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1.
Methods Mol Biol ; 1826: 133-142, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30194598

RESUMO

Serpins function as a trap for serine proteases, presenting the reactive center loop (RCL) as a target for individual proteases. When the protease cleaves the RCL, the serpin and protease become covalently linked leading to a loss of function of both the protease and the serpin; this suicide inhibition is often referred to as a "mouse trap." When the RCL P1-P1' scissile bond is cut by the protease, the resulting bond between the protease and the RCL leads to insertion of the cleaved RCL into the ß-sheet A and relocation of the protease to the opposite pole of the serpin, forming a suicide complex. Only a relatively small part of the serpin molecule can be removed in deletion mutations before the serpin RCL inhibitory function is lost. Serpin RCL peptides have been developed to block formation of serpin aggregates in serpinopathies, genetic serpin mutations wherein the abnormal serpins insert their RCL into adjacent serpins forming aggregates of inactive serpins.We have further posited that this natural cleavage site in the serpin RCL may form active serpin metabolites with potential to add to the serpin's inhibitory functions. We have developed RCL peptides based upon predicted serpin RCL cleavage (or metabolism) sites and tested these serpins for inhibitory function. In this chapter we describe the development of RCL-derived peptides, peptides derived based upon the RCL sequences of two myxomaviral serpins. Methods used to develop peptides are described for RCL-derived peptides from Serp-1, a thrombotic and thrombolytic serine protease inhibitor, and Serp-2, a cross class serine and cysteine protease inhibitor (Subheadings 2.1 and 3.1). Approaches to testing RCL peptide functions, in vitro by molecular assays and in vivo in models of cell migration, MHV-68 infection, and aortic allograft transplant are described (Subheadings 2.2 and 3.2).


Assuntos
Proteínas de Membrana , Peptídeos , Rhadinovirus , Proteínas Virais , Animais , Aorta/metabolismo , Aorta/patologia , Aorta/transplante , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/patologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Transplante de Órgãos , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Rhadinovirus/química , Rhadinovirus/genética , Rhadinovirus/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
2.
J Virol ; 92(19)2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29997213

RESUMO

Like many other large double-stranded DNA (dsDNA) viruses, herpesviruses are known to capture host genes to evade host defenses. Little is known about the detailed natural history of such genes, nor do we fully understand their evolutionary dynamics. A major obstacle is that they are often highly divergent, maintaining very low sequence similarity to host homologs. Here we use the herpesvirus genus Rhadinovirus as a model system to develop an analytical approach that combines complementary evolutionary and bioinformatic techniques, offering results that are both detailed and robust for a range of genes. Using a systematic phylogenetic strategy, we identify the original host lineage of viral genes with high confidence. We show that although host immunomodulatory genes evolve rapidly compared to other host genes, they undergo a clear increase in purifying selection once captured by a virus. To characterize this shift in detail, we developed a novel technique to identify changes in selection pressure that can be attributable to particular domains. These findings will inform us on how viruses develop strategies to evade the immune system, and our synthesis of techniques can be reapplied to other viruses or biological systems with similar analytical challenges.IMPORTANCE Viruses and hosts have been shown to capture genes from one another as part of the evolutionary arms race. Such genes offer a natural experiment on the effects of evolutionary pressure, since the same gene exists in vastly different selective environments. However, sequences of viral homologs often bear little similarity to the original sequence, complicating the reconstruction of their shared evolutionary history with host counterparts. In this study, we use a genus of herpesviruses as a model system to comprehensively investigate the evolution of host-derived viral genes, using a synthesis of genomics, phylogenetics, selection analysis, and nucleotide and amino acid modeling.


