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1.
J Virol ; 93(1)2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30305351

RESUMO

The emergence of Old and New World arenaviruses from rodent reservoirs persistently threatens human health. The GP1 subunit of the envelope-displayed arenaviral glycoprotein spike complex (GPC) mediates host cell recognition and is an important determinant of cross-species transmission. Previous structural analyses of Old World arenaviral GP1 glycoproteins, alone and in complex with a cognate GP2 subunit, have revealed that GP1 adopts two distinct conformational states distinguished by differences in the orientations of helical regions of the molecule. Here, through comparative study of the GP1 glycoprotein architectures of Old World Loei River virus and New World Whitewater Arroyo virus, we show that these rearrangements are restricted to Old World arenaviruses and are not induced solely by the pH change that is associated with virus endosomal trafficking. Our structure-based phylogenetic analysis of arenaviral GP1s provides a blueprint for understanding the discrete structural classes adopted by these therapeutically important targets.IMPORTANCE The genetically and geographically diverse group of viruses within the family Arenaviridae includes a number of zoonotic pathogens capable of causing fatal hemorrhagic fever. The multisubunit GPC glycoprotein spike complex displayed on the arenavirus envelope is a key determinant of species tropism and a primary target of the host humoral immune response. Here, we show that the receptor-binding GP1 subcomponent of the GPC spike from Old World but not New World arenaviruses adopts a distinct, pH-independent conformation in the absence of the cognate GP2. Our analysis provides a structure-based approach to understanding the discrete conformational classes sampled by these therapeutically important targets, informing strategies to develop arenaviral glycoprotein immunogens that resemble GPC as presented on the mature virion surface.


Assuntos
Arenavirus do Novo Mundo/classificação , Arenavirus do Velho Mundo/classificação , Proteínas do Envelope Viral/química , Arenavirus do Novo Mundo/química , Arenavirus do Novo Mundo/metabolismo , Arenavirus do Velho Mundo/química , Arenavirus do Velho Mundo/metabolismo , Endossomos/virologia , Evolução Molecular , Concentração de Íons de Hidrogênio , Modelos Moleculares , Filogenia , Estrutura Secundária de Proteína
2.
mSphere ; 3(3)2018.
Artigo em Inglês | MEDLINE | ID: mdl-29720525

RESUMO

Arenaviruses pose a major public health threat and cause numerous infections in humans each year. Although most viruses belonging to this family do not cause disease in humans, some arenaviruses, such as Lassa virus and Machupo virus, are the etiological agents of lethal hemorrhagic fevers. The absence of a currently licensed vaccine and the highly pathogenic nature of these viruses both make the necessity of developing viable vaccines and therapeutics all the more urgent. Arenaviruses have a single glycoprotein on the surface of virions, the glycoprotein complex (GPC), and this protein can be used as a target for vaccine development. Here, we describe immunization strategies to generate monoclonal antibodies (MAbs) that cross-react between the glycoprotein complexes of both Old World and New World arenaviruses. Several monoclonal antibodies isolated from immunized mice were highly cross-reactive, binding a range of Old World arenavirus glycoproteins, including that of Lassa virus. One such monoclonal antibody, KL-AV-2A1, bound to GPCs of both New World and Old World viruses, including Lassa and Machupo viruses. These cross-reactive antibodies bound to epitopes present on the glycoprotein 2 subunit of the glycoprotein complex, which is relatively conserved among arenaviruses. Monoclonal antibodies binding to these epitopes, however, did not inhibit viral entry as they failed to neutralize a replication-competent vesicular stomatitis virus pseudotyped with the Lassa virus glycoprotein complex in vitro In addition, no protection from virus challenge was observed in in vivo mouse models. Even so, these monoclonal antibodies might still prove to be useful in the development of clinical and diagnostic assays.IMPORTANCE Several viruses in the Arenaviridae family infect humans and cause severe hemorrhagic fevers which lead to high case fatality rates. Due to their pathogenicity and geographic tropisms, these viruses remain very understudied. As a result, an effective vaccine or therapy is urgently needed. Here, we describe efforts to produce cross-reactive monoclonal antibodies that bind to both New and Old World arenaviruses. All of our MAbs seem to be nonneutralizing and nonprotective and target subunit 2 of the glycoprotein. Due to the lack of reagents such as recombinant glycoproteins and antibodies for rapid detection assays, our MAbs could be beneficial as analytic and diagnostic tools.


