat to human health. This review will focus on mammarenavirus genetic diversity and its biological implications. Some mammarenaviruses including lymphocytic choriomeningitis virus (LCMV) are excellent experimental model systems for the investigation of acute and persistent viral infections, whereas others including Lassa (LASV) and Junin (JUNV) viruses, the causative agents of Lassa fever (LF) and Argentine hemorrhagic fever (AHF), respectively, are important human pathogens. Mammarenaviruses were thought to have high degree of intra-and inter-species amino acid sequence identities, but recent evidence has revealed a high degree of mammarenavirus genetic diversity in the field. Moreover, closely related mammarenavirus can display dramatic phenotypic differences in vivo. These findings support a role of genetic variability in mammarenavirus adaptability and pathogenesis. Here, we will review the molecular biology of mammarenaviruses, phylogeny, and evolution, as well as the quasispecies dynamics of mammarenavirus populations and their biological implications.


Assuntos
Arenaviridae , Animais , Humanos , Arenaviridae/genética , Arenaviridae/metabolismo , Roedores , Variação Genética
6.
J Virol ; 95(24): e0139921, 2021 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-34586865

RESUMO

Targeting host factors is a promising strategy to develop broad-spectrum antiviral drugs. Drugs targeting anti-apoptotic Bcl-2 family proteins that were originally developed as tumor suppressors have been reported to inhibit multiplication of different types of viruses. However, the mechanisms whereby Bcl-2 inhibitors exert their antiviral activity remain poorly understood. In this study, we have investigated the mechanisms by which obatoclax (OLX) and ABT-737 Bcl-2 inhibitors exhibited a potent antiviral activity against the mammarenavirus lymphocytic choriomeningitis virus (LCMV). OLX and ABT-737 potent anti-LCMV activity was not associated with their proapoptotic properties but rather with their ability to induce cell arrest at the G0/G1 phase. OLX- and ABT-737-mediated inhibition of Bcl-2 correlated with reduced expression levels of thymidine kinase 1 (TK1), cyclin A2 (CCNA2), and cyclin B1 (CCNB1) cell cycle regulators. In addition, small interfering RNA (siRNA)-mediated knockdown of TK1, CCNA2, and CCNB1 resulted in reduced levels of LCMV multiplication. The antiviral activity exerted by Bcl-2 inhibitors correlated with reduced levels of viral RNA synthesis at early times of infection. Importantly, ABT-737 exhibited moderate efficacy in a mouse model of LCMV infection, and Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals. IMPORTANCE Antiapoptotic Bcl-2 inhibitors have been shown to exert potent antiviral activities against various types of viruses via mechanisms that are currently poorly understood. This study has revealed that Bcl-2 inhibitors' mediation of cell cycle arrest at the G0/G1 phase, rather than their proapoptotic activity, plays a critical role in blocking mammarenavirus multiplication in cultured cells. In addition, we show that Bcl-2 inhibitor ABT-737 exhibited moderate antimammarenavirus activity in vivo and that Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals.


Assuntos
Apoptose , Arenaviridae/efeitos dos fármacos , Tratamento Farmacológico da COVID-19 , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Células A549 , Animais , Antivirais/farmacologia , Proteínas Reguladoras de Apoptose/farmacologia , Compostos de Bifenilo/farmacologia , COVID-19/virologia , Ciclo Celular , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/virologia , Chlorocebus aethiops , Ciclina A2/biossíntese , Ciclina B1/biossíntese , Fase G1 , Humanos , Indóis/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Nitrofenóis/farmacologia , Piperazinas/farmacologia , Pirróis/farmacologia , Fase de Repouso do Ciclo Celular , SARS-CoV-2 , Sulfonamidas/farmacologia , Timidina Quinase/biossíntese , Células Vero
7.
J Virol ; 95(22): e0112621, 2021 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-34495697