Assuntos
Genes Virais/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Interações Hospedeiro-Patógeno , Rhadinovirus/genética , Seleção Genética , Proteínas Virais/genética , Animais , Antígenos CD/química , Antígenos CD/genética , Antígenos CD/imunologia , Atelinae/virologia , Evolução Biológica , Antígenos CD59/química , Antígenos CD59/genética , Antígenos CD59/imunologia , Callithrix/virologia , Quimiocina CCL3/química , Quimiocina CCL3/genética , Quimiocina CCL3/imunologia , Biologia Computacional , Regulação da Expressão Gênica , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/imunologia , Interleucina-17/química , Interleucina-17/genética , Interleucina-17/imunologia , Camundongos , Modelos Moleculares , Filogenia , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Ratos , Rhadinovirus/química , Rhadinovirus/imunologia , Saimiri/virologia , Proteínas Virais/química , Proteínas Virais/imunologia
3.
Acta Virol ; 59(4): 418-22, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26666191

RESUMO

In this study, we investigated the medium of three cell lines transformed with murine herpesvirus 68 (MHV-68) in vitro and in vivo, 68/HDF, 68/NIH3T3, and S11E, for the presence of compounds resembling growth factors of some herpesviruses which have displayed transforming and transformed phenotype suppressing activity in normal and tumor cells. When any of spent medium was added to cell culture we observed the onset of transformed phenotype in baby hamster kidney cells (BHK-21) cells and transformed phenotype suppressing activity in tumor human epithelial cells (HeLa). In media tested, we have identified the presence of putative growth factor related to MHV-68 (MHGF-68). Its bivalent properties have been blocked entirely by antisera against MHV-68 and two monoclonal antibodies against glycoprotein B (gB) of MHV-68 suggesting viral origin of MHGF-68. The results of initial efforts to separate MHGF-68 on FPLC Sephadex G15 column in the absence of salts revealed the loss of its transforming activity but transformed phenotype suppressing activity retained. On the other hand, the use of methanol-water mobile phase on RP-HPLC C18 column allowed separation of MHGF-68 to two compounds. Both separated fractions, had only the transforming activity to normal cells. Further experiments exploring the nature and the structure of hitherto unknown MHGF-68 are now in the progress to characterize its molecular and biological properties.


Assuntos
Transformação Celular Viral , Infecções por Herpesviridae/virologia , Rhadinovirus/química , Rhadinovirus/fisiologia , Animais , Infecções por Herpesviridae/patologia , Camundongos , Células NIH 3T3 , Rhadinovirus/genética
4.
J Gen Virol ; 96(Pt 6): 1436-1445, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26068188

RESUMO

The viral tegument is a layer of proteins between the herpesvirus capsid and its outer envelope. According to phylogenetic studies, only a third of these proteins are conserved amongst the three subfamilies (Alpha-, Beta- and Gammaherpesvirinae) of the family Herpesviridae. Although some of these tegument proteins have been studied in more detail, the structure and function of the majority of them are still poorly characterized. VP22 from Herpes simplex virus 1 (subfamily Alphaherpesvirinae) is a highly interacting tegument protein that has been associated with tegument assembly. We have determined the crystal structure of the conserved core domain of VP22, which reveals an elongated dimer with several potential protein-protein interaction regions and a peptide-binding site. The structure provides us with the structural basics to understand the numerous functional mutagenesis studies of VP22 found in the literature. It also establishes an unexpected structural homology to the tegument protein ORF52 from Murid herpesvirus 68 (subfamily Gammaherpesvirinae). Homologues for both VP22 and ORF52 have been identified in their respective subfamilies. Although there is no obvious sequence overlap in the two subfamilies, this structural conservation provides compelling structural evidence for shared ancestry and functional conservation.


Assuntos
Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/genética , Sequência Conservada , Cristalografia por Raios X , Herpesvirus Humano 1/química , Herpesvirus Humano 1/genética , Modelos Moleculares , Conformação Proteica , Rhadinovirus/química , Rhadinovirus/genética
5.
Structure ; 22(10): 1385-98, 2014 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-25220471

RESUMO

Like many double-stranded DNA viruses, tumor gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus withstand high internal pressure. Bacteriophage HK97 uses covalent chainmail for this purpose, but how this is achieved noncovalently in the much larger gammaherpesvirus capsid is unknown. Our cryoelectron microscopy structure of a gammaherpesvirus capsid reveals a hierarchy of four levels of organization: (1) Within a hexon capsomer, each monomer of the major capsid protein (MCP), 1,378 amino acids and six domains, interacts with its neighboring MCPs at four sites. (2) Neighboring capsomers are linked in pairs by MCP dimerization domains and in groups of three by heterotrimeric triplex proteins. (3) Small (∼280 amino acids) HK97-like domains in MCP monomers alternate with triplex heterotrimers to form a belt that encircles each capsomer. (4) One hundred sixty-two belts concatenate to form noncovalent chainmail. The triplex heterotrimer orchestrates all four levels and likely drives maturation to an angular capsid that can withstand pressurization.


