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1.
Arch Virol ; 165(3): 557-570, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32036428

RESUMO

Codon usage bias (CUB) arises from the preference for a codon over codons for the same amino acid. The major factors contributing to CUB are evolutionary forces, compositional properties, gene expression, and protein properties. The present analysis was performed to investigate the compositional properties and the extent of CUB across the genomes of members of the family Hepadnaviridae, as previously no work using bioinformatic tools has been reported. The viral genes were found to be AT rich with low CUB. Analysis of relative synonymous codon usage (RSCU) was used to identify overrepresented and underrepresented codons for each amino acid. Correlation analysis of overall nucleotide composition and its composition at the third codon position suggested that mutation pressure might influence the CUB. A highly significant correlation was observed between GC12 and GC3 (r = 0.910, p < 0.01), indicating that directional mutation affected all three codon positions across the genome. Translational selection (P2) and mutational responsive index (MRI) values of genes suggested that mutation plays a more important role than translational selection in members of the family Hepadnaviridae.


Assuntos
Regulação Viral da Expressão Gênica/fisiologia , Genoma Viral/fisiologia , Hepadnaviridae/metabolismo , Proteínas Virais/metabolismo , Evolução Biológica , Hepadnaviridae/genética , Mutação , RNA Mensageiro , RNA Viral , Especificidade da Espécie , Proteínas Virais/genética
2.
PLoS Pathog ; 15(12): e1008192, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31809522

RESUMO

The hypoxia-inducible factor 1 alpha (HIF1α) protein and the hypoxic microenvironment are critical for infection and pathogenesis by the oncogenic gammaherpesviruses (γHV), Kaposi sarcoma herpes virus (KSHV) and Epstein-Barr virus (EBV). However, understanding the role of HIF1α during the virus life cycle and its biological relevance in the context of host has been challenging due to the lack of animal models for human γHV. To study the role of HIF1α, we employed the murine gammaherpesvirus 68 (MHV68), a rodent pathogen that readily infects laboratory mice. We show that MHV68 infection induces HIF1α protein and HIF1α-responsive gene expression in permissive cells. siRNA silencing or drug-inhibition of HIF1α reduce virus production due to a global downregulation of viral gene expression. Most notable was the marked decrease in many viral genes bearing hypoxia-responsive elements (HREs) such as the viral G-Protein Coupled Receptor (vGPCR), which is known to activate HIF1α transcriptional activity during KSHV infection. We found that the promoter of MHV68 ORF74 is responsive to HIF1α and MHV-68 RTA. Moreover, Intranasal infection of HIF1αLoxP/LoxP mice with MHV68 expressing Cre- recombinase impaired virus expansion during early acute infection and affected lytic reactivation in the splenocytes explanted from mice. Low oxygen concentrations accelerated lytic reactivation and enhanced virus production in MHV68 infected splenocytes. Thus, we conclude that HIF1α plays a critical role in promoting virus replication and reactivation from latency by impacting viral gene expression. Our results highlight the importance of the mutual interactions of the oxygen-sensing machinery and gammaherpesviruses in viral replication and pathogenesis.


Assuntos
Regulação Viral da Expressão Gênica/fisiologia , Infecções por Herpesviridae/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Latência Viral/fisiologia , Replicação Viral/fisiologia , Animais , Camundongos , Rhadinovirus/metabolismo
3.
PLoS Pathog ; 15(12): e1008180, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31841560

RESUMO

Adenoviral vectors have shown significant promise as vaccine delivery vectors due to their ability to elicit both innate and adaptive immune responses. α-defensins are effector molecules of the innate immune response and have been shown to modulate natural infection with adenoviruses, but the majority of α-defensin-adenovirus interactions studied to date have only been analyzed in vitro. In this study, we evaluated the role of α-defensin 5 (HD5) in modulating adenovirus vaccine immunogenicity using various serotype adenovirus vectors in mice. We screened a panel of human adenoviruses including Ad5 (species C), Ad26 (species D), Ad35 (species B), Ad48 (species D) and a chimeric Ad5HVR48 for HD5 sensitivity. HD5 inhibited transgene expression from Ad5 and Ad35 but augmented transgene expression from Ad26, Ad48, and Ad5HVR48. HD5 similarly suppressed antigen-specific IgG and CD8+ T cell responses elicited by Ad5 vectors in mice, but augmented IgG and CD8+ T cell responses and innate cytokine responses elicited by Ad26 vectors in mice. Moreover, HD5 suppressed the protective efficacy of Ad5 vectors but enhanced the protective efficacy of Ad26 vectors expressing SIINFEKL against a surrogate Listeria-OVA challenge in mice. These data demonstrate that HD5 differentially modulates adenovirus vaccine delivery vectors in a species-specific manner in vivo.


