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1.
Nature ; 619(7969): 338-347, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37380775

RESUMO

Spillover events of avian influenza A viruses (IAVs) to humans could represent the first step in a future pandemic1. Several factors that limit the transmission and replication of avian IAVs in mammals have been identified. There are several gaps in our understanding to predict which virus lineages are more likely to cross the species barrier and cause disease in humans1. Here, we identified human BTN3A3 (butyrophilin subfamily 3 member A3)2 as a potent inhibitor of avian IAVs but not human IAVs. We determined that BTN3A3 is expressed in human airways and its antiviral activity evolved in primates. We show that BTN3A3 restriction acts primarily at the early stages of the virus life cycle by inhibiting avian IAV RNA replication. We identified residue 313 in the viral nucleoprotein (NP) as the genetic determinant of BTN3A3 sensitivity (313F or, rarely, 313L in avian viruses) or evasion (313Y or 313V in human viruses). However, avian IAV serotypes, such as H7 and H9, that spilled over into humans also evade BTN3A3 restriction. In these cases, BTN3A3 evasion is due to substitutions (N, H or Q) in NP residue 52 that is adjacent to residue 313 in the NP structure3. Thus, sensitivity or resistance to BTN3A3 is another factor to consider in the risk assessment of the zoonotic potential of avian influenza viruses.


Assuntos
Aves , Interações entre Hospedeiro e Microrganismos , Vírus da Influenza A , Influenza Aviária , Influenza Humana , Zoonoses Virais , Animais , Humanos , Aves/virologia , Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza A/isolamento & purificação , Influenza Aviária/transmissão , Influenza Aviária/virologia , Influenza Humana/prevenção & controle , Influenza Humana/transmissão , Influenza Humana/virologia , Primatas , Sistema Respiratório/metabolismo , Sistema Respiratório/virologia , Medição de Risco , Zoonoses Virais/prevenção & controle , Zoonoses Virais/transmissão , Zoonoses Virais/virologia , Replicação Viral
2.
PLoS Pathog ; 19(5): e1011357, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37146066

RESUMO

Synonymous recoding of RNA virus genomes is a promising approach for generating attenuated viruses to use as vaccines. Problematically, recoding typically hinders virus growth, but this may be rectified using CpG dinucleotide enrichment. CpGs are recognised by cellular zinc-finger antiviral protein (ZAP), and so in principle, removing ZAP sensing from a virus propagation system will reverse attenuation of a CpG-enriched virus, enabling high titre yield of a vaccine virus. We tested this using a vaccine strain of influenza A virus (IAV) engineered for increased CpG content in genome segment 1. Virus attenuation was mediated by the short isoform of ZAP, correlated with the number of CpGs added, and was enacted via turnover of viral transcripts. The CpG-enriched virus was strongly attenuated in mice, yet conveyed protection from a potentially lethal challenge dose of wildtype virus. Importantly for vaccine development, CpG-enriched viruses were genetically stable during serial passage. Unexpectedly, in both MDCK cells and embryonated hens' eggs that are used to propagate live attenuated influenza vaccines, the ZAP-sensitive virus was fully replication competent. Thus, ZAP-sensitive CpG enriched viruses that are defective in human systems can yield high titre in vaccine propagation systems, providing a realistic, economically viable platform to augment existing live attenuated vaccines.


Assuntos
Vírus da Influenza A , Vacinas contra Influenza , Vacinas Virais , Animais , Feminino , Humanos , Camundongos , Vírus da Influenza A/genética , Vacinas Atenuadas , Galinhas , Vacinas Virais/genética , Desenvolvimento de Vacinas , Replicação Viral
3.
PLoS Pathog ; 17(1): e1009255, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33508041