Assuntos
Anticorpos Antivirais/imunologia , Arenavirus do Novo Mundo/imunologia , Arenavirus do Velho Mundo/imunologia , Reações Cruzadas , Glicoproteínas/imunologia , Proteínas Estruturais Virais/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/isolamento & purificação , Infecções por Arenaviridae/imunologia , Infecções por Arenaviridae/prevenção & controle , Modelos Animais de Doenças , Mapeamento de Epitopos , Epitopos de Linfócito B/imunologia , Camundongos
3.
J Virol ; 91(20)2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28794024

RESUMO

The arenavirus family consists of several highly pathogenic viruses, including the Old World (OW) arenavirus Lassa fever virus (LASV) and the New World (NW) Junin virus (JUNV) and Machupo virus (MACV). Host response to infection by these pathogenic arenaviruses is distinct in many aspects. JUNV and MACV infections readily induce an interferon (IFN) response in human cells, while LASV infection usually triggers an undetectable or weak IFN response. JUNV induces an IFN response through RIG-I, suggesting that the host non-self RNA sensor readily detects JUNV viral RNAs (vRNAs) during infection and activates IFN response. Double-stranded-RNA (dsRNA)-activated protein kinase R (PKR) is another host non-self RNA sensor classically known for its vRNA recognition activity. Here we report that infection with NW arenaviruses JUNV and MACV, but not OW LASV, activated PKR, concomitant with elevated phosphorylation of the translation initiation factor α subunit of eukaryotic initiation factor 2 (eIF2α). Host protein synthesis was substantially suppressed in MACV- and JUNV-infected cells but was only marginally reduced in LASV-infected cells. Despite the antiviral activity known for PKR against many other viruses, the replication of JUNV and MACV was not impaired but was slightly augmented in wild-type (wt) cells compared to that in PKR-deficient cells, suggesting that PKR or PKR activation did not negatively affect JUNV and MACV infection. Additionally, we found an enhanced IFN response in JUNV- or MACV-infected PKR-deficient cells, which was inversely correlated with virus replication.IMPORTANCE The detection of viral RNA by host non-self RNA sensors, including RIG-I and MDA5, is critical to the initiation of the innate immune response to RNA virus infection. Among pathogenic arenaviruses, the OW LASV usually does not elicit an interferon response. However, the NW arenaviruses JUNV and MACV readily trigger an IFN response in a RIG-I-dependent manner. Here, we demonstrate for the first time that pathogenic NW arenaviruses JUNV and MACV, but not the OW arenavirus LASV, activated the dsRNA-dependent PKR, another host non-self RNA sensor, during infection. Interestingly, the replication of JUNV and MACV was not restricted but was rather slightly augmented in the presence of PKR. Our data provide new evidence for a distinct interplay between host non-self RNA sensors and pathogenic arenaviruses, which also provides insights into the pathogenesis of arenaviruses and may facilitate the design of vaccines and treatments against arenavirus-caused diseases.


Assuntos
Arenavirus do Novo Mundo/patogenicidade , Arenavirus do Velho Mundo/patogenicidade , Imunidade Inata , Vírus Junin/patogenicidade , Receptores de Reconhecimento de Padrão/metabolismo , Replicação Viral , eIF-2 Quinase/metabolismo , Células A549 , Arenavirus do Novo Mundo/fisiologia , Arenavirus do Velho Mundo/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Interferons/biossíntese , Interferons/imunologia , Vírus Junin/fisiologia , Fosforilação , Receptores de Reconhecimento de Padrão/genética , Fatores de Transcrição/metabolismo , eIF-2 Quinase/genética
4.
Mol Ecol ; 26(19): 5173-5188, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28779541

RESUMO

The Old World (OW) arenavirus complex includes several species of rodent-borne viruses, some of which (i.e., Lassa virus, LASV and Lymphocytic choriomeningitis virus, LCMV) cause human diseases. Most LCMV and LASV infections are caused by rodent-to-human transmissions. Thus, viral evolution is largely determined by events that occur in the wildlife reservoirs. We used a set of human- and rodent-derived viral sequences to investigate the evolutionary history underlying OW arenavirus speciation, as well as the more recent selective events that accompanied LASV spread in West Africa. We show that the viral RNA polymerase (L protein) was a major positive selection target in OW arenaviruses and during LASV out-of-Nigeria migration. No evidence of selection was observed for the glycoprotein, whereas positive selection acted on the nucleoprotein (NP) during LCMV speciation. Positively selected sites in L and NP are surrounded by highly conserved residues, and the bulk of the viral genome evolves under purifying selection. Several positively selected sites are likely to modulate viral replication/transcription. In both L and NP, structural features (solvent exposed surface area) are important determinants of site-wise evolutionary rate variation. By incorporating several rodent-derived sequences, we also performed an analysis of OW arenavirus codon adaptation to the human host. Results do not support a previously hypothesized role of codon adaptation in disease severity for non-Nigerian strains. In conclusion, L and NP represent the major selection targets and possible determinants of disease presentation; these results suggest that field surveys and experimental studies should primarily focus on these proteins.