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and has been responsible for the still ongoing coronavirus disease 2019 (COVID-19) pandemic. Prophylactic vaccines have been authorized by the U.S. Food and Drug Administration (FDA) for the prevention of COVID-19. Identification of SARS-CoV-2-neutralizing antibodies (NAbs) is important to assess vaccine protection efficacy, including their ability to protect against emerging SARS-CoV-2 variants of concern (VoC). Here, we report the generation and use of a recombinant (r)SARS-CoV-2 USA/WA1/2020 (WA-1) strain expressing Venus and an rSARS-CoV-2 strain expressing mCherry and containing mutations K417N, E484K, and N501Y found in the receptor binding domain (RBD) of the spike (S) glycoprotein of the South African (SA) B.1.351 (beta [ß]) VoC in bifluorescent-based assays to rapidly and accurately identify human monoclonal antibodies (hMAbs) able to neutralize both viral infections in vitro and in vivo. Importantly, our bifluorescent-based system accurately recapitulated findings observed using individual viruses. Moreover, fluorescent-expressing rSARS-CoV-2 strain and the parental wild-type (WT) rSARS-CoV-2 WA-1 strain had similar viral fitness in vitro, as well as similar virulence and pathogenicity in vivo in the K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model of SARS-CoV-2 infection. We demonstrate that these new fluorescent-expressing rSARS-CoV-2 can be used in vitro and in vivo to easily identify hMAbs that simultaneously neutralize different SARS-CoV-2 strains, including VoC, for the rapid assessment of vaccine efficacy or the identification of prophylactic and/or therapeutic broadly NAbs for the treatment of SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2 is responsible of the COVID-19 pandemic that has warped daily routines and socioeconomics. There is still an urgent need for prophylactics and therapeutics to treat SARS-CoV-2 infections. In this study, we demonstrate the feasibility of using bifluorescent-based assays for the rapid identification of hMAbs with neutralizing activity against SARS-CoV-2, including VoC in vitro and in vivo. Importantly, results obtained with these bifluorescent-based assays recapitulate those observed with individual viruses, demonstrating their feasibility to rapidly advance our understanding of vaccine efficacy and to identify broadly protective human NAbs for the therapeutic treatment of SARS-CoV-2.


Assuntos
Anticorpos Neutralizantes/imunologia , Testes de Neutralização/métodos , SARS-CoV-2/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Amplamente Neutralizantes/imunologia , Anticorpos Amplamente Neutralizantes/uso terapêutico , COVID-19/terapia , COVID-19/virologia , Genes Reporter , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/virologia , Camundongos , Mutação , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
9.
J Virol ; 94(7)2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-31941776

RESUMO

Influenza A (IAV) and influenza B (IBV) viruses are highly contagious pathogens that cause fatal respiratory disease every year, with high economic impact. In addition, IAV can cause pandemic infections with great consequences when new viruses are introduced into humans. In this study, we evaluated 10 previously described compounds with antiviral activity against mammarenaviruses for their ability to inhibit IAV infection using our recently described bireporter influenza A/Puerto Rico/8/34 (PR8) H1N1 (BIRFLU). Among the 10 tested compounds, eight (antimycin A [AmA], brequinar [BRQ], 6-azauridine, azaribine, pyrazofurin [PF], AVN-944, mycophenolate mofetil [MMF], and mycophenolic acid [MPA]), but not obatoclax or Osu-03012, showed potent anti-influenza virus activity under posttreatment conditions [median 50% effective concentration (EC50) = 3.80 nM to 1.73 µM; selective index SI for 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, >28.90 to 13,157.89]. AmA, 6-azauridine, azaribine, and PF also showed potent inhibitory effect in pretreatment (EC50 = 0.14 µM to 0.55 µM; SI-MTT = 70.12 to >357.14) or cotreatment (EC50 = 34.69 nM to 7.52 µM; SI-MTT = 5.24 to > 1,441.33) settings. All of the compounds tested inhibited viral genome replication and gene transcription, and none of them affected host cellular RNA polymerase II activities. The antiviral activity of the eight identified compounds against BIRFLU was further confirmed with seasonal IAVs (A/California/04/2009 H1N1 and A/Wyoming/3/2003 H3N2) and an IBV (B/Brisbane/60/2008, Victoria lineage), demonstrating their broad-spectrum prophylactic and therapeutic activity against currently circulating influenza viruses in humans. Together, our results identified a new set of antiviral compounds for the potential treatment of influenza viral infections.IMPORTANCE Influenza viruses are highly contagious pathogens and are a major threat to human health. Vaccination remains the most effective tool to protect humans against influenza infection. However, vaccination does not always guarantee complete protection against drifted or, more noticeably, shifted influenza viruses. Although U.S. Food and Drug Administration (FDA) drugs are approved for the treatment of influenza infections, influenza viruses resistant to current FDA antivirals have been reported and continue to emerge. Therefore, there is an urgent need to find novel antivirals for the treatment of influenza viral infections in humans, a search that could be expedited by repurposing currently approved drugs. In this study, we assessed the influenza antiviral activity of 10 compounds previously shown to inhibit mammarenavirus infection. Among them, eight drugs showed antiviral activities, providing a new battery of drugs that could be used for the treatment of influenza infections.