RESUMO

Cytomegalovirus (CMV) causes clinically important diseases in immune compromised and immune immature individuals. Based largely on work in the mouse model of murine (M)CMV, there is a consensus that myeloid cells are important for disseminating CMV from the site of infection. In theory, such dissemination should expose CMV to cell-mediated immunity and thus necessitate evasion of T cells and NK cells. However, this hypothesis remains untested. We constructed a recombinant MCMV encoding target sites for the hematopoietic specific miRNA miR-142-3p in the essential viral gene IE3. This virus disseminated poorly to the salivary gland following intranasal or footpad infections but not following intraperitoneal infection in C57BL/6 mice, demonstrating that dissemination by hematopoietic cells is essential for specific routes of infection. Remarkably, depletion of NK cells or T cells restored dissemination of this virus in C57BL/6 mice after intranasal infection, while dissemination occurred normally in BALB/c mice, which lack strong NK cell control of MCMV. These data show that cell-mediated immunity is responsible for restricting MCMV to hematopoietic cell-mediated dissemination. Infected hematopoietic cells avoided cell-mediated immunity via three immune evasion genes that modulate class I MHC and NKG2D ligands (m04, m06 and m152). MCMV lacking these 3 genes spread poorly to the salivary gland unless NK cells were depleted, but also failed to replicate persistently in either the nasal mucosa or salivary gland unless CD8+ T cells were depleted. Surprisingly, CD8+ T cells primed after intranasal infection required CD4+ T cell help to expand and become functional. Together, our data suggest that MCMV can use both hematopoietic cell-dependent and -independent means of dissemination after intranasal infection and that cell mediated immune responses restrict dissemination to infected hematopoietic cells, which are protected from NK cells during dissemination by viral immune evasion. In contrast, viral replication within mucosal tissues depends on evasion of T cells.


Assuntos
Infecções por Herpesviridae/imunologia , Evasão da Resposta Imune , Imunidade Celular , Muromegalovirus/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/virologia , Infecções por Herpesviridae/virologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Muromegalovirus/genética , Muromegalovirus/fisiologia , Replicação Viral
4.
BMC Biol ; 20(1): 14, 2022 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-35027054

RESUMO

BACKGROUND: Infectious diseases of farmed and wild animals pose a recurrent threat to food security and human health. The macrophage, a key component of the innate immune system, is the first line of defence against many infectious agents and plays a major role in shaping the adaptive immune response. However, this phagocyte is a target and host for many pathogens. Understanding the molecular basis of interactions between macrophages and pathogens is therefore crucial for the development of effective strategies to combat important infectious diseases. RESULTS: We explored how porcine pluripotent stem cells (PSCs) can provide a limitless in vitro supply of genetically and experimentally tractable macrophages. Porcine PSC-derived macrophages (PSCdMs) exhibited molecular and functional characteristics of ex vivo primary macrophages and were productively infected by pig pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV) and African swine fever virus (ASFV), two of the most economically important and devastating viruses in pig farming. Moreover, porcine PSCdMs were readily amenable to genetic modification by CRISPR/Cas9 gene editing applied either in parental stem cells or directly in the macrophages by lentiviral vector transduction. CONCLUSIONS: We show that porcine PSCdMs exhibit key macrophage characteristics, including infection by a range of commercially relevant pig pathogens. In addition, genetic engineering of PSCs and PSCdMs affords new opportunities for functional analysis of macrophage biology in an important livestock species. PSCs and differentiated derivatives should therefore represent a useful and ethical experimental platform to investigate the genetic and molecular basis of host-pathogen interactions in pigs, and also have wider applications in livestock.


Assuntos
Vírus da Febre Suína Africana , Doenças Transmissíveis , Vírus da Febre Suína Africana/genética , Animais , Interações Hospedeiro-Patógeno/genética , Macrófagos , Células-Tronco , Suínos
5.
PLoS Pathog ; 16(9): e1008844, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32886716

RESUMO

The genomes of RNA and small DNA viruses of vertebrates display significant suppression of CpG dinucleotide frequencies. Artificially increasing dinucleotide frequencies results in substantial attenuation of virus replication, suggesting that these compositional changes may facilitate recognition of non-self RNA sequences. Recently, the interferon inducible protein ZAP, was identified as the host factor responsible for sensing CpG in viral RNA, through direct binding and possibly downstream targeting for degradation. Using an arrayed interferon stimulated gene expression library screen, we identified ZAPS, and its associated factor TRIM25, as inhibitors of human cytomegalovirus (HCMV) replication. Exogenous expression of ZAPS and TRIM25 significantly reduced virus replication while knockdown resulted in increased virus replication. HCMV displays a strikingly heterogeneous pattern of CpG representation with specific suppression of CpG motifs within the IE1 major immediate early transcript which is absent in subsequently expressed genes. We demonstrated that suppression of CpG dinucleotides in the IE1 gene allows evasion of inhibitory effects of ZAP. We show that acute virus replication is mutually exclusive with high levels of cellular ZAP, potentially explaining the higher levels of CpG in viral genes expressed subsequent to IE1 due to the loss of pressure from ZAP in infected cells. Finally, we show that TRIM25 regulates alternative splicing between the ZAP short and long isoforms during HCMV infection and interferon induction, with knockdown of TRIM25 resulting in decreased ZAPS and corresponding increased ZAPL expression. These results demonstrate for the first time that ZAP is a potent host restriction factor against large DNA viruses and that HCMV evades ZAP detection through suppression of CpG dinucleotides within the major immediate early 1 transcript. Furthermore, TRIM25 is required for efficient upregulation of the interferon inducible short isoform of ZAP through regulation of alternative splicing.