Assuntos
Arenavirus do Velho Mundo/genética , Evolução Biológica , RNA Polimerases Dirigidas por DNA/genética , Seleção Genética , Proteínas Virais/genética , África Ocidental , Sequência de Aminoácidos , Arenavirus do Velho Mundo/enzimologia , Vírus Lassa/enzimologia , Vírus Lassa/genética , Vírus da Coriomeningite Linfocítica/enzimologia , Vírus da Coriomeningite Linfocítica/genética , Filogenia , Estrutura Terciária de Proteína
5.
PLoS Pathog ; 13(4): e1006337, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28448640

RESUMO

Cell entry of many enveloped viruses occurs by engagement with cellular receptors, followed by internalization into endocytic compartments and pH-induced membrane fusion. A previously unnoticed step of receptor switching was found to be critical during cell entry of two devastating human pathogens: Ebola and Lassa viruses. Our recent studies revealed the functional role of receptor switching to LAMP1 for triggering membrane fusion by Lassa virus and showed the involvement of conserved histidines in this switching, suggesting that other viruses from this family may also switch to LAMP1. However, when we investigated viruses that are genetically close to Lassa virus, we discovered that they cannot bind LAMP1. A crystal structure of the receptor-binding module from Morogoro virus revealed structural differences that allowed mapping of the LAMP1 binding site to a unique set of Lassa residues not shared by other viruses in its family, illustrating a key difference in the cell-entry mechanism of Lassa virus that may contribute to its pathogenicity.


Assuntos
Infecções por Arenaviridae/virologia , Arenavirus do Velho Mundo/metabolismo , Febre Lassa/virologia , Vírus Lassa/metabolismo , Glicoproteínas de Membrana Associadas ao Lisossomo/química , Sequência de Aminoácidos , Animais , Arenavirus do Velho Mundo/química , Arenavirus do Velho Mundo/genética , Sítios de Ligação , Humanos , Vírus Lassa/química , Vírus Lassa/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Fusão de Membrana , Modelos Moleculares , Modelos Estruturais , Ligação Proteica , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Alinhamento de Sequência , Especificidade da Espécie
6.
J Virol ; 90(22): 10259-10270, 2016 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-27605671

RESUMO

The family Arenaviridae includes several important human pathogens that can cause severe hemorrhagic fever and greatly threaten public health. As a major component of the innate immune system, the RLR/MAVS signaling pathway is involved in recognizing viral components and initiating antiviral activity. It has been reported that arenavirus infection can suppress the innate immune response, and NP and Z proteins of pathogenic arenaviruses can disrupt RLR/MAVS signaling, thus inhibiting production of type I interferon (IFN-I). However, recent studies have shown elevated IFN-I levels in certain arenavirus-infected cells. The mechanism by which arenavirus infection induces IFN-I responses remains unclear. In this study, we determined that the L polymerase (Lp) of Mopeia virus (MOPV), an Old World (OW) arenavirus, can activate the RLR/MAVS pathway and thus induce the production of IFN-I. This activation is associated with the RNA-dependent RNA polymerase activity of Lp. This study provides a foundation for further studies of interactions between arenaviruses and the innate immune system and for the elucidation of arenavirus pathogenesis. IMPORTANCE: Distinct innate immune responses are observed when hosts are infected with different arenaviruses. It has been widely accepted that NP and certain Z proteins of arenaviruses inhibit the RLR/MAVS signaling pathway. The viral components responsible for the activation of the RLR/MAVS signaling pathway remain to be determined. In the current study, we demonstrate for the first time that the Lp of MOPV, an OW arenavirus, can activate the RLR/MAVS signaling pathway and thus induce the production of IFN-I. Based on our results, we proposed that dynamic interactions exist among Lp-produced RNA, NP, and the RLR/MAVS signaling pathway, and the outcome of these interactions may determine the final IFN-I response pattern: elevated or reduced. Our study provides a possible explanation for how IFN-I can become activated during arenavirus infection and may help us gain insights into the interactions that form between different arenavirus components and the innate immune system.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Infecções por Arenaviridae/metabolismo , Arenavirus do Velho Mundo/metabolismo , Transdução de Sinais/fisiologia , Proteínas Virais/metabolismo , Animais , Infecções por Arenaviridae/imunologia , Infecções por Arenaviridae/virologia , Arenavirus/imunologia , Arenavirus/metabolismo , Arenavirus do Velho Mundo/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , RNA Polimerases Dirigidas por DNA/metabolismo , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno/imunologia , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Imunidade Inata/imunologia , Interferon Tipo I/metabolismo , Células Vero
7.
J Gen Virol ; 95(Pt 1): 1-15, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24068704