Assuntos
Adenoviridae/imunologia , Regulação Viral da Expressão Gênica/fisiologia , alfa-Defensinas , Células A549 , Adenoviridae/genética , Animais , Vetores Genéticos , Humanos , Camundongos
4.
PLoS Pathog ; 15(6): e1007827, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31181119

RESUMO

P10 is a small, abundant baculovirus protein that accumulates to high levels in the very late stages of the infection cycle. It is associated with a number of intracellular structures and implicated in diverse processes from occlusion body maturation to nuclear stability and lysis. However, studies have also shown that it is non-essential for virus replication, at least in cell culture. Here, we describe the use of serial block-face scanning electron microscopy to achieve high-resolution 3D characterisation of P10 structures within Trichoplusia ni TN-368 cells infected with Autographa californica multiple nucleopolyhedrovirus. This has enabled unparalleled visualisation of P10 and determined the independent formation of dynamic perinuclear and nuclear vermiform fibrous structures. Our 3D data confirm the sequence of ultrastructural changes that create a perinuclear cage from thin angular fibrils within the cytoplasm. Over the course of infection in cultured cells, the cage remodels to form a large polarised P10 mass and we suggest that these changes are critical for nuclear lysis to release occlusion bodies. In contrast, nuclear P10 forms a discrete vermiform structure that was observed in close spatial association with both electron dense spacers and occlusion bodies; supporting a previously suggested role for P10 and electron dense spacers in the maturation of occlusion bodies. We also demonstrate that P10 hyper-expression is critical for function. Decreasing levels of p10 expression, achieved by manipulation of promoter length, correlated with reduced P10 production, a lack of formation of P10 structures and a concomitant decrease in nuclear lysis.


Assuntos
Núcleo Celular/metabolismo , Regulação Viral da Expressão Gênica/fisiologia , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Núcleo Celular/virologia , Mariposas , /genética , Domínios Proteicos , Proteínas Virais/química , Proteínas Virais/genética
5.
PLoS Pathog ; 15(6): e1007849, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31166996

RESUMO

Virus-host interactions are frequently studied in bulk cell populations, obscuring cell-to-cell variation. Here we investigate endogenous herpesvirus gene expression at the single-cell level, combining a sensitive and robust fluorescent in situ hybridization platform with multiparameter flow cytometry, to study the expression of gammaherpesvirus non-coding RNAs (ncRNAs) during lytic replication, latent infection and reactivation in vitro. This method allowed robust detection of viral ncRNAs of murine gammaherpesvirus 68 (γHV68), Kaposi's sarcoma associated herpesvirus and Epstein-Barr virus, revealing variable expression at the single-cell level. By quantifying the inter-relationship of viral ncRNA, viral mRNA, viral protein and host mRNA regulation during γHV68 infection, we find heterogeneous and asynchronous gene expression during latency and reactivation, with reactivation from latency identified by a distinct gene expression profile within rare cells. Further, during lytic replication with γHV68, we find many cells have limited viral gene expression, with only a fraction of cells showing robust gene expression, dynamic RNA localization, and progressive infection. Lytic viral gene expression was enhanced in primary fibroblasts and by conditions associated with enhanced viral replication, with multiple subpopulations of cells present in even highly permissive infection conditions. These findings, powered by single-cell analysis integrated with automated clustering algorithms, suggest inefficient or abortive γHV infection in many cells, and identify substantial heterogeneity in viral gene expression at the single-cell level.


Assuntos
Gammaherpesvirinae/fisiologia , Regulação Viral da Expressão Gênica/fisiologia , Infecções por Herpesviridae/metabolismo , RNA Mensageiro/biossíntese , RNA não Traduzido/biossíntese , RNA Viral/biossíntese , Replicação Viral/fisiologia , Animais , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/patologia , Humanos , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , RNA não Traduzido/genética , RNA Viral/genética
7.
EMBO J ; 38(12)2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31068361

RESUMO

Several autoimmune diseases including multiple sclerosis (MS) cause increased transcription of endogenous retroviruses (HERVs) normally repressed by heterochromatin. In parallel, HERV-derived sequences were reported to drive gene expression. Here, we have examined a possible link between promoter and enhancer divergent transcription and the production of HERV transcripts. We find that HERV-derived sequences are in general counter-selected at regulatory regions, a counter-selection that is strongest in brain tissues while very moderate in stem cells. By exposing T cells to the pesticide dieldrin, we further found that a series of HERV-driven enhancers otherwise active only at stem cell stages can be reactivated by stress. This in part relies on peptidylarginine deiminase activity, possibly participating in the reawakening of silenced enhancers. Likewise, usage of HERV-driven enhancers was increased in myelin-reactive T cells from patients with MS, correlating with activation of nearby genes at several sites. Altogether, we propose that HERV-driven enhancers constitute a reservoir of auxiliary enhancers transiently induced by stress while chronically active in diseases like MS.