Assuntos
Proteínas do Capsídeo/química , Capsídeo/química , Rhadinovirus/química , Sequência de Aminoácidos , Animais , Microscopia Crioeletrônica , Dimerização , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína
6.
PLoS Pathog ; 9(10): e1003640, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24146614

RESUMO

Kaposi sarcoma-associated herpesvirus (KSHV) establishes a lifelong latent infection and causes several malignancies in humans. Murine herpesvirus 68 (MHV-68) is a related γ2-herpesvirus frequently used as a model to study the biology of γ-herpesviruses in vivo. The KSHV latency-associated nuclear antigen (kLANA) and the MHV68 mLANA (orf73) protein are required for latent viral replication and persistence. Latent episomal KSHV genomes and kLANA form nuclear microdomains, termed 'LANA speckles', which also contain cellular chromatin proteins, including BRD2 and BRD4, members of the BRD/BET family of chromatin modulators. We solved the X-ray crystal structure of the C-terminal DNA binding domains (CTD) of kLANA and MHV-68 mLANA. While these structures share the overall fold with the EBNA1 protein of Epstein-Barr virus, they differ substantially in their surface characteristics. Opposite to the DNA binding site, both kLANA and mLANA CTD contain a characteristic lysine-rich positively charged surface patch, which appears to be a unique feature of γ2-herpesviral LANA proteins. Importantly, kLANA and mLANA CTD dimers undergo higher order oligomerization. Using NMR spectroscopy we identified a specific binding site for the ET domains of BRD2/4 on kLANA. Functional studies employing multiple kLANA mutants indicate that the oligomerization of native kLANA CTD dimers, the characteristic basic patch and the ET binding site on the kLANA surface are required for the formation of kLANA 'nuclear speckles' and latent replication. Similarly, the basic patch on mLANA contributes to the establishment of MHV-68 latency in spleen cells in vivo. In summary, our data provide a structural basis for the formation of higher order LANA oligomers, which is required for nuclear speckle formation, latent replication and viral persistence.


Assuntos
Antígenos Virais/metabolismo , Cromatina/metabolismo , Herpesvirus Humano 8/fisiologia , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Rhadinovirus/fisiologia , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismo , Animais , Antígenos Virais/química , Antígenos Virais/genética , Proteínas de Ciclo Celular , Cromatina/genética , Cromatina/virologia , Proteínas Cromossômicas não Histona , Cristalografia por Raios X , Células HEK293 , Células HeLa , Herpesvirus Humano 8/química , Humanos , Camundongos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Multimerização Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Estrutura Quaternária de Proteína , Rhadinovirus/química , Baço/metabolismo , Baço/virologia , Relação Estrutura-Atividade , Fatores de Transcrição/química , Fatores de Transcrição/genética , Proteínas Virais/química , Proteínas Virais/genética , Latência Viral/fisiologia
7.
PLoS Pathog ; 9(10): e1003673, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24146618

RESUMO

Latency-associated nuclear antigen (LANA) mediates γ2-herpesvirus genome persistence and regulates transcription. We describe the crystal structure of the murine gammaherpesvirus-68 LANA C-terminal domain at 2.2 Šresolution. The structure reveals an alpha-beta fold that assembles as a dimer, reminiscent of Epstein-Barr virus EBNA1. A predicted DNA binding surface is present and opposite this interface is a positive electrostatic patch. Targeted DNA recognition substitutions eliminated DNA binding, while certain charged patch mutations reduced bromodomain protein, BRD4, binding. Virus containing LANA abolished for DNA binding was incapable of viable latent infection in mice. Virus with mutations at the charged patch periphery exhibited substantial deficiency in expansion of latent infection, while central region substitutions had little effect. This deficiency was independent of BRD4. These results elucidate the LANA DNA binding domain structure and reveal a unique charged region that exerts a critical role in viral latent infection, likely acting through a host cell protein(s).