RESUMO

Arenaviruses can cause fatal human haemorrhagic fever (HF) diseases for which vaccines and therapies are extremely limited. Both the New World (NW) and Old World (OW) groups of arenaviruses contain HF-causing pathogens. Although these two groups share many similarities, important differences with regard to pathogenicity and molecular mechanisms of virus infection exist. These closely related pathogens share many characteristics, including genome structure, viral assembly, natural host selection and the ability to interfere with innate immune signalling. However, members of the NW and OW viruses appear to use different receptors for cellular entry, as well as different mechanisms of virus internalization. General differences in disease signs and symptoms and pathological lesions in patients infected with either NW or OW arenaviruses are also noted and discussed herein. Whilst both the OW Lassa virus (LASV) and the NW Junin virus (JUNV) can cause disruption of the vascular endothelium, which is an important pathological feature of HF, the immune responses to these related pathogens seem to be quite distinct. Whereas LASV infection results in an overall generalized immune suppression, patients infected with JUNV seem to develop a cytokine storm. Additionally, the type of immune response required for recovery and clearance of the virus is different between NW and OW infections. These differences may be important to allow the viruses to evade host immune detection. Understanding these differences will aid the development of new vaccines and treatment strategies against deadly HF viral infections.


Assuntos
Infecções por Arenaviridae/patologia , Infecções por Arenaviridae/virologia , Arenavirus do Novo Mundo/genética , Arenavirus do Velho Mundo/genética , Febres Hemorrágicas Virais/patologia , Febres Hemorrágicas Virais/virologia , Animais , Infecções por Arenaviridae/imunologia , Arenavirus do Novo Mundo/classificação , Arenavirus do Novo Mundo/imunologia , Arenavirus do Novo Mundo/patogenicidade , Arenavirus do Velho Mundo/classificação , Arenavirus do Velho Mundo/imunologia , Arenavirus do Velho Mundo/patogenicidade , Febres Hemorrágicas Virais/imunologia , Humanos
8.
J Mol Biol ; 425(24): 4893-903, 2013 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-24075870

RESUMO

Arenaviruses are enveloped, negative-stranded RNA viruses that belong to the family Arenaviridae. This diverse family can be further classified into OW (Old World) and NW (New World) arenaviruses based on their antigenicity, phylogeny, and geographical distribution. Many of the NW arenaviruses are highly pathogenic viruses that cause systemic human infections characterized by hemorrhagic fever and/or neurological manifestations, constituting public health problems in their endemic regions. NW arenavirus infection induces a variety of host innate immune responses, which could contribute to the viral pathogenesis and/or influence the final outcome of virus infection in vitro and in vivo. On the other hand, NW arenaviruses have also developed several strategies to counteract the host innate immune response. We will review current knowledge regarding the interplay between the host innate immune response and NW arenavirus infection in vitro and in vivo, with emphasis on viral-encoded proteins and their effect on the type I interferon response.


Assuntos
Infecções por Arenaviridae/imunologia , Arenavirus do Novo Mundo/imunologia , Imunidade Inata/imunologia , Interferon Tipo I/metabolismo , Proteínas Virais/metabolismo , Animais , Infecções por Arenaviridae/virologia , Arenavirus do Novo Mundo/genética , Arenavirus do Novo Mundo/fisiologia , Arenavirus do Velho Mundo/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Evasão da Resposta Imune , Interferon Tipo I/genética , Camundongos , Modelos Moleculares , Proteínas Virais/genética
9.
J Virol ; 87(11): 6406-14, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23536681

RESUMO

The arenaviruses are an important family of emerging viruses that includes several causative agents of severe hemorrhagic fevers in humans that represent serious public health problems. A crucial step of the arenavirus life cycle is maturation of the envelope glycoprotein precursor (GPC) by the cellular subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P). Comparison of the currently known sequences of arenavirus GPCs revealed the presence of a highly conserved aromatic residue at position P7 relative to the SKI-1/S1P cleavage side in Old World and clade C New World arenaviruses but not in New World viruses of clades A and B or cellular substrates of SKI-1/S1P. Using a combination of molecular modeling and structure-function analysis, we found that residue Y285 of SKI-1/S1P, distal from the catalytic triad, is implicated in the molecular recognition of the aromatic "signature residue" at P7 in the GPC of Old World Lassa virus. Using a quantitative biochemical approach, we show that Y285 of SKI-1/S1P is crucial for the efficient processing of peptides derived from Old World and clade C New World arenavirus GPCs but not of those from clade A and B New World arenavirus GPCs. The data suggest that during coevolution with their mammalian hosts, GPCs of Old World and clade C New World viruses expanded the molecular contacts with SKI-1/S1P beyond the classical four-amino-acid recognition sequences and currently occupy an extended binding pocket.