Assuntos
Retrovirus Endógenos/genética , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Sequências Reguladoras de Ácido Nucleico/genética , Linfócitos T/metabolismo , Adulto , Estudos de Casos e Controles , Células Cultivadas , Feminino , Regulação Viral da Expressão Gênica/fisiologia , Humanos , Células Jurkat , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/metabolismo , Esclerose Múltipla/virologia , Linfócitos T/patologia
8.
PLoS One ; 14(4): e0215394, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30998737

RESUMO

The oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) has two distinct life cycles with lifelong latent/non-productive and a sporadic lytic-reactivating/productive phases in the infected immune compromised human hosts. The virus reactivates from latency in response to various chemical or environmental stimuli, which triggers the lytic cascade and leads to the expression of immediate early gene, i.e. Replication and Transcription Activator (K-RTA). K-RTA, the latent-to-lytic switch protein, activates the expression of early (E) and late (L) lytic genes by transactivating multiple viral promoters. Expression of K-RTA is shown to be sufficient and essential to switch the latent virus to enter into the lytic phase of infection. Similarly, the virus-encoded bZIP family of protein, K8 also plays an important role in viral lytic DNA replication. Although, both K-RTA and K8 are found to be the ori-Lyt binding proteins and are required for lytic DNA replication, the detailed DNA-binding profile of these proteins in the KSHV and host genomes remains uncharacterized. In this study, using chromatin immunoprecipitation combined with high-throughput sequencing (ChIP-seq) assay, we performed a comprehensive analysis of K-RTA and K8 binding sites in the KSHV and human genomes in order to identify specific DNA binding sequences/motifs. We identified two novel K-RTA binding motifs, (i.e. AGAGAGAGGA/motif RB and AGAAAAATTC/motif RV) and one K8 binding motif (i.e. AAAATGAAAA/motif KB), respectively. The binding of K-RTA/K8 proteins with these motifs and resulting transcriptional modulation of downstream genes was further confirmed by DNA electrophoretic gel mobility shift assay (EMSA), reporter promoter assay, Chromatin Immunoprecipitation (ChIP) assay and mRNA quantitation assay. Our data conclusively shows that K-RTA/K8 proteins specifically bind to these motifs on the host/viral genomes to modulate transcription of host/viral genes during KSHV lytic reactivation.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Cromatina/metabolismo , Regulação Viral da Expressão Gênica/fisiologia , Herpesvirus Humano 8/fisiologia , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Proteínas Virais/metabolismo , Ativação Viral/fisiologia , Fatores de Transcrição de Zíper de Leucina Básica/genética , Cromatina/genética , Cromatina/virologia , Células HEK293 , Humanos , Proteínas Repressoras/genética , Proteínas Virais/genética
9.
J Neuroinflammation ; 16(1): 86, 2019 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-30981282

RESUMO

BACKGROUND: Impairment of the blood-brain barrier (BBB) has been associated with cognitive decline in many CNS diseases, including HIV-associated neurocognitive disorders (HAND). Recent research suggests an important role for the Sonic hedgehog (Shh) signaling pathway in the maintenance of BBB integrity under both physiological and pathological conditions. METHODS: In the present study, we sought to examine the expression of Shh and its downstream effectors in relation to brain pericytes and BBB integrity in HIV-infected humans and rhesus macaques infected with simian immunodeficiency virus (SIV), an animal model of HIV infection and CNS disease. Cortical brain tissues from uninfected (n = 4) and SIV-infected macaques with (SIVE, n = 6) or without encephalitis (SIVnoE, n = 4) were examined using multi-label, semi-quantitative immunofluorescence microscopy of Shh, netrin-1, tight junction protein zona occludens 1 (ZO1), glial fibrillary acidic protein, CD163, platelet-derived growth factor receptor b (PDGFRB), glucose transporter 1, fibrinogen, and SIV Gag p28. RESULTS: While Shh presence in the brain persisted during HIV/SIV infection, both netrin-1 immunoreactivity and the size of PDGFRB+ pericytes, a cellular source of netrin-1, were increased around non-lesion-associated vessels in encephalitis compared to uninfected brain or brain without encephalitis, but were completely absent in encephalitic lesions. Hypertrophied pericytes were strongly localized in areas of fibrinogen extravasation and showed the presence of intracellular SIVp28 and HIVp24 by immunofluorescence in all SIV and HIV encephalitis cases examined, respectively. CONCLUSIONS: The lack of pericytes and netrin-1 in encephalitic lesions, in line with downregulation of ZO1 on the fenestrated endothelium, suggests that pericyte loss, despite the strong presence of Shh, contributes to HIV/SIV-induced BBB disruption and neuropathogenesis in HAND.