Assuntos
Antivirais/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza B/efeitos dos fármacos , Células A549 , Animais , Proliferação de Células , Cães , Avaliação Pré-Clínica de Medicamentos , Genoma Viral , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos da radiação , Vírus da Influenza A Subtipo H3N2/fisiologia , Vírus da Influenza B/fisiologia , Células Madin Darby de Rim Canino , Replicação Viral/efeitos dos fármacos
10.
J Virol ; 94(8)2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-31996435

RESUMO

Argentine hemorrhagic fever is a potentially lethal disease that is caused by Junin virus (JUNV). There are currently around 5 million individuals at risk of infection within regions of endemicity in Argentina. The live attenuated vaccine strain Candid #1 (Can) is approved for use in regions of endemicity and has substantially decreased the number of annual Argentine hemorrhagic fever (AHF) cases. The glycoprotein (GPC) gene is primarily responsible for attenuation of the Can strain, and we have shown that the absence of an N-linked glycosylation motif in the subunit G1 of the glycoprotein complex of Can, which is otherwise present in the wild-type pathogenic JUNV, causes GPC retention in the endoplasmic reticulum (ER). Here, we show that Can GPC aggregates in the ER of infected cells, forming incorrect cross-chain disulfide bonds, which results in impaired GPC processing into G1 and G2. The GPC fails to cleave into its G1 and G2 subunits and is targeted for degradation within lysosomes. Cells infected with the wild-type Romero (Rom) strain do not produce aggregates that are observed in Can infection, and the stress on the ER remains minimal. While the mutation of the N-linked glycosylation motif (T168A) is primarily responsible for the formation of aggregates, other mutations within G1 that occurred earlier in the passage history of the Can strain also contribute to aggregation of the GPC within the ER.IMPORTANCE The development of vaccines and therapeutics to combat viral hemorrhagic fevers remains a top priority within the Implementation Plan of the U.S. Department of Health and Human Services Public Health Emergency Medical Countermeasures Enterprise. The Can strain, derived from the pathogenic XJ strain of JUNV, has been demonstrated to be both safe and protective against AHF. While the vaccine strain is approved for use in regions of endemicity within Argentina, the mechanisms of Can attenuation have not been elucidated. A better understanding of the viral genetic determinants of attenuation will improve our understanding of the mechanisms contributing to disease pathogenesis and provide critical information for the rational design of live attenuated vaccine candidates for other viral hemorrhagic fevers.


Assuntos
Estresse do Retículo Endoplasmático/imunologia , Glicoproteínas/imunologia , Vírus Junin/imunologia , Lisossomos/metabolismo , Vacinas Atenuadas/imunologia , Vacinas Virais/imunologia , Animais , Autofagia , Encéfalo/metabolismo , Chlorocebus aethiops , Retículo Endoplasmático/imunologia , Glicoproteínas/genética , Glicosilação , Células HEK293 , Febre Hemorrágica Americana/virologia , Febres Hemorrágicas Virais/prevenção & controle , Humanos , Vírus Junin/genética , Camundongos , Mutação , Células Vero , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia
11.
PLoS Pathog ; 14(2): e1006892, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29462184

RESUMO

Several mammalian arenaviruses (mammarenaviruses) cause hemorrhagic fevers in humans and pose serious public health concerns in their endemic regions. Additionally, mounting evidence indicates that the worldwide-distributed, prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV), is a neglected human pathogen of clinical significance. Concerns about human-pathogenic mammarenaviruses are exacerbated by of the lack of licensed vaccines, and current anti-mammarenavirus therapy is limited to off-label use of ribavirin that is only partially effective. Detailed understanding of virus/host-cell interactions may facilitate the development of novel anti-mammarenavirus strategies by targeting components of the host-cell machinery that are required for efficient virus multiplication. Here we document the generation of a recombinant LCMV encoding a nucleoprotein (NP) containing an affinity tag (rLCMV/Strep-NP) and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na+/K+ transporting subunit alpha 1 (ATP1A1) and prohibitin (PHB) as pro-viral factors. Cell-based assays revealed that ATP1A1 and PHB are involved in different steps of the virus life cycle. Accordingly, we observed a synergistic inhibitory effect on LCMV multiplication with a combination of ATP1A1 and PHB inhibitors. We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans.