Assuntos
DNA Viral/química , Dobramento de Proteína , Rhadinovirus/química , Proteínas Virais/química , Latência Viral , DNA Viral/genética , DNA Viral/metabolismo , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Rhadinovirus/fisiologia , Proteínas Virais/genética , Proteínas Virais/metabolismo
8.
J Virol ; 87(24): 13676-93, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24109218

RESUMO

The complete sequence of retroperitoneal fibromatosis-associated herpesvirus Macaca nemestrina (RFHVMn), the pig-tailed macaque homolog of Kaposi's sarcoma-associated herpesvirus (KSHV), was determined by next-generation sequence analysis of a Kaposi's sarcoma (KS)-like macaque tumor. Colinearity of genes was observed with the KSHV genome, and the core herpesvirus genes had strong sequence homology to the corresponding KSHV genes. RFHVMn lacked homologs of open reading frame 11 (ORF11) and KSHV ORFs K5 and K6, which appear to have been generated by duplication of ORFs K3 and K4 after the divergence of KSHV and RFHV. RFHVMn contained positional homologs of all other unique KSHV genes, although some showed limited sequence similarity. RFHVMn contained a number of candidate microRNA genes. Although there was little sequence similarity with KSHV microRNAs, one candidate contained the same seed sequence as the positional homolog, kshv-miR-K12-10a, suggesting functional overlap. RNA transcript splicing was highly conserved between RFHVMn and KSHV, and strong sequence conservation was noted in specific promoters and putative origins of replication, predicting important functional similarities. Sequence comparisons indicated that RFHVMn and KSHV developed in long-term synchrony with the evolution of their hosts, and both viruses phylogenetically group within the RV1 lineage of Old World primate rhadinoviruses. RFHVMn is the closest homolog of KSHV to be completely sequenced and the first sequenced RV1 rhadinovirus homolog of KSHV from a nonhuman Old World primate. The strong genetic and sequence similarity between RFHVMn and KSHV, coupled with similarities in biology and pathology, demonstrate that RFHVMn infection in macaques offers an important and relevant model for the study of KSHV in humans.


Assuntos
Genoma Viral , Herpesvirus Humano 8/genética , Macaca nemestrina/virologia , Doenças dos Primatas/virologia , Sarcoma de Kaposi/veterinária , Sequência de Aminoácidos , Animais , Sequência de Bases , Feminino , Herpesvirus Humano 8/química , Herpesvirus Humano 8/classificação , Herpesvirus Humano 8/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Rhadinovirus/química , Rhadinovirus/classificação , Rhadinovirus/genética , Sarcoma de Kaposi/virologia , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/genética
9.
Phys Rev Lett ; 101(15): 158101, 2008 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-18999646

RESUMO

We report the recording and reconstruction of x-ray diffraction patterns from single, unstained viruses, for the first time. By separating the diffraction pattern of the virus particles from that of their surroundings, we performed quantitative and high-contrast imaging of a single virion. The structure of the viral capsid inside a virion was visualized. This work opens the door for quantitative x-ray imaging of a broad range of specimens from protein machineries and viruses to cellular organelles. Moreover, our experiment is directly transferable to the use of x-ray free electron lasers, and represents an experimental milestone towards the x-ray imaging of large protein complexes.


Assuntos
Rhadinovirus/química , Vírion/química , Difração de Raios X/métodos , Algoritmos , Animais , Camundongos , Microscopia de Força Atômica/métodos , Difração de Raios X/instrumentação
10.
J Gen Virol ; 89(Pt 6): 1352-1363, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18474550

RESUMO

Herpesviruses are ancient pathogens that infect all vertebrates. The most conserved component of their entry machinery is glycoprotein B (gB), yet how gB functions is unclear. A striking feature of the murid herpesvirus 4 (MuHV-4) gB is its resistance to neutralization. Here, we show by direct visualization of infected cells that the MuHV-4 gB changes its conformation between extracellular virions and those in late endosomes, where capsids are released. Specifically, epitopes on its N-terminal cell-binding domain become inaccessible, whilst non-N-terminal epitopes are revealed, consistent with structural changes reported for the vesicular stomatitis virus glycoprotein G. Inhibitors of endosomal acidification blocked the gB conformation switch. They also blocked capsid release and the establishment of infection, implying that the gB switch is a key step in entry. Neutralizing antibodies could only partially inhibit the switch. Their need to engage a less vulnerable, upstream form of gB, because its fusion form is revealed only in endosomes, helps to explain why gB-directed MuHV-4 neutralization is so difficult.