Assuntos
Processamento Alternativo , Ilhas de CpG , Infecções por Citomegalovirus/metabolismo , Citomegalovirus/fisiologia , Regulação Viral da Expressão Gênica , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Replicação Viral , Linhagem Celular , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/patologia , Humanos , Proteínas Imediatamente Precoces , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
6.
J Gen Virol ; 102(5)2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34020727

RESUMO

Viral infections activate the powerful interferon (IFN) response that induces the expression of several hundred IFN stimulated genes (ISGs). The principal role of this extensive response is to create an unfavourable environment for virus replication and to limit spread; however, untangling the biological consequences of this large response is complicated. In addition to a seemingly high degree of redundancy, several ISGs are usually required in combination to limit infection as individual ISGs often have low to moderate antiviral activity. Furthermore, what ISG or combination of ISGs are antiviral for a given virus is usually not known. For these reasons, and since the function(s) of many ISGs remains unexplored, genome-wide approaches are well placed to investigate what aspects of this response result in an appropriate, virus-specific phenotype. This review discusses the advances screening approaches have provided for the study of host defence mechanisms, including clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9), ISG expression libraries and RNA interference (RNAi) technologies.


Assuntos
Antivirais/imunologia , Testes Genéticos , Transdução de Sinais/imunologia , Animais , Sistemas CRISPR-Cas , Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Imunidade Inata/genética , Imunidade Inata/imunologia , Interferons/genética , Interferons/imunologia , Interferência de RNA , Transdução de Sinais/genética , Replicação Viral/imunologia
7.
PLoS Pathog ; 13(5): e1006329, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28494016

RESUMO

The human cytomegalovirus major immediate early proteins IE1 and IE2 are critical drivers of virus replication and are considered pivotal in determining the balance between productive and latent infection. IE1 and IE2 are derived from the same primary transcript by alternative splicing and regulation of their expression likely involves a complex interplay between cellular and viral factors. Here we show that knockdown of the host ubiquitin-dependent segregase VCP/p97, results in loss of IE2 expression, subsequent suppression of early and late gene expression and, ultimately, failure in virus replication. RNAseq analysis showed increased levels of IE1 splicing, with a corresponding decrease in IE2 splicing following VCP knockdown. Global analysis of viral transcription showed the expression of a subset of viral genes is not reduced despite the loss of IE2 expression, including UL112/113. Furthermore, Immunofluorescence studies demonstrated that VCP strongly colocalised with the viral replication compartments in the nucleus. Finally, we show that NMS-873, a small molecule inhibitor of VCP, is a potent HCMV antiviral with potential as a novel host targeting therapeutic for HCMV infection.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Infecções por Citomegalovirus/virologia , Citomegalovirus/fisiologia , Replicação do DNA , Proteínas Imediatamente Precoces/metabolismo , Transativadores/metabolismo , Replicação Viral , Acetanilidas/farmacologia , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/farmacologia , Antivirais/farmacologia , Benzotiazóis/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/farmacologia , Núcleo Celular/metabolismo , Citomegalovirus/genética , Infecções por Citomegalovirus/tratamento farmacológico , Técnicas de Silenciamento de Genes , Humanos , Proteínas Imediatamente Precoces/genética , Glicoproteínas de Membrana/metabolismo , Análise de Sequência de RNA , Transativadores/genética , Ubiquitina/metabolismo , Proteína com Valosina
8.
J Gen Virol ; 96(Pt 4): 739-751, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25406174

RESUMO

The identification of virally encoded microRNAs (miRNAs) has had a major impact on the field of herpes virology. Given their ability to target cellular and viral transcripts, and the lack of immune response to small RNAs, miRNAs represent an ideal mechanism of gene regulation during viral latency and persistence. In this review, we discuss the role of miRNAs in virus latency and persistence, specifically focusing on herpesviruses. We cover the current knowledge on miRNAs in establishing and maintaining virus latency and promoting survival of infected cells through targeting of both viral and cellular transcripts, highlighting key publications in the field. We also discuss potential areas of future research and how novel technologies may aid in determining how miRNAs shape virus latency in the context of herpesvirus infections.