Assuntos
Encéfalo/patologia , Regulação Viral da Expressão Gênica/fisiologia , Proteínas Hedgehog/metabolismo , Infecções por Lentivirus/patologia , Pericitos/metabolismo , Pericitos/patologia , Transdução de Sinais/fisiologia , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Encéfalo/virologia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Infecções por HIV/patologia , Humanos , Macaca mulatta , Masculino , Netrina-1/metabolismo , Ocludina/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Vírus da Imunodeficiência Símia/patogenicidade , Proteína da Zônula de Oclusão-1/metabolismo
10.
Vet Res ; 50(1): 22, 2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30894203

RESUMO

Nuclear localization of paramyxovirus proteins is crucial for virus life cycle, including the regulation of viral replication and the evasion of host immunity. We previously showed that a recombinant Newcastle disease virus (NDV) with nuclear localization signal mutation in the matrix (M) protein results in a pathotype change and attenuates viral pathogenicity in chickens. However, little is known about the nuclear localization functions of NDV M protein. In this study, the potential functions of the M protein in the nucleus were investigated. We first demonstrate that nuclear localization of the M protein could not only promote the cytopathogenicity of NDV but also increase viral RNA synthesis and transcription efficiency in DF-1 cells. Using microarray analysis, we found that nuclear localization of the M protein might inhibit host cell transcription, represented by numerous up-regulating genes associated with transcriptional repressor activity and down-regulating genes associated with transcriptional activator activity. The role of representative up-regulated gene prospero homeobox 1 (PROX1) and down-regulated gene aryl hydrocarbon receptor (AHR) in the replication of NDV was then evaluated. The results show that siRNA-mediated knockdown of PROX1 or AHR significantly reduced or increased the viral RNA synthesis and viral replication, respectively, demonstrating the important roles of the expression changes of these genes in NDV replication. Together, our findings demonstrate for the first time that nuclear localization of NDV M protein promotes virus replication by affecting viral RNA synthesis and transcription and inhibiting host cell transcription, improving our understanding of the molecular mechanism of NDV replication and pathogenesis.


Assuntos
Fibroblastos/virologia , Proteínas Associadas à Matriz Nuclear/fisiologia , Transporte Proteico/fisiologia , RNA Viral/metabolismo , Transcrição Genética , Replicação Viral/fisiologia , Animais , Linhagem Celular , Galinhas , Regulação Viral da Expressão Gênica/fisiologia , Vírus da Doença de Newcastle , RNA Viral/genética
11.
J Virol ; 93(8)2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30728268

RESUMO

Upon virus infection of a cell, the uncoated DNA is usually blocked by the host intrinsic immune system inside the nucleus. Although it is crucial for the virus to counteract the host intrinsic immune system and access its genome, little is known about how viruses can knock down host restriction and identify their blocked genomes for later viral gene activation and replication. We found that upon baculovirus transduction into Vero E6 cells, the invading viral DNA is trapped by the cellular death domain-associated protein (Daxx) and histone H3.3 in the nucleus, resulting in gene inactivation. IE2, a baculovirus transactivator, targets host Daxx through IE2 SUMO-interacting motifs (SIMs) to indirectly access viral DNA and forms unique nuclear body structures, which we term clathrate cage-like apparatus (CCLAs), at the early transduction stage. At the later transduction stage, CCLAs gradually enlarge, and IE2 continues to closely interact with viral DNA but no longer associates with Daxx. The association with Daxx is essential for IE2 CCLA formation, and the enlarged CCLAs are capable of transactivating viral but not chromosomal DNA of Vero E6 cells. Our study reveals that baculovirus IE2 counteracts the cellular intrinsic immune system by specifically targeting Daxx and H3.3 to associate with viral DNA indirectly and efficiently. IE2 then utilizes this association with viral DNA to establish a unique CCLA cellular nanomachinery, which is visible under light microscopy as an enclosed environment for proper viral gene expression.IMPORTANCE The major breakthrough of this work is that viral protein IE2 localizes and transactivates its own viral DNA through a most unlikely route, i.e., host proteins Daxx and H3.3, which are designed to efficiently restrict viral DNA from expression. By interacting with these host intrinsic immune factors, IE2 can thus target the viral DNA and then form a unique spherical nuclear body, which we name the CCLA, to enclose the viral DNA and necessary factors to assist in high-level transactivation. Our study represents one of the most complete investigations of nuclear body formation. In addition, so far only RNA or protein molecules have been reported as potential nucleators for initiating nuclear body formation; our study may represent the first example showing that DNA can be a nucleator for a new class of nuclear body formation.