Assuntos
Infecções por Arenaviridae/enzimologia , Arenavirus do Novo Mundo/metabolismo , Arenavirus do Velho Mundo/metabolismo , Pró-Proteína Convertases/metabolismo , Serina Endopeptidases/metabolismo , Proteínas do Envelope Viral/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Infecções por Arenaviridae/genética , Infecções por Arenaviridae/virologia , Arenavirus do Novo Mundo/classificação , Arenavirus do Novo Mundo/genética , Arenavirus do Velho Mundo/classificação , Arenavirus do Velho Mundo/genética , Células CHO , Cricetinae , Humanos , Dados de Sequência Molecular , Pró-Proteína Convertases/química , Pró-Proteína Convertases/genética , Processamento de Proteína Pós-Traducional , Alinhamento de Sequência , Serina Endopeptidases/química , Serina Endopeptidases/genética , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética
10.
J Theor Biol ; 317: 55-61, 2013 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-23041432

RESUMO

Well-established theoretical models predict host density thresholds for invasion and persistence of parasites with a density-dependent transmission. Studying such thresholds in reality, however, is not obvious because it requires long-term data for several fluctuating populations of different size. We developed a spatially explicit and individual-based SEIR model of Mopeia virus in multimammate mice Mastomys natalensis. This is an interesting model system for studying abundance thresholds because the host is the most common African rodent, populations fluctuate considerably and the virus is closely related to Lassa virus but non-pathogenic to humans so can be studied safely in the field. The simulations show that, while host density clearly is important, sharp thresholds are only to be expected for persistence (and not for invasion), since at short time-spans (as during invasion), stochasticity is determining. Besides host density, also the spatial extent of the host population is important. We observe the repeated local occurrence of herd immunity, leading to a decrease in transmission of the virus, while even a limited amount of dispersal can have a strong influence in spreading and re-igniting the transmission. The model is most sensitive to the duration of the infectious stage, the size of the home range and the transmission coefficient, so these are important factors to determine experimentally in the future.


Assuntos
Arenavirus do Velho Mundo/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Murinae/virologia , Doenças dos Roedores/epidemiologia , Doenças dos Roedores/virologia , Animais , Simulação por Computador , Progressão da Doença , Camundongos , Modelos Biológicos , Densidade Demográfica , Fatores de Risco , Análise de Sobrevida
11.
Fed Regist ; 77(194): 61083-115, 2012 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-23038847

RESUMO

In accordance with the Public Health Security and Bioterrorism Preparedness and Response Act of 2002, the Centers for Disease Control and Prevention (CDC) located within the Department of Health and Human Services (HHS) has reviewed the list of biological agents and toxins that have the potential to pose a severe threat to public health and safety and is republishing that list. As a result of our review, we have added Chapare virus, Lujo virus, and SARS-associated coronavirus (SARS-CoV) to the list of HHS select agents and toxins. We have also removed from the list of HHS and overlap select agents and toxins, or excluded from compliance with part 73, the agents and toxins described in the Executive Summary. Further, in accordance with Executive Order 13546, "Optimizing the Security of Biological Select Agents and Toxins in the United States," HHS/CDC has designated those select agents and toxins that present the greatest risk of deliberate misuse with the most significant potential for mass casualties or devastating effects to the economy, critical infrastructure; or public confidence as "Tier 1" agents; established new security requirements for entities possessing Tier 1 agents, including the requirement to conduct pre-access assessments and on-going monitoring of personnel with access to Tier 1 agents and toxins; and made revisions to the regulations to clarify regulatory language concerning security, training, biosafety, and incident response. In a companion document published in this issue of the Federal Register, the United States Department of Agriculture (USDA) has made parallel regulatory changes.


Assuntos
Produtos Biológicos , Bioterrorismo/legislação & jurisprudência , Surtos de Doenças/legislação & jurisprudência , Pandemias/legislação & jurisprudência , Medidas de Segurança/legislação & jurisprudência , Toxinas Biológicas , Arenavirus do Novo Mundo , Arenavirus do Velho Mundo , Centers for Disease Control and Prevention, U.S. , Regulamentação Governamental , Humanos , Vírus da SARS , Estados Unidos
12.
J Gen Virol ; 93(Pt 10): 2247-51, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22815269

RESUMO

In order to survey arenaviruses in the Republic of Zambia, we captured 335 rodents from three cities between 2010 and 2011. Eighteen Luna virus (LUNV) and one lymphocytic choriomeningitis virus (LCMV)-related virus RNAs were detected by one-step RT-PCR from Mastomys natalensis and Mus minutoides, respectively. Four LUNV strains and one LCMV-related virus were isolated, and the whole genome nucleotide sequence was determined by pyrosequencing. Phylogenetic analyses revealed that the LUNV clade consists of two branches that are distinguished by geographical location and that the LCMV-related virus belongs to the LCMV clade, but diverges from the typical LCMVs. Comparison of nucleoprotein amino acid sequences indicated that the LCMV-related virus could be designated a novel arenavirus, which was tentatively named as the Lunk virus. Amino acid sequences of the GP, NP, Z and L proteins showed poor similarity among the three Zambian arenavirus strains, i.e. Luna, Lunk and Lujo virus.