Assuntos
Herpesviridae/genética , MicroRNAs/genética , Animais , Regulação Viral da Expressão Gênica , Herpesviridae/fisiologia , Humanos , RNA Viral/genética , Latência Viral/genética
9.
PLoS Pathog ; 9(12): e1003820, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24385903

RESUMO

Recent advances in microRNA target identification have greatly increased the number of putative targets of viral microRNAs. However, it is still unclear whether all targets identified are biologically relevant. Here, we use a combined approach of RISC immunoprecipitation and focused siRNA screening to identify targets of HCMV encoded human cytomegalovirus that play an important role in the biology of the virus. Using both a laboratory and clinical strain of human cytomegalovirus, we identify over 200 putative targets of human cytomegalovirus microRNAs following infection of fibroblast cells. By comparing RISC-IP profiles of miRNA knockout viruses, we have resolved specific interactions between human cytomegalovirus miRNAs and the top candidate target transcripts and validated regulation by western blot analysis and luciferase assay. Crucially we demonstrate that miRNA target genes play important roles in the biology of human cytomegalovirus as siRNA knockdown results in marked effects on virus replication. The most striking phenotype followed knockdown of the top target ATP6V0C, which is required for endosomal acidification. siRNA knockdown of ATP6V0C resulted in almost complete loss of infectious virus production, suggesting that an HCMV microRNA targets a crucial cellular factor required for virus replication. This study greatly increases the number of identified targets of human cytomegalovirus microRNAs and demonstrates the effective use of combined miRNA target identification and focused siRNA screening for identifying novel host virus interactions.


Assuntos
Citomegalovirus/fisiologia , Interações Hospedeiro-Patógeno/genética , MicroRNAs/genética , ATPases Vacuolares Próton-Translocadoras/fisiologia , Replicação Viral/genética , Células Cultivadas , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/genética , Perfilação da Expressão Gênica , Células HEK293 , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Análise em Microsséries , Organismos Geneticamente Modificados , RNA Interferente Pequeno/farmacologia , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , Proteínas Virais/genética , Replicação Viral/efeitos dos fármacos
10.
J Immunol ; 190(7): 3410-6, 2013 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-23455500

RESUMO

The unique ability of CMV to drive the expansion of virus-specific T cell populations during the course of a lifelong, persistent infection has generated interest in the virus as a potential vaccine strategy. When designing CMV-based vaccine vectors to direct immune responses against HIV or tumor Ags, it becomes important to understand how and why certain CMV-specific populations are chosen to inflate over time. To investigate this, we designed recombinant murine CMVs (MCMVs) encoding a SIINFEKL-enhanced GFP fusion protein under the control of endogenous immediate early promoters. When mice were infected with these viruses, T cells specific for the SIINFEKL epitope inflated and profoundly dominated T cells specific for nonrecombinant (i.e., MCMV-derived) Ags. Moreover, when the virus encoded SIINFEKL, T cells specific for nonrecombinant Ags displayed a phenotype indicative of less frequent exposure to Ag. The immunodominance of SIINFEKL-specific T cells could not be altered by decreasing the number of SIINFEKL-specific cells available to respond, or by increasing the number of cells specific for endogenous MCMV Ags. In contrast, coinfection with viruses expressing and lacking SIINFEKL enabled coinflation of T cells specific for both SIINFEKL and nonrecombinant Ags. Because coinfection allows presentation of SIINFEKL and MCMV-derived Ags by different cells within the same animal, these data reveal that competition for, or availability of, Ag at the level of the APC determines the composition of the inflationary response to MCMV. SIINFEKL's strong affinity for H-2K(b), as well as its early and abundant expression, may provide this epitope's competitive advantage.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Antígenos/imunologia , Infecções por Herpesviridae/imunologia , Epitopos Imunodominantes/imunologia , Memória Imunológica , Muromegalovirus/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Expressão Gênica , Antígenos H-2/imunologia , Antígenos H-2/metabolismo , Imunofenotipagem , Camundongos , Muromegalovirus/genética , Ovalbumina/química , Ovalbumina/genética , Ovalbumina/imunologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fenótipo
11.
Proc Natl Acad Sci U S A ; 109(1): 279-84, 2012 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-22184245