Assuntos
DNA Viral/metabolismo , Regulação Viral da Expressão Gênica/fisiologia , Chaperonas Moleculares/metabolismo , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Animais , DNA Viral/genética , Histonas/genética , Histonas/metabolismo , Chaperonas Moleculares/genética , Células Sf9 , Spodoptera , Células Vero , Proteínas Virais/genética
12.
Virology ; 529: 205-215, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30735904

RESUMO

Herpesvirus capsid assembly involves cleavage and packaging of the viral genome. The Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 43 (orf43) encodes a putative portal protein. The portal complex functions as a gate through which DNA is packaged into the preformed procapsids, and is injected into the cell nucleus upon infection. The amino acid sequence of the portal proteins is conserved among herpesviruses. Here, we generated an antiserum to ORF43 and determined late expression kinetics of ORF43 along with its nuclear localization. We generated a recombinant KSHV mutant, which fails to express ORF43 (BAC16-ORF43-null). Assembled capsids were observed upon lytic induction of this virus; however, the released virions lacked viral DNA and thus could not establish infection. Ectopic expression of ORF43 rescued the ability to produce infectious particles. ORF43 antiserum and the recombinant ORF43-null virus can provide an experimental system for further studies of the portal functions and its interactions.


Assuntos
Regulação Viral da Expressão Gênica/fisiologia , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Proteínas do Capsídeo , DNA Viral , Genoma Viral , Células HEK293 , Humanos , Fases de Leitura Aberta , Proteínas Virais/genética , Vírion , Replicação Viral
13.
J Virol ; 93(8)2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30728255

RESUMO

The Kaposi's sarcoma-associated herpesvirus (KSHV) alkaline exonuclease SOX, encoded by open reading frame 37 (ORF37), is a bifunctional early-lytic-phase protein that possesses alkaline 5'-to-3' DNase activity and promotes host shutoff at the mRNA level during productive lytic infection. While the SOX protein is well characterized for drastically impairing cellular gene expression, little is known about the impact of its DNase activity on the KSHV genome and life cycle and the biology of KSHV infections. Here, we introduced a previously described DNase-inactivating Glu129His (Q129H) mutation into the ORF37 gene of the viral genome to generate ORF37-Q129H recombinant virus (the Q129H mutant) and investigated the effects of loss or inactivation of DNase activity on viral genome replication, cleavage, and packaging. For the first time, we provide experimental evidence that the DNase activity of the SOX protein does not affect viral latent/lytic DNA synthesis but is required for cleavage and processing of the KSHV genome during lytic replication. Interestingly, the Q129H mutation severely impaired intranuclear processing of progeny virions compared to the wild-type ORF37, as assessed by pulsed-field and Gardella gel electrophoresis, electron microscopy, and single-molecule analysis of replicating DNA (SMARD) assays. Complementation with ORF37-wt (wild type) or BGLF5 (the KSHV protein homolog in Epstein-Barr virus) in 293L/Q129H cells restored the viral genome encapsidation defects. Together, these results indicated that ORF37's proposed DNase activity is essential for viral genome processing and encapsidation and, hence, can be targeted for designing antiviral agents to block KSHV virion production.IMPORTANCE Kaposi's sarcoma (KS)-associated herpesvirus is the causative agent of multiple malignancies, predominantly in immunocompromised individuals, including HIV/AIDS patients. Reduced incidence of KS in HIV/AIDS patients receiving antiherpetic drugs to block lytic replication confirms the role of lytic DNA replication and gene products in KSHV-mediated tumorigenesis. Herpesvirus lytic replication results in the production of complex concatemeric DNA, which is cleaved into unit length viral DNA for packaging into the infectious virions. The conserved herpesviral alkaline exonucleases play an important role in viral genome cleavage and packaging. Here, by using the previously described Q129H mutant virus that selectively lacks DNase activity but retains host shutoff activity, we provide experimental evidence confirming that the DNase function of the KSHV SOX protein is essential for viral genome processing and packaging and capsid maturation into the cytoplasm during lytic replication in infected cells. This led to the identification of ORF37's DNase activity as a potential target for antiviral therapeutics.


Assuntos
Exodesoxirribonucleases/metabolismo , Regulação Viral da Expressão Gênica/fisiologia , Genoma Viral/fisiologia , Infecções por Herpesviridae/enzimologia , Herpesvirus Humano 8/fisiologia , Ativação Transcricional/fisiologia , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Substituição de Aminoácidos , Exodesoxirribonucleases/genética , Células HEK293 , Infecções por Herpesviridae/genética , Humanos , Mutação de Sentido Incorreto , Proteínas Virais/genética
14.
Invest Ophthalmol Vis Sci ; 60(2): 704-711, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30786281