Assuntos
Arenavirus do Velho Mundo/classificação , Genoma Viral , Nucleoproteínas/genética , Sequência de Aminoácidos , Animais , Arenavirus do Velho Mundo/genética , Sequência de Bases , Dados de Sequência Molecular , Filogenia , RNA Viral/genética , Roedores/genética , Roedores/virologia , Zâmbia
13.
Vaccine ; 30(8): 1445-52, 2012 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-22234266

RESUMO

Lassa fever (LF) is one of the most prevalent viral hemorrhagic fevers in West Africa responsible for thousands of deaths annually. The BSL-4 containment requirement and lack of small animal model to evaluate Lassa virus (LASV)-specific cell-mediated immunity (CMI) complicate development of effective LF vaccines. Here we have described a CBA/J-ML29 model allowing evaluation of LASV-specific CMI responses in mice. This model is based on Mopeia virus reassortant clone ML29, an attractive immunogenic surrogate for LASV. A single intraperitoneal (i.p.) immunization of CBA/J mice with ML29 protected animals against a lethal homologous intracerebral (i.c.) challenge with 588 LD(50). The ML29-immunized mice displayed negligible levels of LASV-specific antibody titers, but LASV-specific CMI responses were detectable early and peaked on day 8-10 after immunization. A T cell cytotoxicity assay in vivo showed a correlation between LASV-specific cytotoxicity and the timing of protection induced by the ML29 immunization. Notably, CBA/J mice that received CD8+ T cell-depleted splenocytes from ML29-immunized donors all succumbed to a lethal i.c. challenge, demonstrating that CD8+ T cells are critical in protection. The CBA/J-ML29 model can be useful immunological tool for the preliminary evaluation of immunogenicity and efficacy of vaccine candidates against LASV outside of BSL-4 containment facilities.


Assuntos
Arenavirus do Velho Mundo/imunologia , Febre Lassa/prevenção & controle , Linfócitos T Citotóxicos/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Modelos Animais de Doenças , Injeções Intraperitoneais , Depleção Linfocítica/métodos , Camundongos , Camundongos Endogâmicos CBA , Vírus Reordenados/imunologia , Análise de Sobrevida
14.
Virology ; 423(1): 14-22, 2012 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-22154237

RESUMO

The cellular protease subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) is implicated in the proteolytic processing of the viral envelope glycoprotein precursor (GPC) of arenaviruses, a step strictly required for production of infectious progeny. The small molecule SKI-1/S1P inhibitor PF-429242 was shown to have anti-viral activity against Old World arenaviruses. Here we extended these studies and show that PF-429242 also inhibits GPC processing and productive infection of New World arenaviruses, making PF-429242 a broadly active anti-arenaviral drug. In combination therapy, PF-429242 potentiated the anti-viral activity of ribavirin, indicating a synergism between the two drugs. A hallmark of arenaviruses is their ability to establish persistent infection in vitro and in vivo. Notably, PF-429242 was able to efficiently and rapidly clear persistent infection by arenaviruses. Interruption of drug treatment did not result in re-emergence of infection, indicating that PF-429242 treatment leads to virus extinction.


Assuntos
Infecções por Arenaviridae/tratamento farmacológico , Infecções por Arenaviridae/enzimologia , Arenavirus do Velho Mundo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Pró-Proteína Convertases/antagonistas & inibidores , Pirrolidinas/farmacologia , Sequência de Aminoácidos , Infecções por Arenaviridae/virologia , Arenavirus do Velho Mundo/metabolismo , Sequência de Bases , Linhagem Celular , Humanos , Dados de Sequência Molecular , Pró-Proteína Convertases/genética , Pró-Proteína Convertases/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo
15.
Viruses ; 3(5): 613-9, 2011 05.
Artigo em Inglês | MEDLINE | ID: mdl-21994748

RESUMO

The continuing use of high-throughput assays to investigate cellular responses to infection is providing a large repository of information. Due to the large number of differentially expressed transcripts, often running into the thousands, the majority of these data have not been thoroughly investigated. Advances in techniques for the downstream analysis of high-throughput datasets are providing additional methods for the generation of additional hypotheses for further investigation. The large number of experimental observations, combined with databases that correlate particular genes and proteins with canonical pathways, functions and diseases, allows for the bioinformatic exploration of functional networks that may be implicated in replication or pathogenesis. Herein, we provide an example of how analysis of published high-throughput datasets of cellular responses to hemorrhagic fever virus infection can generate additional functional data. We describe enrichment of genes involved in metabolism, post-translational modification and cardiac damage; potential roles for specific transcription factors and a conserved involvement of a pathway based around cyclooxygenase-2. We believe that these types of analyses can provide virologists with additional hypotheses for continued investigation.