RESUMO

Individual microRNAs (miRNAs) are rapidly down-regulated during conditions of cellular activation and infection, but factors mediating miRNA turnover are poorly understood. Infection of mouse cells with murine cytomegalovirus (MCMV) induces the rapid down-regulation of an antiviral cellular miRNA, miR-27. Here, we identify a transcript produced by MCMV that binds to miR-27 and mediates its degradation. UV-crosslinking and high-throughput sequencing [CRAC (UV-crosslinking and analysis of cDNA)] identified MCMV RNA segments associated with the miRNA-binding protein Argonaute 2 (Ago2). A cluster of hits mapped to a predicted miR-27-binding site in the 3'UTR of the previously uncharacterized ORF, m169. The expression kinetics of the m169 transcript correlated with degradation of miR-27 during infection, and m169 expression inhibited miR-27 functional activity in a reporter assay. siRNA knockdown of m169 demonstrated its requirement for miR-27 degradation following infection and did not affect other host miRNAs. Substitution of the miR-27-binding site in m169 to create complementarity to a different cellular miRNA, miR-24, resulted in down-regulation of only miR-24 following infection. The m169 transcript is cytoplasmic, capped, polyadenylated, and interacts with miRNA-27 through seed pairing: characteristic features of the normal messenger RNA (mRNA) targets of miRNAs. This virus-host interaction reveals a mode of miRNA regulation in which a mRNA directs the degradation of a miRNA. We speculate that RNA-mediated miRNA degradation could be a more general viral strategy for manipulating host cells.


Assuntos
MicroRNAs/antagonistas & inibidores , Muromegalovirus/genética , Regiões 3' não Traduzidas/genética , Animais , Proteínas Argonautas/metabolismo , Sequência de Bases , Sítios de Ligação , Reagentes de Ligações Cruzadas/metabolismo , Citoplasma/metabolismo , DNA Complementar/genética , Regulação da Expressão Gênica/efeitos da radiação , Ensaios de Triagem em Larga Escala , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Dados de Sequência Molecular , Muromegalovirus/efeitos da radiação , Células NIH 3T3 , Nucleotídeos/genética , Estabilidade de RNA/genética , Estabilidade de RNA/efeitos da radiação , Transporte de RNA/efeitos da radiação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , Transcriptoma/genética , Raios Ultravioleta
12.
J Gen Virol ; 95(Pt 2): 472-480, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24172907

RESUMO

Herpesviruses encode microRNAs (miRNAs) that target both virus and host genes; however, their role in herpesvirus biology is understood poorly. We identified previously eight miRNAs encoded by ovine herpesvirus-2 (OvHV-2), the causative agent of malignant catarrhal fever (MCF), and have now investigated the role of these miRNAs in regulating expression of OvHV-2 genes that play important roles in virus biology. ORF20 (cell cycle inhibition), ORF50 (reactivation) and ORF73 (latency maintenance) each contain predicted targets for several OvHV-2 miRNAs. Co-transfection of miRNA mimics with luciferase reporter constructs containing the predicted targets showed the 5' UTRs of ORF20 and ORF73 contain functional targets for ovhv-miR-2 and ovhv2-miR-8, respectively, and the 3' UTR of ORF50 contains a functional target for ovhv2-miR-5. Transfection of BJ1035 cells (an OvHV-2-infected bovine T-cell line) with the relevant miRNA mimic resulted in a significant decrease in ORF50 and a smaller but non-significant decrease in ORF20. However, we were unable to demonstrate a decrease in ORF73. MCF is a disease of dysregulated lymphocyte proliferation; miRNA inhibition of ORF20 expression may play a role in this aberrant lymphocyte proliferation. The proteins encoded by ORF50 and ORF73 play opposing roles in latency. It has been hypothesized that miRNA-induced inhibition of virus genes acts to ensure that fluctuations in virus mRNA levels do not result in reactivation under conditions that are unfavourable for viral replication and our data supported this hypothesis.


Assuntos
Regulação Viral da Expressão Gênica , Herpesviridae/genética , Herpesviridae/fisiologia , MicroRNAs/genética , Proteínas Virais/genética , Latência Viral , Animais , Bovinos , Linhagem Celular , MicroRNAs/metabolismo , Linfócitos T/virologia , Proteínas Virais/metabolismo
13.
Eur J Immunol ; 43(5): 1252-63, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23404526

RESUMO

Both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV) establish persistent infections that induce the accumulation of virus-specific T cells over time in a process called memory inflation. It has been proposed that T cells expressing T-cell receptors (TCRs) with high affinity for HCMV-derived peptides are preferentially selected after acute HCMV infection. To test this in the murine model, small numbers of OT-I transgenic T cells, which express a TCR with high affinity for the SIINFEKL peptide, were transferred into congenic mice and recipients were challenged with recombinant MCMV expressing SIINFEKL. OT-I T cells were selectively enriched during the first 3 weeks of infection. Similarly, in the absence of OT-I T cells, the functional avidity of SIINFEKL-specific T cells increased from early to late times postinfection. However, even when exceedingly small numbers of OT-I T cells were transferred, their inflation limited the inflation of host-derived T cells specific for SIINFEKL. Importantly, subtle minor histocompatibility differences led to late rejection of the transferred OT-I T cells in some mice, which allowed host-derived T cells to inflate substantially. Thus, T cells with a high functional avidity are selected shortly after MCMV infection and continuously sustain their clonal dominance in a competitive manner.


