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1.
J Virol ; 87(14): 8064-74, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23678180

RESUMO

Influenza virus defective interfering (DI) particles are naturally occurring noninfectious virions typically generated during in vitro serial passages in cell culture of the virus at a high multiplicity of infection. DI particles are recognized for the role they play in inhibiting viral replication and for the impact they have on the production of infectious virions. To date, influenza virus DI particles have been reported primarily as a phenomenon of cell culture and in experimentally infected embryonated chicken eggs. They have also been isolated from a respiratory infection of chickens. Using a sequencing approach, we characterize several subgenomic viral RNAs from human nasopharyngeal specimens infected with the influenza A(H1N1)pdm09 virus. The distribution of these in vivo-derived DI-like RNAs was similar to that of in vitro DIs, with the majority of the defective RNAs generated from the PB2 (segment 1) of the polymerase complex, followed by PB1 and PA. The lengths of the in vivo-derived DI-like segments also are similar to those of known in vitro DIs, and the in vivo-derived DI-like segments share internal deletions of the same segments. The presence of identical DI-like RNAs in patients linked by direct contact is compatible with transmission between them. The functional role of DI-like RNAs in natural infections remains to be established.


Assuntos
Vírus Defeituosos/genética , Vírus da Influenza A Subtipo H1N1/genética , RNA Viral/genética , Sequência de Bases , Clonagem Molecular , Biologia Computacional , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNA
2.
Infect Immun ; 79(3): 1044-56, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21199910

RESUMO

We previously demonstrated that plasmid-deficient Chlamydia muridarum retains the ability to infect the murine genital tract but does not elicit oviduct pathology because it fails to activate Toll-like receptor 2 (TLR2). We derived a plasmid-cured derivative of the human genital isolate Chlamydia trachomatis D/UW-3/Cx, strain CTD153, which also fails to activate TLR2, indicating this virulence phenotype is associated with plasmid loss in both C. trachomatis and C. muridarum. As observed with plasmid-deficient C. muridarum, CTD153 displayed impaired accumulation of glycogen within inclusions. Transcriptional profiling of the plasmid-deficient strains by using custom microarrays identified a conserved group of chromosomal loci, the expression of which was similarly controlled in plasmid-deficient C. muridarum strains CM972 and CM3.1 and plasmid-deficient C. trachomatis CTD153. However, although expression of glycogen synthase, encoded by glgA, was greatly reduced in CTD153, it was unaltered in plasmid-deficient C. muridarum strains. Thus, additional plasmid-associated factors are required for glycogen accumulation by this chlamydial species. Furthermore, in C. trachomatis, glgA and other plasmid-responsive chromosomal loci (PRCLs) were transcriptionally responsive to glucose limitation, indicating that additional regulatory elements may be involved in the coordinated expression of these candidate virulence effectors. Glucose-limited C. trachomatis displayed reduced TLR2 stimulation in an in vitro assay. During human chlamydial infection, glucose limitation may decrease chlamydial virulence through its effects on plasmid-responsive chromosomal genes.


Assuntos
Infecções por Chlamydia/genética , Chlamydia muridarum/genética , Chlamydia trachomatis/genética , Regulação Bacteriana da Expressão Gênica/genética , Plasmídeos/genética , Receptor 2 Toll-Like/metabolismo , Animais , Linhagem Celular , Infecções por Chlamydia/metabolismo , Chlamydia muridarum/metabolismo , Chlamydia muridarum/patogenicidade , Chlamydia trachomatis/metabolismo , Chlamydia trachomatis/patogenicidade , Cromossomos Bacterianos/genética , Expressão Gênica , Loci Gênicos , Glucose/metabolismo , Glicogênio/metabolismo , Glicogênio Sintase/biossíntese , Glicogênio Sintase/genética , Humanos , Corpos de Inclusão/metabolismo , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Virulência/genética
3.
J Virol ; 83(8): 3977-81, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19176627

RESUMO

The bICP0 protein encoded by bovine herpesvirus 1 stimulates productive infection and viral gene expression but inhibits interferon (IFN)-dependent transcription. bICP0 inhibits beta IFN (IFN-beta) promoter activity and induces degradation of IFN regulatory factor 3 (IRF3). Although bICP0 inhibits the trans-activation activity of IRF7, IRF7 protein levels are not reduced. In this study, we demonstrate that bICP0 is associated with IRF7. Furthermore, bICP0 inhibits the ability of IRF7 to trans-activate the IFN-beta promoter in the absence of IRF3 expression. The interaction between bICP0 and IRF7 correlates with reduced trans-activation of the IFN-beta promoter by IRF7.