Assuntos
Infecções por Arenaviridae/metabolismo , Arenavirus do Velho Mundo/fisiologia , Bases de Dados Genéticas , Ebolavirus/fisiologia , Febres Hemorrágicas Virais/metabolismo , Animais , Infecções por Arenaviridae/genética , Infecções por Arenaviridae/virologia , Arenavirus do Velho Mundo/genética , Ebolavirus/genética , Febres Hemorrágicas Virais/genética , Febres Hemorrágicas Virais/virologia , Humanos , Transdução de Sinais
16.
J Virol ; 85(23): 12518-28, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21917982

RESUMO

Lassa virus (LASV) causing hemorrhagic Lassa fever in West Africa, Mopeia virus (MOPV) from East Africa, and lymphocytic choriomeningitis virus (LCMV) are the main representatives of the Old World arenaviruses. Little is known about how the components of the arenavirus replication machinery, i.e., the genome, nucleoprotein (NP), and L protein, interact. In addition, it is unknown whether these components can function across species boundaries. We established minireplicon systems for MOPV and LCMV in analogy to the existing LASV system and exchanged the components among the three systems. The functional and physical integrity of the resulting complexes was tested by reporter gene assay, Northern blotting, and coimmunoprecipitation studies. The minigenomes, NPs, and L proteins of LASV and MOPV could be exchanged without loss of function. LASV and MOPV L protein was also active in conjunction with LCMV NP, while the LCMV L protein required homologous NP for activity. Analysis of LASV/LCMV NP chimeras identified a single LCMV-specific NP residue (Ile-53) and the C terminus of NP (residues 340 to 558) as being essential for LCMV L protein function. The defect of LASV and MOPV NP in supporting transcriptional activity of LCMV L protein was not caused by a defect in physical NP-L protein interaction. In conclusion, components of the replication complex of Old World arenaviruses have the potential to functionally and physically interact across species boundaries. Residue 53 and the C-terminal domain of NP are important for function of L protein during genome replication and transcription.


Assuntos
Arenavirus do Velho Mundo/classificação , Arenavirus do Velho Mundo/genética , Replicação do DNA , DNA Viral/genética , Nucleoproteínas/metabolismo , Replicon/genética , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Infecções por Arenaviridae/genética , Infecções por Arenaviridae/metabolismo , Infecções por Arenaviridae/virologia , Northern Blotting , Western Blotting , Chlorocebus aethiops , Imunoprecipitação , Dados de Sequência Molecular , Nucleoproteínas/genética , RNA Viral/genética , Elementos Reguladores de Transcrição , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Ativação Transcricional , Células Vero , Proteínas Virais/genética
17.
J Virol ; 85(16): 8293-306, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21632749

RESUMO

The events leading to death in severe cases of Lassa fever (LF) are unknown. Fatality seems to be linked to high viremia and immunosuppression, and cellular immunity, rather than neutralizing antibodies, appears to be essential for survival. We previously compared Lassa virus (LV) with its genetically close but nonpathogenic homolog Mopeia virus (MV), which was used to model nonfatal LF. We showed that strong and early activation of antigen-presenting cells (APC) may play a crucial role in controlling infection. Here we developed an in vitro model of dendritic-cell (DC)-T-cell coculture in order to characterize human T-cell responses induced by MV- or LV-infected DCs. Our results show very different responses to infection with LV and MV. MV strongly and durably stimulated CD8(+) and CD4(+) T cells, showing early and high activation, a strong proliferative response, and acquisition of effector and memory phenotypes. Furthermore, robust and functional CD4(+) and CD8(+) cytotoxic T lymphocytes (CTL) were generated. LV, however, induced only weak memory responses. Thus, this study allows an improved understanding of the pathogenesis and immune mechanisms involved in the control of human LV.


Assuntos
Arenavirus do Velho Mundo/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/virologia , Linfócitos T Citotóxicos/imunologia , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Citocinas/biossíntese , Células Dendríticas/imunologia , Ensaio de Imunoadsorção Enzimática , ELISPOT , Citometria de Fluxo , Humanos , Memória Imunológica , Vírus Lassa/imunologia , Ativação Linfocitária , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
J Virol ; 85(7): 3631-41, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21248028

RESUMO

During virus particle assembly, the arenavirus nucleoprotein (NP) associates with the viral genome to form nucleocapsids, which ultimately become incorporated into new virions at the cell membrane. Virion release is facilitated by the viral matrix Z protein through its interaction with the cellular endosomal sorting complex required for transport (ESCRT) machinery. However, the mechanism of nucleocapsid incorporation into virions is not well understood. Here, we demonstrate that ALIX/AIP1, an ESCRT-associated host protein, is required for the incorporation of the NP of Mopeia virus, a close relative of Lassa virus, into Z-induced virus-like particles (VLPs). Furthermore, we show that the Bro1 domain of ALIX/AIP1 interacts with the NP and Z proteins simultaneously, facilitating their interaction, and we identify residues 342 to 399 of NP as being necessary for its interaction with ALIX/AIP1. Our observations suggest a potential role for ALIX/AIP1 in linking Mopeia virus NP to Z and the budding apparatus, thereby promoting NP incorporation into virions.