Assuntos
Infecções por Herpesviridae/imunologia , Epitopos Imunodominantes/imunologia , Memória Imunológica , Muromegalovirus , Peptídeos/imunologia , Linfócitos T/imunologia , Transferência Adotiva , Animais , Rastreamento de Células , Células Clonais , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/virologia , Epitopos Imunodominantes/genética , Camundongos , Camundongos Transgênicos , Peptídeos/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/citologia , Linfócitos T/transplante , Fatores de Tempo
14.
Biochem Biophys Res Commun ; 437(2): 287-91, 2013 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-23811402

RESUMO

Cytomegalovirus (CMV) induces strong and long-lasting immune responses, which make it an attractive candidate for a cancer vaccine vector. In this study, we tested whether a tumor antigen expressed in CMV can induce a strong anti-tumor effect. We expressed an unmodified melanoma antigen, mouse tyrosinase-related protein 2 (TRP2), in mouse cytomegalovirus (MCMV). Prophylactic vaccination of the mice with a single dose of MCMV-TRP2 induced rejection of B16 melanoma challenge; therapeutic vaccination with MCMV-TRP2 prolonged the survival of the mice challenged with B16 cells. Additionally, vaccination with MCMV-TRP2 five months before tumor challenge still induced tumor rejection, which indicated that the vaccine induced long-term protection. Furthermore, MCMV-TRP2 protected mice against B16 melanoma challenge regardless of the pre-existing CMV infection. We found that vaccination with MCMV-TRP2 induced long-lasting TRP2 specific antibodies but not CD8 T cells. In addition, depletion of CD4 and CD8 T cells did not compromise the antitumor effect by MCMV-TRP2; while in B cell deficient (µMT) mice, the vaccine lost its antitumor effect. These results indicate that antibodies, not T cells, are important in mediating the antitumor effect during the effector phase by the vaccine. We also made a spread deficient MCMV-TRP2 lacking the essential glycoprotein gL, which showed a similar antitumor effect. In conclusion, our study indicates that tumor antigen (TRP2) expressed in MCMV induces a strong and long-lasting anti-melanoma effect through an antibody-dependent mechanism. Our findings demonstrate that CMV might be a promising vector for the development of cancer vaccines.


Assuntos
Vacinas Anticâncer/uso terapêutico , Oxirredutases Intramoleculares/genética , Melanoma Experimental/prevenção & controle , Animais , Vacinas Anticâncer/genética , Feminino , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL
15.
J Virol ; 86(9): 5278-87, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22345437

RESUMO

MicroRNAs (miRNAs) are a class of noncoding small RNAs that regulate multiple cellular processes, as well as the replication and pathogenesis of many DNA viruses and some RNA viruses. Examination of cellular miRNA profiles in West Nile virus (WNV)-infected HEK293 and SK-N-MC cells revealed increased expression of multiple miRNA species. One of these miRNAs, Hs_154, was significantly induced not only in WNV-infected neuronal cells in culture but also in the central nervous system tissues of infected mice and, upon transfection, caused a significant reduction in viral replication. Analysis of mRNA transcripts enriched through immunoprecipitation of the RNA-induced silencing complex identified several transcripts that contain seed sequence matches to Hs_154 in their 3' untranslated regions (UTRs). Two of these targets, the CCCTC-binding factor (CTCF) and the epidermal growth factor receptor (EGFR)-coamplified and overexpressed protein (ECOP/VOPP1) proteins display reduced expression in WNV-infected cells, and the 3' UTRs of these transcripts were sufficient to cause downregulation of expression in infected cells or in cells transfected with Hs_154, findings consistent with miRNA targeting of these transcripts. CTCF and ECOP have been shown to be associated with cell survival, implicating miRNA-directed repression of these targets in WNV-induced cell death. Consistent with this hypothesis, expression of these genes in WNV-infected cells results in a reduction in the number of cells undergoing apoptosis. These observations suggest that induction of Hs_154 expression after WNV infection modulates the apoptotic response to WNV and that cellular miRNA expression can be quickly altered during WNV infection to control aspects of the host response.