Assuntos
Herpesvirus Bovino 1/imunologia , Fator Regulador 7 de Interferon/antagonistas & inibidores , Fator Regulador 7 de Interferon/metabolismo , Interferon beta/biossíntese , Regiões Promotoras Genéticas , Transativadores/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular Tumoral , Herpesvirus Bovino 1/fisiologia , Fator Regulador 3 de Interferon/metabolismo , Camundongos , Ligação Proteica
4.
PLoS Biol ; 5(1): e4, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17194211

RESUMO

Selenocysteine (Sec) is cotranslationally inserted into protein in response to UGA codons and is the 21st amino acid in the genetic code. However, the means by which Sec is synthesized in eukaryotes is not known. Herein, comparative genomics and experimental analyses revealed that the mammalian Sec synthase (SecS) is the previously identified pyridoxal phosphate-containing protein known as the soluble liver antigen. SecS required selenophosphate and O-phosphoseryl-tRNA([Ser]Sec) as substrates to generate selenocysteyl-tRNA([Ser]Sec). Moreover, it was found that Sec was synthesized on the tRNA scaffold from selenide, ATP, and serine using tRNA([Ser]Sec), seryl-tRNA synthetase, O-phosphoseryl-tRNA([Ser]Sec) kinase, selenophosphate synthetase, and SecS. By identifying the pathway of Sec biosynthesis in mammals, this study not only functionally characterized SecS but also assigned the function of the O-phosphoseryl-tRNA([Ser]Sec) kinase. In addition, we found that selenophosphate synthetase 2 could synthesize monoselenophosphate in vitro but selenophosphate synthetase 1 could not. Conservation of the overall pathway of Sec biosynthesis suggests that this pathway is also active in other eukaryotes and archaea that synthesize selenoproteins.


Assuntos
Células Eucarióticas/química , Aminoacil-RNA de Transferência/biossíntese , Aminoacil-RNA de Transferência/genética , RNA de Transferência/genética , Selenocisteína/biossíntese , Selenocisteína/genética , Trifosfato de Adenosina/metabolismo , Animais , Proteínas Arqueais/biossíntese , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biologia Computacional/métodos , Dipeptídeos/metabolismo , Células Eucarióticas/enzimologia , Genômica/métodos , Hidrólise , Espectroscopia de Ressonância Magnética , Camundongos , Fosforilação , Fosfosserina/química , Fosfosserina/metabolismo , Fosfotransferases/química , Fosfotransferases/metabolismo , Ligação Proteica/genética , RNA de Transferência/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Selênio/química , Selênio/metabolismo , Selenocisteína/química , Selenocisteína/metabolismo , Transferases/biossíntese , Transferases/genética , Transferases/metabolismo
5.
J Virol ; 82(24): 12060-8, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18842710

RESUMO

Bovine herpesvirus 1 (BHV-1) infected cell protein 0 (bICP0) stimulates productive infection, in part by activating viral gene expression. The C(3)HC(4) zinc RING finger of bICP0 is crucial for activating viral transcription and productive infection. In this study, we used a bacterial artificial chromosome containing a wild-type (wt) virulent BHV-1 strain to generate a single amino acid mutation in the C(3)HC(4) zinc RING finger of bICP0. This virus (the 51g mutant) contains a cysteine-to-glycine mutation (51st amino acid) in the C(3)HC(4) zinc RING finger of bICP0. A plasmid expressing the 51g mutant protein did not transactivate viral promoter activity as efficiently as wt bICP0. The 51g mutant virus expressed higher levels of the bICP0 protein than did the 51g rescued virus (51gR) but yielded reduced virus titers following infection of permissive bovine cells. The 51g mutant virus, but not the 51gR virus, grew poorly in bovine cells pretreated with imiquimod to stimulate interferon production. During acute infection of calves, levels of infectious virus were 2 to 3 logs lower in ocular or nasal swabs with 51g than with 51gR. Calves latently infected with the 51g mutant did not reactivate from latency because virus shedding did not occur in ocular or nasal cavities. As expected, calves latently infected with 51gR reactivated from latency following dexamethasone treatment. These studies demonstrate that mutation of a single well-conserved cysteine residue in the C(3)HC(4) zinc RING finger of bICP0 has dramatic effects on the growth properties of BHV-1.