Assuntos
Coriomeningite Linfocítica/metabolismo , Vírus da Coriomeningite Linfocítica/metabolismo , Nucleoproteínas/metabolismo , Mapas de Interação de Proteínas , Proteoma/análise , Proteínas Repressoras/fisiologia , ATPase Trocadora de Sódio-Potássio/fisiologia , Células A549 , Animais , Arenaviridae/fisiologia , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/fisiologia , Camundongos , Proibitinas , Ligação Proteica , Proteínas Repressoras/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Células Vero
12.
PLoS Pathog ; 14(7): e1007172, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30028868

RESUMO

The interferon inducible protein, BST-2 (or, tetherin), plays an important role in the innate antiviral defense system by inhibiting the release of many enveloped viruses. Consequently, viruses have evolved strategies to counteract the anti-viral activity of this protein. While the mechanisms by which BST-2 prevents viral dissemination have been defined, less is known about how this protein shapes the early viral distribution and immunological defense against pathogens during the establishment of persistence. Using the lymphocytic choriomeningitis virus (LCMV) model of infection, we sought insights into how the in vitro antiviral activity of this protein compared to the immunological defense mounted in vivo. We observed that BST-2 modestly reduced production of virion particles from cultured cells, which was associated with the ability of BST-2 to interfere with the virus budding process mediated by the LCMV Z protein. Moreover, LCMV does not encode a BST-2 antagonist, and viral propagation was not significantly restricted in cells that constitutively expressed BST-2. In contrast to this very modest effect in cultured cells, BST-2 played a crucial role in controlling LCMV in vivo. In BST-2 deficient mice, a persistent strain of LCMV was no longer confined to the splenic marginal zone at early times post-infection, which resulted in an altered distribution of LCMV-specific T cells, reduced T cell proliferation / function, delayed viral control in the serum, and persistence in the brain. These data demonstrate that BST-2 is important in shaping the anatomical distribution and adaptive immune response against a persistent viral infection in vivo.


Assuntos
Antígenos CD/imunologia , Coriomeningite Linfocítica/imunologia , Linfócitos T/imunologia , Animais , Antígenos CD/metabolismo , Proliferação de Células , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Humanos , Ativação Linfocitária , Coriomeningite Linfocítica/metabolismo , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Endogâmicos C57BL
13.
J Infect Dis ; 219(11): 1818-1822, 2019 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-30517671

RESUMO

Lassa fever (LF) survivors develop various clinical manifestations including polyserositis, myalgia, epididymitis, and hearing loss weeks to months after recovery from acute infection. We demonstrate a systemic lymphoplasmacytic and histiocytic arteritis and periarteritis in guinea pigs more than 2 months after recovery from acute Lassa virus (LASV) infection. LASV was detected in the arterial tunica media smooth muscle cells by immunohistochemistry, in situ hybridization, and transmission electron microscopy. Our results suggest that the sequelae of LASV infection may be due to virus persistence resulting in systemic vascular damage. These findings shed light on the pathogenesis of LASV sequelae in convalescent human survivors.