Assuntos
Glicoproteínas/metabolismo , Infecções por Herpesviridae/virologia , Rhadinovirus/fisiologia , Infecções Tumorais por Vírus/virologia , Proteínas Virais/metabolismo , Animais , Anticorpos Antivirais/imunologia , Linhagem Celular , Cricetinae , Cricetulus , Humanos , Camundongos , Testes de Neutralização , Conformação Proteica , Rhadinovirus/química , Ligação Viral
11.
PLoS One ; 3(2): e1669, 2008 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-18301747

RESUMO

The first contact a virus makes with cells is an important determinant of its tropism. Murid Herpesvirus-4 (MuHV-4) is highly dependent on glycosaminoglycans (GAGs) for cell binding. Its first contact is therefore likely to involve a GAG-binding virion glycoprotein. We have previously identified two such proteins, gp70 and gp150. Gp70 binds strongly to GAGs. However, deleting it makes little difference to MuHV-4 cell binding or GAG-dependence. Deleting gp150, by contrast, frees MuHV-4 from GAG dependence. This implies that GAGs normally displace gp150 to allow GAG-independent cell binding. But the gp150 GAG interaction is weak, and so would seem unlikely to make an effective first contact. Since neither gp70 nor gp150 matches the expected profile of a first contact glycoprotein, our understanding of MuHV-4 GAG interactions must be incomplete. Here we relate the seemingly disconnected gp70 and gp150 GAG interactions by showing that the MuHV-4 gH/gL also binds to GAGs. gH/gL-blocking and gp70-blocking antibodies individually had little effect on cell binding, but together were strongly inhibitory. Thus, there was redundancy in GAG binding between gp70 and gH/gL. Gp150-deficient MuHV-4 largely resisted blocks to gp70 and gH/gL binding, consistent with its GAG independence. The failure of wild-type MuHV-4 to do the same argues that gp150 is normally engaged only down-stream of gp70 or gH/gL. MuHV-4 GAG dependence is consequently two-fold: gp70 or gH/gL binding provides virions with a vital first foothold, and gp150 is then engaged to reveal GAG-independent binding.


Assuntos
Glicosaminoglicanos/metabolismo , Receptores Virais , Rhadinovirus/química , Proteínas do Envelope Viral/metabolismo , Glicoproteínas/metabolismo , Ligação Proteica
13.
EMBO J ; 26(24): 5131-42, 2007 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-18034158

RESUMO

Herpesviruses characteristically transmit infection from immune hosts. Although their success in escaping neutralization by pre-formed antibody is indisputable, the underlying molecular mechanisms remain largely unknown. Glycoprotein B (gB) is the most conserved component of the herpesvirus entry machinery and its N terminus (gB-NT) is a common neutralization target. We used murid herpesvirus-4 to determine how gB-NT contributes to the virus-antibody interaction. Deleting gB-NT had no obvious impact on virus replication, but paradoxically increased virion neutralization by immune sera. This reflected greater antibody access to neutralization epitopes on gH/gL, with which gB was associated. gB-NT itself was variably protected against antibody by O-linked glycans; on virions from epithelial cells it was protected almost completely. gB-NT therefore provides a protective and largely protected cover for a vulnerable part of gH/gL. The conservation of predicted glycosylation sites in other mammalian herpesvirus gB-NTs suggests that this evasion mechanism is widespread. Interestingly, the gB-NT glycans that blocked antibody binding could be targeted for neutralization instead by a lectin, suggesting a means of therapeutic counterattack.


Assuntos
Modulação Antigênica , Infecções por Herpesviridae/imunologia , Rhadinovirus/metabolismo , Proteínas do Envelope Viral/imunologia , Adjuvantes Imunológicos/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular , Feminino , Genoma Viral , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mutação , Testes de Neutralização , Lectinas de Plantas/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Rhadinovirus/química , Rhadinovirus/genética , Alinhamento de Sequência , Proteínas do Envelope Viral/genética , Vírion/genética , Vírion/metabolismo , Replicação Viral
14.
J Virol ; 81(8): 4166-76, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17287274