Assuntos
Apoptose/genética , MicroRNAs/biossíntese , Vírus do Nilo Ocidental/fisiologia , Animais , Sequência de Bases , Fator de Ligação a CCCTC , Caspases/metabolismo , Linhagem Celular , Análise por Conglomerados , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Interferons/metabolismo , Cinética , Camundongos , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Replicação Viral/genética
16.
Sci Rep ; 13(1): 10342, 2023 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-37604847

RESUMO

African swine fever virus (ASFV) is a lethal animal pathogen that enters its host cells through endocytosis. So far, host factors specifically required for ASFV replication have been barely identified. In this study a genome-wide CRISPR/Cas9 knockout screen in porcine cells indicated that the genes RFXANK, RFXAP, SLA-DMA, SLA-DMB, and CIITA are important for productive ASFV infection. The proteins encoded by these genes belong to the major histocompatibility complex II (MHC II), or swine leucocyte antigen complex II (SLA II). RFXAP and CIITA are MHC II-specific transcription factors, whereas SLA-DMA/B are subunits of the non-classical MHC II molecule SLA-DM. Targeted knockout of either of these genes led to severe replication defects of different ASFV isolates, reflected by substantially reduced plating efficiency, cell-to-cell spread, progeny virus titers and viral DNA replication. Transgene-based reconstitution of SLA-DMA/B fully restored the replication capacity demonstrating that SLA-DM, which resides in late endosomes, plays a crucial role during early steps of ASFV infection.


Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Traumatismos Craniocerebrais , Animais , Suínos , Vírus da Febre Suína Africana/genética , Replicação do DNA , DNA Viral , Replicação Viral/genética , Antígenos de Histocompatibilidade Classe II/genética , Proteínas de Membrana , Complexo Principal de Histocompatibilidade , Febre Suína Africana/genética
17.
J Virol ; 85(22): 11938-44, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21900172

RESUMO

Human cytomegalovirus (HCMV) encodes at least 14 microRNAs (miRNAs) that act posttranscriptionally to repress gene expression. Although several HCMV miRNA targets of both cellular and viral origin have been identified, our knowledge of their function remains limited. HCMV miRNA targets, as well as phenotypes associated with HCMV miRNA mutants, have been difficult to identify since the downregulation of targets by a single miRNA is often less than 2-fold. Several factors can contribute to the strength of repression, including the mechanism of translational inhibition, the degree of complementarity between the miRNA and target mRNA, the number of binding sites for one miRNA, and cooperativity or antagonism between miRNAs. To determine the effect of multiple miRNAs on one gene, we examined the repression of a viral gene, US7. Here we demonstrate that the HCMV-encoded miRNAs miR-US5-1 and miR-US5-2 function in a highly synergistic manner to regulate US7, even at very low miRNA concentrations. Regulation of US7 involves three functional miRNA binding sites: two that are completely complementary to the 3' untranslated region (3'UTR) and one that is imperfectly matched. Surprisingly, we observed equal contributions to inhibition from both complete and partially complementary sites, and repression was not completely abrogated until all three sites were mutated simultaneously. We also observed that the miRNA binding sites did not follow the spacing constraints for corepressive miRNAs observed in earlier reports. These results underscore the importance of evaluating the contribution of multiple miRNAs on gene regulation and shed new insight into miRNA:mRNA interactions.


Assuntos
Citomegalovirus/fisiologia , Regulação Viral da Expressão Gênica , Glicoproteínas de Membrana/biossíntese , MicroRNAs/metabolismo , Proteínas Virais/biossíntese , Regiões 3' não Traduzidas , Sítios de Ligação , Humanos
18.
J Virol ; 85(1): 378-89, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20980502

RESUMO

MicroRNAs (miRNAs) are a class of small noncoding RNAs involved in posttranscriptional regulation. miRNAs are utilized in organisms ranging from plants to higher mammals, and data have shown that DNA viruses also use this method for host and viral gene regulation. Here, we report the sequencing of the small RNAs in rat cytomegalovirus (RCMV)-infected fibroblasts and persistently infected salivary glands. We identified 24 unique miRNAs that mapped to hairpin structures found within the viral genome. While most miRNAs were detected in both samples, four were detected exclusively in the infected fibroblasts and two were specific for the infected salivary glands. The RCMV miRNAs are distributed across the viral genome on both the positive and negative strands, with clusters of miRNAs at a number of locations, including near viral genes r1 and r111. The RCMV miRNAs have a genomic positional orientation similar to that of the miRNAs described for mouse cytomegalovirus, but they do not share any substantial sequence conservation. Similar to other reported miRNAs, the RCMV miRNAs had considerable variation at their 3' and 5' ends. Interestingly, we found a number of specific examples of differential isoform usage between the fibroblast and salivary gland samples. We determined by real-time PCR that expression of the RCMV miRNA miR-r111.1-2 is highly expressed in the salivary glands and that miR-R87-1 is expressed in most tissues during the acute infection phase. Our study identified the miRNAs expressed by RCMV in vitro and in vivo and demonstrated that expression is tissue specific and associated with a stage of viral infection.