Assuntos
Febre Lassa/virologia , Vírus Lassa/imunologia , Animais , Convalescença , Modelos Animais de Doenças , Progressão da Doença , Feminino , Cobaias , Humanos , Imuno-Histoquímica , Inflamação , Febre Lassa/patologia , Masculino
14.
J Virol ; 92(11)2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29593035

RESUMO

Mammarenaviruses cause chronic infections in their natural rodent hosts. Infected rodents shed infectious virus into excreta. Humans are infected through mucosal exposure to aerosols or direct contact of abraded skin with fomites, resulting in a wide range of manifestations from asymptomatic or mild febrile illness to severe life-threatening hemorrhagic fever. The mammarenavirus matrix Z protein has been shown to be a main driving force of virus budding and to act as a negative regulator of viral RNA synthesis. To gain a better understanding of how the Z protein exerts its several different functions, we investigated the interaction between Z and viral polymerase L protein using the prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV). We found that in the presence of an active viral ribonucleoprotein (vRNP), the Z protein translocated from nonionic detergent-resistant, membrane-rich structures to a subcellular compartment with a different membrane composition susceptible to disruption by nonionic detergents. Alanine (A) substitution of a highly conserved leucine (L) at position 72 in LCMV Z protein abrogated Z-L interaction. The L72A mutation did not affect the stability or budding activity of Z when expressed alone, but in the presence of an active vRNP, mutation L72A promoted rapid degradation of Z via a proteasome- and lysosome-independent pathway. Accordingly, L72A mutation in the Z protein resulted in nonviable LCMV. Our findings have uncovered novel aspects of the dynamics of the Z protein for which a highly conserved L residue was strictly required.IMPORTANCE Several mammarenaviruses, chiefly Lassa virus (LASV), cause hemorrhagic fever disease in humans and pose important public health concerns in their regions of endemicity. Moreover, mounting evidence indicates that the worldwide-distributed, prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV), is a neglected human pathogen of clinical significance. The mammarenavirus matrix Z protein plays critical roles in different steps of the viral life cycle by interacting with viral and host cellular components. Here we report that alanine substitution of a highly conserved leucine residue, located at position 72 in LCMV Z protein, abrogated Z-L interaction. The L72A mutation did not affect Z budding activity but promoted its rapid degradation in the presence of an active viral ribonucleoprotein (vRNP). Our findings have uncovered novel aspects of the dynamics of the Z protein for which a highly conserved L residue was strictly required.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Vírus da Coriomeningite Linfocítica/crescimento & desenvolvimento , RNA Polimerase Dependente de RNA/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Virais/metabolismo , Liberação de Vírus/genética , Alanina/genética , Substituição de Aminoácidos/genética , Cloreto de Amônio/farmacologia , Animais , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Leucina/genética , Leupeptinas/farmacologia , Vírus da Coriomeningite Linfocítica/genética , Lisossomos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , RNA Polimerase Dependente de RNA/genética , Células Vero , Proteínas Virais/genética
15.
J Virol ; 90(22): 10102-10112, 2016 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-27581982

RESUMO

Several arenaviruses, chiefly Lassa virus (LASV), cause hemorrhagic fever disease in humans and pose serious public health concerns in their regions of endemicity. Moreover, mounting evidence indicates that the worldwide-distributed prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), is a neglected human pathogen of clinical significance. We have documented that a recombinant LCMV containing the glycoprotein (GPC) gene of LASV within the backbone of the immunosuppressive clone 13 (Cl-13) variant of the Armstrong strain of LCMV (rCl-13/LASV-GPC) exhibited Cl-13-like growth properties in cultured cells, but in contrast to Cl-13, rCl-13/LASV-GPC was unable to establish persistence in immunocompetent adult mice, which prevented its use for some in vivo experiments. Recently, V459K and K461G mutations within the GP2 cytoplasmic domain (CD) of rCl-13/LASV-GPC were shown to increase rCl-13/LASV-GPC infectivity in mice. Here, we generated rCl-13(GPC/VGKS) by introducing the corresponding revertant mutations K465V and G467K within GP2 of rCl-13 and we show that rCl-13(GPC/VGKS) was unable to persist in mice. K465V and G467K mutations did not affect GPC processing, virus RNA replication, or gene expression. In addition, rCl-13(GPC/VGKS) grew to high titers in cultured cell lines and in immunodeficient mice. Further analysis revealed that rCl-13(GPC/VGKS) infected fewer splenic plasmacytoid dendritic cells than rCl-13, yet the two viruses induced similar type I interferon responses in mice. Our findings have identified novel viral determinants of Cl-13 persistence and also revealed that virus GPC-host interactions yet to be elucidated critically contribute to Cl-13 persistence. IMPORTANCE: The prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), provides investigators with a superb experimental model system to investigate virus-host interactions. The Armstrong strain (ARM) of LCMV causes an acute infection, whereas its derivative, clone 13 (Cl-13), causes a persistent infection. Mutations F260L and K1079Q within GP1 and L polymerase, respectively, have been shown to play critical roles in Cl-13's ability to persist in mice. However, there is an overall lack of knowledge about other viral determinants required for Cl-13's persistence. Here, we report that mutations K465V and G467K within the cytoplasmic domain of Cl-13 GP2 resulted in a virus, rCl-13(GPC/VGKS), that failed to persist in mice despite exhibiting Cl-13 wild-type-like fitness in cultured cells and immunocompromised mice. This finding has uncovered novel viral determinants of viral persistence, and a detailed characterization of rCl-13(GPC/VGKS) can provide novel insights into the mechanisms underlying persistent viral infection.