RESUMO

The diversity of viral strategies to modulate complement activation indicates that this component of the immune system has significant antiviral potential. One example is the Kaposi's sarcoma-associated herpesvirus (KSHV) complement control protein (KCP), which inhibits progression of the complement cascade. Rhesus rhadinovirus (RRV), like KSHV, is a member of the subfamily Gammaherpesvirinae and currently provides the only in vivo model of KSHV pathobiology in primates. In the present study, we characterized the KCP homologue encoded by RRV, RRV complement control protein (RCP). Two strains of RRV have been sequenced to date (H26-95 and 17577), and the RCPs they encode differ substantially in structure: RCP from strain H26-95 has four complement control protein (CCP) domains, whereas RCP from strain 17577 has eight CCP domains. Transcriptional analyses of the RCP gene (ORF4, referred to herein as RCP) in infected rhesus macaque fibroblasts mapped the ends of the transcripts of both strains. They revealed that H26-95 encodes a full-length, unspliced RCP transcript, while 17577 RCP generates a full-length unspliced mRNA and two alternatively spliced transcripts. Western blotting confirmed that infected cells express RCP, and immune electron microscopy disclosed this protein on the surface of RRV virions. Functional studies of RCP encoded by both RRV strains revealed their ability to suppress complement activation by the classical (antibody-mediated) pathway. These data provide the foundation for studies into the biological significance of gammaherpesvirus complement regulatory proteins in a tractable, non-human primate model.


Assuntos
Genes Virais , Rhadinovirus/química , Rhadinovirus/genética , Proteínas Virais/genética , Proteínas Virais/fisiologia , Animais , Sequência de Bases , Células CHO , Células Cultivadas , Ativação do Complemento , Via Clássica do Complemento , Cricetinae , Cricetulus , Fibroblastos/virologia , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologia , Humanos , Macaca mulatta , Dados de Sequência Molecular , Fases de Leitura Aberta , Estrutura Terciária de Proteína , Splicing de RNA , RNA Mensageiro/biossíntese , RNA Viral/biossíntese , Vírion/química
15.
J Virol ; 81(6): 2957-69, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17215283

RESUMO

Rhesus rhadinovirus (RRV) is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) and causes KSHV-like diseases in immunocompromised rhesus macaques (RM) that resemble KSHV-associated diseases including multicentric Castleman's disease and non-Hodgkin's lymphoma. RRV retains a majority of open reading frames (ORFs) postulated to be involved in the pathogenesis of KSHV and is the closest available animal model to KSHV infection in humans. Here we describe the generation of a recombinant clone of RRV strain 17577 (RRV(17577)) utilizing bacterial artificial chromosome (BAC) technology. Characterization of the RRV BAC demonstrated that it is a pathogenic molecular clone of RRV(17577), producing virus that behaves like wild-type RRV both in vitro and in vivo. Specifically, BAC-derived RRV displays wild-type growth properties in vitro and readily infects simian immunodeficiency virus-infected RM, inducing B cell hyperplasia, persistent lymphadenopathy, and persistent infection in these animals. This RRV BAC will allow for rapid genetic manipulation of the RRV genome, facilitating the creation of recombinant versions of RRV that harbor specific alterations and/or deletions of viral ORFs. This system will provide insights into the roles of specific RRV genes in various aspects of the viral life cycle and the RRV-associated pathogenesis in vivo in an RM model of infection. Furthermore, the generation of chimeric versions of RRV containing KSHV genes will allow analysis of the function and contributions of KSHV genes to viral pathogenesis by using a relevant primate model system.


Assuntos
Cromossomos Artificiais Bacterianos , Herpesvirus Humano 8/genética , Macaca mulatta/virologia , Rhadinovirus/genética , Sarcoma de Kaposi/etiologia , Animais , Células Cultivadas , DNA Viral/análise , Fibroblastos/virologia , Herpesvirus Humano 8/isolamento & purificação , Humanos , Hibridização de Ácido Nucleico , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Rhadinovirus/química , Sarcoma de Kaposi/virologia , Análise de Sequência de DNA
16.
Curr Top Microbiol Immunol ; 312: 43-69, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17089793