RESUMO

Purpose: While VZV DNA and antigen have been detected in acute and chronic VZV keratitis, it is unclear whether productive infection of corneal cells is ongoing or whether residual, noninfectious VZV antigens elicit inflammation. Herein, we examined VZV-infected primary human corneal epithelial cells (HCECs) and keratocytes (HKs) to elucidate the pathogenesis of VZV keratitis. Methods: HCECs and HKs were mock- or VZV infected. Seven days later, cells were examined for morphology, proinflammatory cytokine and matrix metalloproteinase (MMP) release, ability to recruit peripheral blood mononuclear cells (PBMCs) and neutrophils, and MMP substrate cleavage. Results: Both cell types synthesized infectious virus. VZV-infected HCECs proliferated, whereas VZV-infected HKs died. Compared to mock-infected cells, VZV-infected HCECs secreted significantly more IL-6, IL-8, IL-10, and IL-12p70 that were confirmed at the transcript level, and MMP-1 and MMP-9; conditioned supernatant attracted PBMCs and neutrophils and cleaved MMP substrates. In contrast, VZV-infected HKs suppressed cytokine secretion except for IL-8, which attracted neutrophils, and suppressed MMP release and substrate cleavage. Conclusions: Overall, VZV-infected HCECs recapitulate findings of VZV keratitis with respect to epithelial cell proliferation, pseudodendrite formation and creation of a proinflammatory environment, providing an in vitro model for VZV infection of corneal epithelial cells. Furthermore, the proliferation and persistence of VZV-infected HCECs suggest that these cells may serve as viral reservoirs if immune clearance is incomplete. Finally, the finding that VZV-infected HKs die and suppress most proinflammatory cytokines and MMPs may explain the widespread death of these cells with unchecked viral spread due to ineffective recruitment of PBMCs.


Assuntos
Ceratócitos da Córnea/virologia , Epitélio Anterior/virologia , Regulação Viral da Expressão Gênica/fisiologia , Herpesvirus Humano 3/fisiologia , Transcrição Genética , Apoptose/fisiologia , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Ceratócitos da Córnea/metabolismo , Citocinas/metabolismo , Técnicas Eletroquímicas , Epitélio Anterior/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Metaloproteinases da Matriz/metabolismo , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
J Virol ; 93(8)2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30700606

RESUMO

The Epstein-Barr virus (EBV) lytic phase, like those of all herpesviruses, proceeds via an orderly cascade that integrates DNA replication and gene expression. EBV early genes are expressed independently of viral DNA amplification, and several early gene products facilitate DNA amplification. On the other hand, EBV late genes are defined by their dependence on viral DNA replication for expression. Recently, a set of orthologous genes found in beta- and gammaherpesviruses have been determined to encode a viral preinitiation complex (vPIC) that mediates late gene expression. The EBV vPIC requires an origin of lytic replication in cis, implying that the vPIC mediates transcription from newly replicated DNA. In agreement with this implication, EBV late gene mRNAs localize to replication factories. Notably, these factories exclude canonical histones. In this review, we compare and contrast the mechanisms and epigenetics of EBV early and late gene expression. We summarize recent findings, propose a model explaining the dependence of EBV late gene expression on lytic DNA amplification, and suggest some directions for future study.


Assuntos
Epigênese Genética/fisiologia , Regulação Viral da Expressão Gênica/fisiologia , Herpesvirus Humano 4/fisiologia , Histonas/metabolismo , Transcrição Genética/fisiologia , Replicação Viral/fisiologia , Replicação do DNA/fisiologia , DNA Viral/biossíntese , Humanos
16.
Virology ; 529: 152-159, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30710799

RESUMO

Little is known about Epstein Barr Virus (EBV) proteins that participate in viral DNA cleavage and packaging. Genes encoding potential terminase subunit and portal protein homologs include BGRF1/BDRF1, BALF3, BFRF1A and BBRF1 respectively. EBV mutants with deletions in one or more of these genes were impaired for DNA packaging (Pavlova et al., 2013). In the current study, BBRF1 oligomers were purified from recombinant baculovirus infected insect cell extracts. Transmission electron microscopy revealed that purified EBV portals retained features typically found in other portals including a central channel with clip, stem and wing/crown domains. Although compounds have been identified that target DNA encapsidation in human cytomegalovirus, herpes simplex viruses and varicella-zoster virus, the identification of new EBV targets has lagged significantly. Characterization of the EBV portal will direct studies aimed at developing potential small molecular inhibitors of the EBV encapsidation process.