Assuntos
Glicoproteínas/genética , Vírus da Coriomeningite Linfocítica/genética , Vírus da Coriomeningite Linfocítica/patogenicidade , Células A549 , Animais , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Citoplasma , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Humanos , Hospedeiro Imunocomprometido/fisiologia , Interferon Tipo I/metabolismo , Vírus Lassa/genética , Vírus Lassa/patogenicidade , Coriomeningite Linfocítica/metabolismo , Coriomeningite Linfocítica/virologia , Camundongos , Mutação/genética , Células Vero , Replicação Viral/genética
16.
J Virol ; 90(6): 3187-97, 2016 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-26739049

RESUMO

UNLABELLED: Hemorrhagic fever arenaviruses (HFAs) pose important public health problems in regions where they are endemic. Concerns about human-pathogenic arenaviruses are exacerbated because of the lack of FDA-licensed arenavirus vaccines and because current antiarenaviral therapy is limited to an off-label use of ribavirin that is only partially effective. We have recently shown that the noncoding intergenic region (IGR) present in each arenavirus genome segment, the S and L segments (S-IGR and L-IGR, respectively), plays important roles in the control of virus protein expression and that this knowledge could be harnessed for the development of live-attenuated vaccine strains to combat HFAs. In this study, we further investigated the sequence plasticity of the arenavirus IGR. We demonstrate that recombinants of the prototypic arenavirus lymphocytic choriomeningitis virus (rLCMVs), whose S-IGRs were replaced by the S-IGR of Lassa virus (LASV) or an entirely nonviral S-IGR-like sequence (Ssyn), are viable, indicating that the function of S-IGR tolerates a high degree of sequence plasticity. In addition, rLCMVs whose L-IGRs were replaced by Ssyn or S-IGRs of the very distantly related reptarenavirus Golden Gate virus (GGV) were viable and severely attenuated in vivo but able to elicit protective immunity against a lethal challenge with wild-type LCMV. Our findings indicate that replacement of L-IGR by a nonviral Ssyn could serve as a universal molecular determinant of arenavirus attenuation. IMPORTANCE: Hemorrhagic fever arenaviruses (HFAs) cause high rates of morbidity and mortality and pose important public health problems in regions where they are endemic. Implementation of live-attenuated vaccines (LAVs) will represent a major step to combat HFAs. Here we document that the arenavirus noncoding intergenic region (IGR) has a high degree of plasticity compatible with virus viability. This observation led us to generate recombinant LCMVs containing nonviral synthetic IGRs. These rLCMVs were severely attenuated in vivo but able to elicit protective immunity against a lethal challenge with wild-type LCMV. These nonviral synthetic IGRs can be used as universal molecular determinants of arenavirus attenuation for the rapid development of safe and effective, as well as stable, LAVs to combat HFA.


Assuntos
DNA Intergênico , Vírus da Coriomeningite Linfocítica/genética , Vírus da Coriomeningite Linfocítica/patogenicidade , Mutagênese Insercional , Recombinação Genética , Vacinas Virais/imunologia , Animais , Infecções por Arenaviridae/patologia , Infecções por Arenaviridae/prevenção & controle , Modelos Animais de Doenças , Vírus Lassa/genética , Vírus da Coriomeningite Linfocítica/fisiologia , Camundongos Endogâmicos C57BL , Viabilidade Microbiana , Análise de Sobrevida , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética
17.
Curr Top Microbiol Immunol ; 392: 231-76, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26472215