Assuntos
Fibroblastos/virologia , Infecções por Herpesviridae/virologia , MicroRNAs/metabolismo , Muromegalovirus/patogenicidade , Glândulas Salivares/virologia , Animais , Células Cultivadas , Fibroblastos/metabolismo , Transplante de Coração , Camundongos , MicroRNAs/genética , Muromegalovirus/genética , Muromegalovirus/metabolismo , Especificidade de Órgãos , Ratos , Glândulas Salivares/metabolismo , Transplante Homólogo
19.
PLoS Pathog ; 6(6): e1000967, 2010 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-20585629

RESUMO

Global gene expression data combined with bioinformatic analysis provides strong evidence that mammalian miRNAs mediate repression of gene expression primarily through binding sites within the 3' untranslated region (UTR). Using RNA induced silencing complex immunoprecipitation (RISC-IP) techniques we have identified multiple cellular targets for a human cytomegalovirus (HCMV) miRNA, miR-US25-1. Strikingly, this miRNA binds target sites primarily within 5'UTRs, mediating significant reduction in gene expression. Intriguingly, many of the genes targeted by miR-US25-1 are associated with cell cycle control, including cyclin E2, BRCC3, EID1, MAPRE2, and CD147, suggesting that miR-US25-1 is targeting genes within a related pathway. Deletion of miR-US25-1 from HCMV results in over expression of cyclin E2 in the context of viral infection. Our studies demonstrate that a viral miRNA mediates translational repression of multiple cellular genes by targeting mRNA 5'UTRs.


Assuntos
Regiões 5' não Traduzidas/genética , Proteínas de Ciclo Celular/genética , Regulação da Expressão Gênica , MicroRNAs/genética , RNA Mensageiro/genética , RNA Viral/genética , Replicação Viral/genética , Western Blotting , Células Cultivadas , Ciclinas/antagonistas & inibidores , Ciclinas/genética , Ciclinas/metabolismo , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Regulação para Baixo , Humanos , Rim/citologia , Rim/metabolismo , Luciferases/metabolismo , Complexo de Inativação Induzido por RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
20.
Front Vet Sci ; 9: 868912, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35450136

RESUMO

Animal trypanosomiasis (AT) is a significant livestock disease, affecting millions of animals across Sub-Saharan Africa, Central and South America, and Asia, and is caused by the protozoan parasites Trypanosoma brucei, Trypanosoma vivax, and Trypanosoma congolense, with the largest economic impact in cattle. There is over-reliance on presumptive chemotherapy due to inadequate existing diagnostic tests, highlighting the need for improved AT diagnostics. A small RNA species, the 7SL sRNA, is excreted/secreted by trypanosomes in infected animals, and has been previously shown to reliably diagnose active infection. We sought to explore key properties of 7SL sRNA RT-qPCR assays; namely, assessing the potential for cross-reaction with the widespread and benign Trypanosoma theileri, directly comparing assay performance against currently available diagnostic methods, quantitatively assessing specificity and sensitivity, and assessing the rate of decay of 7SL sRNA post-treatment. Results showed that the 7SL sRNA RT-qPCR assays specific for T. brucei, T. vivax, and T. congolense performed better than microscopy and DNA PCR in detecting infection. The 7SL sRNA signal was undetectable or significantly reduced by 96-h post treatment; at 1 × curative dose there was no detectable signal in 5/5 cattle infected with T. congolense, and in 3/5 cattle infected with T. vivax, with the signal being reduced 14,630-fold in the remaining two T. vivax cattle. Additionally, the assays did not cross-react with T. theileri. Finally, by using a large panel of validated infected and uninfected samples, the species-specific assays are shown to be highly sensitive and specific by receiver operating characteristic (ROC) analysis, with 100% sensitivity (95% CI, 96.44-100%) and 100% specificity (95% CI, 96.53-100%), 96.73% (95% CI, 95.54-99.96%) and 99.19% specificity (95% CI, 92.58-99.60%), and 93.42% (95% CI, 85.51-97.16% %) and 82.43% specificity (95% CI, 72.23-89.44% %) for the T brucei, T. congolense and T. vivax assays, respectively, under the conditions used. These findings indicate that the 7SL sRNA has many attributes that would be required for a potential diagnostic marker of AT: no cross-reaction with T. theileri, high specificity and sensitivity, early infection detection, continued signal even in the absence of detectable parasitaemia in blood, and clear discrimination between infected and treated animals.

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