Assuntos
Regulação Viral da Expressão Gênica/fisiologia , Herpesvirus Humano 4/fisiologia , Proteínas Virais/metabolismo , Montagem de Vírus/fisiologia , Animais , Clonagem Molecular , Modelos Moleculares , Conformação Proteica , Células Sf9 , Proteínas Virais/genética
17.
Artigo em Inglês | MEDLINE | ID: mdl-30759770

RESUMO

The hepatitis C virus (HCV) RNA replication cycle is a dynamic intracellular process occurring in three-dimensional space (3D), which is difficult both to capture experimentally and to visualize conceptually. HCV-generated replication factories are housed within virus-induced intracellular structures termed membranous webs (MW), which are derived from the Endoplasmatic Reticulum (ER). Recently, we published 3D spatiotemporal resolved diffusion⁻reaction models of the HCV RNA replication cycle by means of surface partial differential equation (sPDE) descriptions. We distinguished between the basic components of the HCV RNA replication cycle, namely HCV RNA, non-structural viral proteins (NSPs), and a host factor. In particular, we evaluated the sPDE models upon realistic reconstructed intracellular compartments (ER/MW). In this paper, we propose a significant extension of the model based upon two additional parameters: different aggregate states of HCV RNA and NSPs, and population dynamics inspired diffusion and reaction coefficients instead of multilinear ones. The combination of both aspects enables realistic modeling of viral replication at all scales. Specifically, we describe a replication complex state consisting of HCV RNA together with a defined amount of NSPs. As a result of the combination of spatial resolution and different aggregate states, the new model mimics a cis requirement for HCV RNA replication. We used heuristic parameters for our simulations, which were run only on a subsection of the ER. Nevertheless, this was sufficient to allow the fitting of core aspects of virus reproduction, at least qualitatively. Our findings should help stimulate new model approaches and experimental directions for virology.


Assuntos
Hepacivirus/fisiologia , Modelos Biológicos , Replicação Viral/fisiologia , Linhagem Celular Tumoral , Simulação por Computador , Regulação Viral da Expressão Gênica/fisiologia , Humanos , RNA Viral/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
18.
J Virol ; 93(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30602607

RESUMO

Lund human mesencephalic (LUHMES) cells are human embryonic neuronal precursor cells that can be maintained as proliferating cells due to the expression of a tetracycline-regulatable (Tet-off) v-myc transgene. They can be differentiated to postmitotic neurons by the addition of tetracycline, glial cell-derived neurotrophic factor (GDNF), and dibutyryl cAMP. We demonstrate that these cells can be infected with herpes simplex virus 1 (HSV-1) at a multiplicity of infection (MOI) of 3 with the majority of cells surviving. By 6 days postinfection, there is a loss of lytic gene transcription and an increase in the numbers of neurons that express the latency-associated transcripts (LATs). Importantly, the virus can then be reactivated by the addition of a phosphoinositide 3-kinase inhibitor, which has previously been shown to reactivate HSV-1 in rat neuron cultures. While rodent primary culture neuron systems have been described, these are limited by their lack of scalability, as it is difficult to obtain more than 500,000 neurons to employ for a given experiment. Several recent papers have described a human dorsal root ganglion (DRG) neuron culture model and human induced pleuripotent stem cell (iPSC) neuron culture models that are scalable, but they require that the presence of an antiviral suppression be maintained following HSV-1 infection. The human LUHMES cell model of HSV-1 infection described here may be especially useful for studying HSV-1 latency and reactivation on account of its scalability, its amenability to maintenance of latency without the continual use of antiviral inhibitors, and its latent gene expression profile which mirrors many properties observed in vivo, importantly, the heterogeneity of cells expressing the LATs.IMPORTANCE Herpes simplex virus (HSV) is responsible for significant morbidity in humans due to its ability to cause oral and genital lesions, ocular disease, and encephalitis. While antivirals can attenuate the severity and frequency of disease, there is no vaccine or cure. Understanding the molecular details of HSV latency and reactivation is key to the development of new therapies. One of the difficulties in studying HSV latency has been the need to rely on establishment of latent infections in animal models. While rodent primary neuron culture models have shown promise, they yield relatively small numbers of latently infected neurons for biochemical and molecular analyses. Here we present the use of a human central nervous system (CNS)-derived conditionally proliferating cell line that can be differentiated into mature neurons and latently infected with HSV-1. This model shows promise as a scalable tool to study molecular and biochemical aspects of HSV-1 latency and reactivation in human neurons.