RESUMO

The family Arenaviridae currently comprises over 20 viral species, each of them associated with a main rodent species as the natural reservoir and in one case possibly phyllostomid bats. Moreover, recent findings have documented a divergent group of arenaviruses in captive alethinophidian snakes. Human infections occur through mucosal exposure to aerosols or by direct contact of abraded skin with infectious materials. Arenaviruses merit interest both as highly tractable experimental model systems to study acute and persistent infections and as clinically important human pathogens including Lassa (LASV) and Junin (JUNV) viruses, the causative agents of Lassa and Argentine hemorrhagic fevers (AHFs), respectively, for which there are no FDA-licensed vaccines, and current therapy is limited to an off-label use of ribavirin (Rib) that has significant limitations. Arenaviruses are enveloped viruses with a bi-segmented negative strand (NS) RNA genome. Each genome segment, L (ca 7.3 kb) and S (ca 3.5 kb), uses an ambisense coding strategy to direct the synthesis of two polypeptides in opposite orientation, separated by a noncoding intergenic region (IGR). The S genomic RNA encodes the virus nucleoprotein (NP) and the precursor (GPC) of the virus surface glycoprotein that mediates virus receptor recognition and cell entry via endocytosis. The L genome RNA encodes the viral RNA-dependent RNA polymerase (RdRp, or L polymerase) and the small (ca 11 kDa) RING finger protein Z that has functions of a bona fide matrix protein including directing virus budding. Arenaviruses were thought to be relatively stable genetically with intra- and interspecies amino acid sequence identities of 90-95 % and 44-63 %, respectively. However, recent evidence has documented extensive arenavirus genetic variability in the field. Moreover, dramatic phenotypic differences have been documented among closely related LCMV isolates. These data provide strong evidence of viral quasispecies involvement in arenavirus adaptability and pathogenesis. Here, we will review several aspects of the molecular biology of arenaviruses, phylogeny and evolution, and quasispecies dynamics of arenavirus populations for a better understanding of arenavirus pathogenesis, as well as for the development of novel antiviral strategies to combat arenavirus infections.


Assuntos
Infecções por Arenaviridae/virologia , Arenavirus/genética , Evolução Molecular , Animais , Antivirais/farmacologia , Infecções por Arenaviridae/tratamento farmacológico , Arenavirus/classificação , Arenavirus/efeitos dos fármacos , Arenavirus/fisiologia , Variação Genética , Genoma Viral , Humanos , Filogenia , Replicação Viral
18.
J Virol ; 89(23): 12166-77, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26401045

RESUMO

UNLABELLED: Hemorrhagic fever arenaviruses (HFA) pose important public health problems in regions where they are endemic. Thus, Lassa virus (LASV) infects several hundred thousand individuals yearly in West Africa, causing a large number of Lassa fever cases associated with high morbidity and mortality. Concerns about human-pathogenic arenaviruses are exacerbated because of the lack of FDA-licensed arenavirus vaccines and because current antiarenaviral therapy is limited to an off-label use of ribavirin that is only partially effective. The Mopeia virus (MOPV)/LASV reassortant (ML29) is a LASV candidate live-attenuated vaccine (LAV) that has shown promising results in animal models. Nevertheless, the mechanism of ML29 attenuation remains unknown, which raises concerns about the phenotypic stability of ML29 in response to additional mutations. Development of LAVs based on well-defined molecular mechanisms of attenuation will represent a major step in combatting HFA. We used the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) to develop a general molecular strategy for arenavirus attenuation. Our approach involved replacement of the noncoding intergenic region (IGR) of the L genome segment with the IGR of the S genome segment to generate a recombinant LCMV, rLCMV(IGR/S-S), that was highly attenuated in vivo but induced protection against a lethal challenge with wild-type LCMV. Attenuation of rLCMV(IGR/S-S) was associated with a stable reorganization of the control of viral gene expression. This strategy can facilitate the rapid development of LAVs with the antigenic composition of the parental HFA and a mechanism of attenuation that minimizes concerns about increased virulence that could be caused by genetic changes in the LAV. IMPORTANCE: Hemorrhagic fever arenaviruses (HFA) cause high morbidity and mortality, and pose important public health problems in the regions where they are endemic. Implementation of live-attenuated vaccines (LAV) will represent a major step in combatting HFA. Here we have used the prototypic arenavirus LCMV to document a general molecular strategy for arenavirus attenuation that can facilitate the rapid development of safe and effective, as well as stable, LAV to combat HFA.