RESUMO

Rhesus monkey rhadinovirus (RRV) is one of the closest phylogenetic relatives to the human pathogen Kaposi sarcoma-associated herpesvirus (KSHV)-a gamma-2 herpesvirus and the etiologic agent of three malignancies associated with immunosuppression. In contrast to KSHV, RRV displays robust lytic-phase growth in culture, replicating to high titer, and therefore holds promise as an effective model for studying primate gammaherpesvirus lytic gene transcription as well as virion structure, assembly, and proteomics. More recently, investigators have devised complementary latent systems of RRV infection, thereby also enabling the characterization of the more restricted latent transcriptional program. Another benefit of working with RRV as a primate gammaherpesvirus model is that its efficient lytic growth makes genetic manipulation easier than that in its human counterpart. Exploiting this quality, laboratories have already begun to generate mutant RRV, setting the stage for future work investigating the function of individual viral genes. Finally, rhesus macaques support experimental infection with RRV, providing a natural in vivo model of infection, while similar nonhuman systems have remained resistant to prolonged KSHV infection. Recently, dual infection with RRV and a strain of simian immunodeficiency virus (SIV) has led to a lymphoproliferative disorder (LPD) reminiscent of multicentric Castleman disease (MCD)--a clinical manifestation of KSHV infection in a subset of immunosuppressed patients. RRV, in short, shows a high degree of homology with KSHV yet is more amenable to experimental manipulation both in vitro and in vivo. Taken together, these qualities ensure its current position as one of the most relevant viral models of KSHV biology and infection.


Assuntos
Herpesvirus Humano 8/genética , Macaca mulatta/virologia , Rhadinovirus/genética , Animais , Capsídeo/química , Genoma Viral , Herpesvirus Humano 8/química , Herpesvirus Humano 8/fisiologia , Rhadinovirus/química , Rhadinovirus/fisiologia , Transcrição Gênica , Vírion/ultraestrutura
17.
J Gen Virol ; 88(Pt 1): 40-45, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17170434

RESUMO

The rhadinovirus Ovine herpesvirus 2 (OvHV-2) is the causative agent of sheep-associated malignant catarrhal fever. OvHV-2 primarily affects ruminants and has a worldwide distribution. In this study, a composite sequence of OvHV-2 genomic DNA isolated from nasal secretions of sheep experiencing virus-shedding episodes was determined and compared with the sequence of OvHV-2 DNA isolated from a lymphoblastoid cell line derived from a clinically affected cow. The study confirmed the OvHV-2 sequence information determined for the cell line-isolated DNA and showed no apparently significant changes in the OvHV-2 genome during passage through a clinically susceptible species with subsequent maintenance in vitro. Amino acid identity between the predicted open reading frames (ORFs) of the two genomes was 94-100%, except for ORF73, which had an identity of 83%. Polymorphism in ORF73 was due primarily to variability in the G/E-rich repetitive central region of the ORF.


Assuntos
Doenças dos Bovinos/virologia , DNA Viral/análise , Mucosa Nasal/virologia , Rhadinovirus/química , Rhadinovirus/isolamento & purificação , Doenças dos Ovinos/virologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , DNA Viral/sangue , Infecções por Herpesviridae/veterinária , Infecções por Herpesviridae/virologia , Dados de Sequência Molecular , Rhadinovirus/genética , Ovinos
18.
J Virol ; 80(12): 6123-35, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16731951

RESUMO

Gammaherpesviruses subvert eukaryotic signaling pathways to favor latent infections in their cellular reservoirs. To this end, they express proteins that regulate or replace functionally specific signaling proteins of eukaryotic cells. Here we describe a new type of such viral-host interaction that is established through M2, a protein encoded by murine gammaherpesvirus 68. M2 associates with Vav proteins, a family of phosphorylation-dependent Rho/Rac exchange factors that play critical roles in lymphocyte signaling. M2 expression leads to Vav1 hyperphosphorylation and to the subsequent stimulation of its exchange activity towards Rac1, a process mediated by the formation of a trimolecular complex with Src kinases. This heteromolecular complex is coordinated by proline-rich and Src family-dependent phosphorylated regions of M2. Infection of Vav-deficient mice with gammaherpesvirus 68 results in increased long-term levels of latency in germinal center B lymphocytes, corroborating the importance of the M2/Vav cross talk in the process of viral latency. These results reveal a novel strategy used by the murine gammaherpesvirus family to subvert the lymphocyte signaling machinery to its own benefit.