Assuntos
Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Neurônios/virologia , Latência Viral/fisiologia , Linhagem Celular , Gânglios Espinais/virologia , Regulação Viral da Expressão Gênica/fisiologia , Humanos , Transcrição Genética/fisiologia , Ativação Viral/fisiologia , Replicação Viral/fisiologia
19.
Biotechnol Lett ; 41(1): 147-158, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30421092

RESUMO

OBJECTIVES: To analyze the function of Ac34 in Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and elucidate the JNK apoptotic signaling pathway activation in host Spodoptera frugiperda 9 (Sf9) cells induced by the recombinant virus AcMNPV-Ac34-EGFP. RESULTS: AcMNPV is an important species of baculoviruses. First, viral propagation assay indicated that overexpression of Ac34 protein promoted replication of AcMNPV. Quantitative RT-PCR analysis showed that Ac34 increased the transcriptional level of late genes 38k and vp39, which suggested that Ac34 promoted the production of progeny virus by upregulating transcription of late genes. Second, AcMNPV-Ac34-EGFP inhibited the proliferation of Sf9 cells. Moreover, Sf9 cells infected with AcMNPV-Ac34-EGFP resulted in abundant expression of SfP53 and its accumulation in the nucleus. c-Jun N-terminal kinase (JNK) activation requires MKK4 and MKK7 mediated phosphorylation at Thr183 and Tyr185. We found increased levels of p-JNK1/2 in Sf9 cells infected by AcMNPV-Ac34-EGFP, with concomitant induction of Sf9 cell death. Furthermore, treatment of infected Sf9 cells with SP600125 (an inhibitor of JNK pathway) downregulated p-JNK1/2 and influenced the expression of virus-induced apoptosis protein SfP53, as well as Cytochrome C and Bax. CONCLUSION: AcMNPV-Ac34-EGFP virus upregulated the progeny virus production and triggered apoptosis via activation of the JNK pathway in Sf9 cells. In this work, we unveiled an effective virus replication factor-Ac34 and more importantly, developed a recombinant virus that can be used as an improved version of biopesticide.


Assuntos
Apoptose , Regulação Viral da Expressão Gênica/fisiologia , Proteínas Virais/biossíntese , Replicação Viral/fisiologia , Animais , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Células Sf9 , Spodoptera , Proteínas Virais/genética
20.
J Neuroinflammation ; 15(1): 318, 2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30442152

RESUMO

BACKGROUND: Varicella zoster virus (VZV) is a ubiquitous alphaherpesvirus that produces varicella and zoster. VZV can infect multiple cell types in the spinal cord and brain, including astrocytes, producing myelopathy and encephalopathy. While studies of VZV-astrocyte interactions are sparse, a recent report showed that quiescent primary human spinal cord astrocytes (qHA-sps) did not appear activated morphologically during VZV infection. Since astrocytes play a critical role in host defenses during viral infections of the central nervous system, we examined the cytokine responses of qHA-sps and quiescent primary human hippocampal astrocytes (qHA-hps) to VZV infection in vitro, as well as the ability of conditioned supernatant to recruit immune cells. METHODS: At 3 days post-infection, mock- and VZV-infected qHA-sps and qHA-hps were examined for morphological changes by immunofluorescence antibody assay using antibodies directed against glial fibrillary acidic protein and VZV. Conditioned supernatants were analyzed for proinflammatory cytokines [interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, interferon-gamma, and tumor necrosis factor-α] using the Meso Scale Discovery multiplex ELISA platform. Finally, the ability of conditioned supernatants to attract peripheral blood mononuclear cells (PBMCs) was determined using a chemotaxis assay. Quiescent primary human perineurial cells (qHPNCs) served as a control for VZV-induced cytokine production and PBMC migration. To confirm that the astrocytes have the ability to increase cytokine secretion, qHA-sps and qHA-hps were treated with IL-1ß and examined for morphological changes and IL-6 secretion. RESULTS: VZV-infected qHA-sps displayed extensive cellular processes, whereas VZV-infected qHA-hps became swollen and clustered together. Astrocytes had the capacity to secrete IL-6 in response to IL-1ß. Compared to mock-infected cells, VZV-infected qHA-sps showed significantly reduced secretion of IL-2, IL-4, IL-6, IL-12p70, and IL-13, while VZV-infected qHA-hps showed significantly reduced IL-8 secretion. In contrast, levels of all 10 cytokines examined were significantly increased in VZV-infected qHPNCs. Consistent with these results, conditioned supernatant from VZV-infected qHPNCs, but not that from VZV-infected qHA-sps and qHA-hps, recruited PBMCs. CONCLUSIONS: VZV-infected qHA-sps and qHA-hps have distinct morphological alterations and patterns of proinflammatory cytokine suppression that could contribute to ineffective viral clearance in VZV myelopathy and encephalopathy, respectively.


Assuntos
Astrócitos/metabolismo , Astrócitos/virologia , Citocinas/metabolismo , Regulação Viral da Expressão Gênica/fisiologia , Hipocampo/patologia , Medula Espinal/patologia , Idoso , Astrócitos/efeitos dos fármacos , Movimento Celular/fisiologia , Citocinas/genética , Citocinas/farmacologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Herpesvirus Humano 3/metabolismo , Humanos , Masculino , RNA Mensageiro/metabolismo , Infecção pelo Vírus da Varicela-Zoster/patologia
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