Assuntos
Arenaviridae/imunologia , Febre Lassa/prevenção & controle , Vacinas Atenuadas/biossíntese , Vacinas Virais/biossíntese , Animais , Arenaviridae/genética , Northern Blotting , Chlorocebus aethiops , Primers do DNA/genética , Humanos , Vírus da Coriomeningite Linfocítica/genética , Plasmídeos/genética , Vacinas Atenuadas/imunologia , Vacinas Sintéticas/genética , Células Vero , Vacinas Virais/imunologia
19.
J Virol ; 89(10): 5734-8, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25762740

RESUMO

In this study, we document that efficient interaction between arenavirus nucleoprotein (NP) and RNA-dependent RNA polymerase (L protein), the two trans-acting viral factors required for both virus RNA replication and gene transcription, requires the presence of virus-specific RNA sequences located within the untranslated 5' and 3' termini of the viral genome.


Assuntos
Arenavirus/metabolismo , Nucleocapsídeo/metabolismo , Nucleoproteínas/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/metabolismo , Arenavirus/genética , Genoma Viral , Células HEK293 , Humanos , Vírus da Coriomeningite Linfocítica/genética , Vírus da Coriomeningite Linfocítica/fisiologia , Nucleocapsídeo/genética , Nucleoproteínas/genética , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/genética , Replicação Viral
20.
J Virol ; 89(21): 10924-33, 2015 11.
Artigo em Inglês | MEDLINE | ID: mdl-26292327

RESUMO

UNLABELLED: Several arenaviruses cause hemorrhagic fever disease in humans and represent important public health problems in the regions where these viruses are endemic. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an important neglected human pathogen. There are no licensed arenavirus vaccines and current antiarenavirus therapy is limited to the use of ribavirin that is only partially effective. Therefore, there is an unmet need for novel antiarenaviral therapeutics. Here, we report the generation of a novel recombinant LCM virus and its use to develop a cell-based high-throughput screen to rapidly identify inhibitors of LCMV multiplication. We used this novel assay to screen a library of 30,400 small molecules and identified compound F3406 (chemical name: N-[3,5-bis(fluoranyl)phenyl]-2-[5,7-bis(oxidanylidene)-6-propyl-2-pyrrolidin-1-yl-[1,3]thiazolo[4,5-d]pyrimidin-4-yl]ethanamide), which exhibited strong anti-LCMV activity in the absence of cell toxicity. Mechanism-of-action studies revealed that F3406 inhibited LCMV cell entry by specifically interfering with the pH-dependent fusion in the endosome compartment that is mediated by LCMV glycoprotein GP2 and required to release the virus ribonucleoprotein into the cell cytoplasm to initiate transcription and replication of the virus genome. We identified residue M437 within the transmembrane domain of GP2 as critical for virus susceptibility to F3406. IMPORTANCE: Hemorrhagic fever arenaviruses (HFA) are important human pathogens that cause high morbidity and mortality in areas where these viruses are endemic. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. Concerns posed by arenavirus infections are aggravated by the lack of U.S. Food and Drug Administration-licensed arenavirus vaccines and current antiarenaviral therapy being limited to the off-label use of ribavirin that is only partially effective. Here we describe a novel recombinant LCMV and its use to develop a cell-based assay suitable for HTS to rapidly identify inhibitors arenavirus multiplication. The concepts and experimental strategies we describe in this work provide the bases for the rapid identification and characterization of novel anti-HFA therapeutics.


Assuntos
Infecções por Arenaviridae/prevenção & controle , Vírus da Coriomeningite Linfocítica/genética , Vírus da Coriomeningite Linfocítica/fisiologia , Pirimidinonas/farmacologia , Bibliotecas de Moléculas Pequenas/química , Tiazóis/farmacologia , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/fisiologia , Animais , Western Blotting , Chlorocebus aethiops , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Plasmídeos/genética , Pirimidinonas/análise , Tiazóis/análise , Células Vero , Replicação Viral/efeitos dos fármacos
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