Assuntos
Linfócitos B/virologia , Proteínas Proto-Oncogênicas c-vav/metabolismo , Rhadinovirus/fisiologia , Proteínas da Matriz Viral/fisiologia , Latência Viral , Animais , Camundongos , Fosforilação , Receptor Cross-Talk , Rhadinovirus/química , Proteínas rac1 de Ligação ao GTP/metabolismo
19.
J Gen Virol ; 87(Pt 5): 1047-1074, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16603506

RESUMO

The human gamma(1)-herpesvirus Epstein-Barr virus (EBV) and the gamma(2)-herpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV), rhesus rhadinovirus (RRV), herpesvirus saimiri (HVS) and herpesvirus ateles (HVA) all contain genes located adjacent to the terminal-repeat region of their genomes, encoding membrane proteins involved in signal transduction. Designated 'terminal membrane proteins' (TMPs) because of their localization in the viral genome, they interact with a variety of cellular signalling molecules, such as non-receptor protein tyrosine kinases, tumour-necrosis factor receptor-associated factors, Ras and Janus kinase (JAK), thereby initiating further downstream signalling cascades, such as the MAPK, PI3K/Akt, NF-kappaB and JAK/STAT pathways. In the case of TMPs expressed during latent persistence of EBV and HVS (LMP1, LMP2A, Stp and Tip), their modulation of intracellular signalling pathways has been linked to the provision of survival signals to latently infected cells and, hence, a contribution to occasional cellular transformation. In contrast, activation of similar pathways by TMPs of KSHV (K1 and K15) and RRV (R1), expressed during lytic replication, may extend the lifespan of virus-producing cells, alter their migration and/or modulate antiviral immune responses. Whether R1 and K1 contribute to the oncogenic properties of KSHV and RRV has not been established satisfactorily, despite their transforming qualities in experimental settings.


Assuntos
Gammaherpesvirinae/fisiologia , Infecções por Herpesviridae/virologia , Transdução de Sinais , Infecções Tumorais por Vírus/virologia , Proteínas Virais/fisiologia , Animais , Sobrevivência Celular , Transformação Celular Neoplásica , Transformação Celular Viral/fisiologia , Gammaherpesvirinae/química , Gammaherpesvirinae/metabolismo , Infecções por Herpesviridae/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Rhadinovirus/química , Sequências Repetidas Terminais , Infecções Tumorais por Vírus/metabolismo , Proteínas da Matriz Viral/fisiologia , Latência Viral
20.
J Virol ; 80(3): 1574-83, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16415032

RESUMO

The repertoire of proteins that comprise intact gammaherpesviruses, including the human pathogen Kaposi's sarcoma-associated herpesvirus (KSHV), is likely to have critical functions not only in viral structure and assembly but also in the early stages of infection and evasion of the host's rapidly deployed antiviral defenses. To develop a better understanding of these proteins, we analyzed the composition of rhesus monkey rhadinovirus (RRV), a close phylogenetic relative of KSHV. Unlike KSHV, RRV replicates to high titer in cell culture and thus serves as an effective model for studying primate gammaherpesvirus structure and virion proteomics. We employed two complementary mass spectrometric approaches and found that RRV contains at least 33 distinct virally encoded proteins. We have assigned 7 of these proteins to the capsid, 17 to the tegument, and 9 to the envelope. Of the five gammaherpesvirus-specific tegument proteins, three have no known function. We also found three proteins not previously associated with a purified herpesvirus and an additional seven that represent new findings for a member of the gamma-2 herpesviruses. Detergent extraction resulted in particles that contained six distinct tegument proteins in addition to the expected capsid structural proteins, suggesting that this subset of tegument components may interact more directly with or with higher affinity for the underlying capsid and, in turn, may play a role in assembly or transport of viral or subviral particles during entry or egress.


Assuntos
Macaca mulatta/virologia , Rhadinovirus/química , Proteínas Virais/química , Animais , Proteínas do Capsídeo/química , Proteínas do Capsídeo/isolamento & purificação , Cromatografia Líquida , Detergentes , Humanos , Espectrometria de Massas , Microscopia Eletrônica , Peptídeo Hidrolases , Rhadinovirus/isolamento & purificação , Rhadinovirus/ultraestrutura , Proteínas Virais/isolamento & purificação
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