Assuntos
Arenavirus do Velho Mundo/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Nucleoproteínas/metabolismo , Proteínas Estruturais Virais/metabolismo , Virossomos/metabolismo , Montagem de Vírus , Animais , Linhagem Celular , Chlorocebus aethiops , Interações Hospedeiro-Patógeno , Humanos
19.
J Virol ; 84(19): 9947-56, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20668086

RESUMO

Arenaviruses cause severe human disease ranging from aseptic meningitis following lymphocytic choriomeningitis virus (LCMV) infection to hemorrhagic fever syndromes following infection with Guanarito virus (GTOV), Junin virus (JUNV), Lassa virus (LASV), Machupo virus (MACV), Sabia virus (SABV), or Whitewater Arroyo virus (WWAV). Cellular immunity, chiefly the CD8(+) T-cell response, plays a critical role in providing protective immunity following infection with the Old World arenaviruses LASV and LCMV. In the current study, we evaluated whether HLA class I-restricted epitopes that are cross-reactive among pathogenic arenaviruses could be identified for the purpose of developing an epitope-based vaccination approach that would cross-protect against multiple arenaviruses. We were able to identify a panel of HLA-A*0201-restricted peptides derived from the same region of the glycoprotein precursor (GPC) of LASV (GPC spanning residues 441 to 449 [GPC(441-449)]), LCMV (GPC(447-455)), JUNV (GPC(429-437)), MACV (GPC(444-452)), GTOV (GPC(427-435)), and WWAV (GPC(428-436)) that displayed high-affinity binding to HLA-A*0201 and were recognized by CD8(+) T cells in a cross-reactive manner following LCMV infection or peptide immunization of HLA-A*0201 transgenic mice. Immunization of HLA-A*0201 mice with the Old World peptide LASV GPC(441-449) or LCMV GPC(447-455) induced high-avidity CD8(+) T-cell responses that were able to kill syngeneic target cells pulsed with either LASV GPC(441-449) or LCMV GPC(447-455) in vivo and provided significant protection against viral challenge with LCMV. Through this study, we have demonstrated that HLA class I-restricted, cross-reactive epitopes exist among diverse arenaviruses and that individual epitopes can be utilized as effective vaccine determinants for multiple pathogenic arenaviruses.


Assuntos
Infecções por Arenaviridae/imunologia , Infecções por Arenaviridae/prevenção & controle , Arenavirus do Velho Mundo , Vacinas Virais/administração & dosagem , Sequência de Aminoácidos , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/virologia , Antígenos Virais/genética , Infecções por Arenaviridae/genética , Arenavirus do Novo Mundo/genética , Arenavirus do Novo Mundo/imunologia , Arenavirus do Novo Mundo/patogenicidade , Arenavirus do Velho Mundo/genética , Arenavirus do Velho Mundo/imunologia , Arenavirus do Velho Mundo/patogenicidade , Linfócitos T CD8-Positivos/imunologia , Reações Cruzadas , Citotoxicidade Imunológica , Epitopos/administração & dosagem , Epitopos/genética , Antígenos HLA-A/genética , Antígeno HLA-A2 , Humanos , Vírus Lassa/genética , Vírus Lassa/imunologia , Vírus Lassa/patogenicidade , Vírus da Coriomeningite Linfocítica/genética , Vírus da Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/patogenicidade , Camundongos , Camundongos Transgênicos , Vacinas Virais/genética , Vacinas Virais/imunologia
20.
J Virol ; 84(10): 5415-22, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20200234

RESUMO

Arenaviruses are enveloped, negative-strand RNA viruses. For several arenaviruses, virus-like particle (VLP) formation requires the viral matrix Z protein. However, the mechanism by which viral ribonucleoprotein complexes are incorporated into virions is poorly understood. Here, we show that the expression of the Z protein and nucleoprotein (NP) of Mopeia virus, a close relative of the pathogenic Lassa virus, resulted in the highly selective incorporation of the NP protein into Z protein-induced VLPs. Moreover, the Z protein promoted the association of NP with cellular membranes, suggesting that the association of NP, Z, and the cellular membranes may facilitate the efficient incorporation of NP into VLPs. By employing a series of NP deletion constructs and testing their VLP incorporation, we further demonstrated an important role for the C-terminal half of NP in its incorporation into VLPs.


Assuntos
Arenavirus do Velho Mundo/fisiologia , Nucleoproteínas/metabolismo , Proteínas da Matriz Viral/metabolismo , Montagem de Vírus , Animais , Linhagem Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Humanos , Nucleoproteínas/genética , Mapeamento de Interação de Proteínas , Deleção de Sequência , Virossomos/metabolismo
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