Assuntos
Herpesvirus Bovino 1/metabolismo , Herpesvirus Bovino 1/patogenicidade , Transativadores/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Replicação Viral , Dedos de Zinco , Sequência de Aminoácidos , Animais , Bovinos , Linhagem Celular , Sequência Conservada , Herpesvirus Bovino 1/química , Herpesvirus Bovino 1/genética , Rinotraqueíte Infecciosa Bovina/genética , Rinotraqueíte Infecciosa Bovina/metabolismo , Rinotraqueíte Infecciosa Bovina/virologia , Dados de Sequência Molecular , Mutação/genética , Regiões Promotoras Genéticas/genética , Alinhamento de Sequência , Transativadores/química , Transativadores/genética , Ativação Transcricional/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética
6.
Future Virol ; 7(6): 563-573, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-23682295

RESUMO

RNA virus exploration within the field of medical virology has greatly benefited from technological developments in genomics, deepening our understanding of viral dynamics and emergence. Large-scale first-generation technology sequencing projects have expedited molecular epidemiology studies at an unprecedented scale for two pathogenic RNA viruses chosen as models: influenza A virus and dengue. Next-generation sequencing approaches are now leading to a more in-depth analysis of virus genetic diversity, which is greater for RNA than DNA viruses because of high replication rates and the absence of proofreading activity of the RNA-dependent RNA polymerase. In the field of virus discovery, technological advancements and metagenomic approaches are expanding the catalogs of novel viruses by facilitating our probing into the RNA virus world.

7.
J Gen Virol ; 89(Pt 6): 1338-1345, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18474548

RESUMO

Bovine herpesvirus type 1 (BHV-1) is an important pathogen that can initiate bovine respiratory disease complex. Like other members of the subfamily Alphaherpesvirinae, BHV-1 establishes latency in sensory neurons. The latency-related (LR) gene expresses a family of alternatively spliced transcripts in infected sensory neurons that have the potential to encode several LR proteins. An LR mutant virus that contains three stop codons near the 5' terminus of the first open reading frame in the LR gene does not express two LR proteins or reactivate from latency. In addition, the LR mutant virus induces higher levels of apoptosis in trigeminal ganglionic neurons and grows less efficiently in certain tissues of infected calves. In spite of the reduced pathogenesis, the LR mutant virus, wild-type BHV-1 and the LR rescued virus exhibit identical growth properties in cultured bovine cells. In this study, we demonstrated that during early phases of productive infection the LR mutant virus expressed higher levels of LR-RNA relative to the LR rescued virus or wt BHV-1. Bovine kidney cells infected with the LR mutant virus also induced higher levels of beta interferon RNA and interferon response genes. These results suggest that inappropriate expression of LR-RNA, in the absence of LR protein expression, may influence the latency-reactivation cycle and pathogenic potential of BHV-1.


Assuntos
Herpesvirus Bovino 1 , Rinotraqueíte Infecciosa Bovina/imunologia , Rinotraqueíte Infecciosa Bovina/virologia , Interferon beta/metabolismo , Neurônios/metabolismo , Gânglio Trigeminal/metabolismo , Proteínas Virais/fisiologia , Animais , Bovinos , Linhagem Celular , Regulação Viral da Expressão Gênica , Herpesvirus Bovino 1/patogenicidade , Herpesvirus Bovino 1/fisiologia , Humanos , Interferon beta/genética , Tonsila Palatina/metabolismo , Tonsila Palatina/virologia , Mutação Puntual , RNA/análise , RNA/genética , Virulência , Latência Viral
8.
J Virol ; 81(7): 3077-86, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17215277

RESUMO

The ICP0 protein (bICP0) encoded by bovine herpesvirus 1 is the major viral regulatory protein because it stimulates all viral promoters and, consequently, productive infection. Like other ICP0 analogues encoded by Alphaherpesvirinae subfamily members, bICP0 contains a zinc RING finger near its amino terminus that is necessary for activating transcription, regulating subcellular localization, and inhibiting interferon-dependent transcription. In this study, we discovered that sequences near the C terminus, and the zinc RING finger, are necessary for inhibiting the human beta interferon (IFN-beta) promoter. In contrast to herpes simplex virus type 1-encoded ICP0, bICP0 reduces interferon response factor 3 (IRF3), but not IRF7, protein levels in transiently transfected cells. The zinc RING finger and sequences near the C terminus are necessary for bICP0-induced degradation of IRF3. A proteasome inhibitor, lactacystin, interfered with bICP0-induced degradation of IRF3, suggesting that bICP0, directly or indirectly, targets IRF3 for proteasome-dependent degradation. IRF3, but not IRF7, is not readily detectable in the nuclei of productively infected bovine cells during the late stages of infection. In the context of productive infection, IRF3 and IRF7 are detected in the nucleus at early times after infection. At late times after infection, IRF7, but not IRF3, is still detectable in the nuclei of infected cells. Collectively, these studies suggest that the ability of bICP0 to reduce IRF3 protein levels is important with respect to disarming the IFN response during productive infection.


Assuntos
Herpesvirus Bovino 1/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/genética , Regiões Promotoras Genéticas/genética , Transativadores/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Linhagem Celular , Regulação da Expressão Gênica , Herpesvirus Bovino 1/genética , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 7 de Interferon/metabolismo , Dados de Sequência Molecular , Mutação/genética , Transativadores/genética , Ubiquitina-Proteína Ligases/genética , Proteínas Virais/genética , Dedos de Zinco
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