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1.
Cell ; 181(7): 1502-1517.e23, 2020 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-32559462

RESUMO

RNA viruses are a major human health threat. The life cycles of many highly pathogenic RNA viruses like influenza A virus (IAV) and Lassa virus depends on host mRNA, because viral polymerases cleave 5'-m7G-capped host transcripts to prime viral mRNA synthesis ("cap-snatching"). We hypothesized that start codons within cap-snatched host transcripts could generate chimeric human-viral mRNAs with coding potential. We report the existence of this mechanism of gene origination, which we named "start-snatching." Depending on the reading frame, start-snatching allows the translation of host and viral "untranslated regions" (UTRs) to create N-terminally extended viral proteins or entirely novel polypeptides by genetic overprinting. We show that both types of chimeric proteins are made in IAV-infected cells, generate T cell responses, and contribute to virulence. Our results indicate that during infection with IAV, and likely a multitude of other human, animal and plant viruses, a host-dependent mechanism allows the genesis of hybrid genes.


Assuntos
Capuzes de RNA/genética , Infecções por Vírus de RNA/genética , Proteínas Recombinantes de Fusão/genética , Regiões 5' não Traduzidas/genética , Animais , Bovinos , Linhagem Celular , Cricetinae , Cães , Humanos , Vírus da Influenza A/metabolismo , Camundongos , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Fases de Leitura Aberta/genética , Capuzes de RNA/metabolismo , Infecções por Vírus de RNA/metabolismo , Vírus de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transcrição Gênica/genética , Proteínas Virais/metabolismo , Replicação Viral/genética
2.
J Virol ; 89(10): 5525-35, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25740985

RESUMO

UNLABELLED: The Bunyamwera (BUNV) orthobunyavirus NSs protein has proven a challenge to study in the context of viral infection. NSs is encoded in a reading frame that overlaps that of the viral nucleocapsid (N) protein thus limiting options for mutagenesis. In addition, NSs is poorly immunogenic, and antibodies only work in certain techniques while the protein itself is subject to proteasomal degradation. In order to generate a virus that expresses NSs independently of N, an ambisense S RNA segment was designed by mutating the 5'- and 3'-terminal nucleotide sequences. These mutations were previously shown to alter promoter activity so that both replication and transcription were promoted from both the genome and the antigenome RNAs (J. N. Barr et al., J. Virol. 79: 12602-12607, 2005). As proof of principle, a recombinant BUNV was created that expressed green fluorescent protein (GFP) in the ambisense orientation. GFP expression was detected throughout at least 10 passages. Recombinant BUNV encoding epitope-tagged versions of NSs in the ambisense orientation expressed NSs via a subgenomic mRNA, and two viruses grew to titers only modestly lower than parental rBUNdelNSs2 virus. The ambisense viruses were temperature sensitive, and NSs was shown to localize to both the nucleus and the cytoplasm during infection. These viruses will be useful in further studies on structure-function relationships of the orthobunyavirus NSs protein. IMPORTANCE: Bunyamwera virus (BUNV) is the type species and model system for both the family Bunyaviridae and the genus Orthobunyavirus, a group that includes many significant human and animal pathogens. Studying the basic molecular biology of these viruses is of great importance to underpin research into vaccines and antivirals. We demonstrate here the plasticity of the BUNV genome by generating recombinant viruses where the normal negative-sense S segment has been converted into an ambisense segment, allowing independent expression of either a foreign gene (green fluorescent protein) or the viral nonstructural NSs protein. These new reagents will allow detailed investigation of NSs, the orthobunyavirus interferon antagonist.


Assuntos
Genoma Viral , Orthobunyavirus/genética , Aedes , Animais , Sequência de Bases , Linhagem Celular , Chlorocebus aethiops , Proteínas de Fluorescência Verde/genética , Humanos , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo/genética , Fases de Leitura Aberta , Orthobunyavirus/patogenicidade , Orthobunyavirus/fisiologia , RNA Antissenso/genética , RNA Viral/genética , Proteínas Recombinantes/genética , Recombinação Genética , Células Vero , Proteínas não Estruturais Virais/genética
3.
PLoS One ; 8(5): e64137, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23667701

RESUMO

The NSs proteins of bunyaviruses are the viral interferon antagonists, counteracting the host's antiviral response to infection. During high-multiplicity infection of cultured mammalian cells with Bunyamwera orthobunyavirus (BUNV), NSs is rapidly degraded after reaching peak levels of expression at 12hpi. Through the use of inhibitors this was shown to be the result of proteasomal degradation. A recombinant virus (rBUN4KR), in which all four lysine residues in NSs were replaced by arginine residues, expresses an NSs protein (NSs4KR) that is resistant to degradation, confirming that degradation is lysine-dependent. However, despite repeated attempts, no direct ubiquitylation of NSs in infected cells could be demonstrated. This suggests that degradation of NSs, although lysine-dependent, may be achieved through an indirect mechanism. Infection of cultured mammalian cells or mice indicated no disadvantage for the virus in having a non-degradable NSs protein: in fact rBUN4KR had a slight growth advantage over wtBUNV in interferon-competent cells, presumably due to the increased and prolonged presence of NSs. In cultured mosquito cells there was no difference in growth between wild-type BUNV and rBUN4KR, but surprisingly NSs4KR was not stabilised compared to the wild-type NSs protein.


Assuntos
Vírus Bunyamwera/metabolismo , Proteólise , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/fisiologia , Aedes , Animais , Northern Blotting , Western Blotting , Vírus Bunyamwera/genética , Vírus Bunyamwera/patogenicidade , Células Cultivadas , Fragmentação do DNA , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Análise de Sequência de DNA , Ubiquitinação , Proteínas não Estruturais Virais/genética , Virulência , Replicação Viral/genética
4.
J Gen Virol ; 94(Pt 4): 851-859, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23255627

RESUMO

Schmallenberg virus (SBV) is a newly emerged orthobunyavirus that has caused widespread disease in cattle, sheep and goats in Europe. Like other orthobunyaviruses, SBV is characterized by a tripartite negative-sense RNA genome that encodes four structural and two non-structural proteins. This study showed that SBV has a wide in vitro host range, and that BHK-21 cells are a convenient host for both SBV propagation and assay by plaque titration. The SBV genome segments were cloned as cDNA and a three-plasmid rescue system was established to recover infectious virus. Recombinant virus behaved similarly in cell culture to authentic virus. The ORF for the non-structural NSs protein, encoded on the smallest genome segment, was disrupted by introduction of translation stop codons in the appropriate cDNA, and when this plasmid was used in reverse genetics, a recombinant virus that lacked NSs expression was recovered. This virus had reduced capacity to shut-off host-cell protein synthesis compared with the wild-type virus. In addition, the NSs-deleted virus induced interferon (IFN) in cells, indicating that, like other orthobunyaviruses, NSs functions as an IFN antagonist, most probably by globally inhibiting host-cell metabolism. The development of a robust reverse genetics system for SBV will facilitate investigation of its pathogenic mechanisms as well as the creation of attenuated strains that could be candidate vaccines.


Assuntos
Orthobunyavirus/genética , Genética Reversa/métodos , Virologia/métodos , Animais , Linhagem Celular , Cricetinae , Especificidade de Hospedeiro , Orthobunyavirus/fisiologia , Plasmídeos , Ensaio de Placa Viral , Cultura de Vírus
5.
J Gen Virol ; 91(Pt 8): 2002-2006, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20427562

RESUMO

Bunyamwera virus NSs protein is involved in the inhibition of cellular transcription and the interferon (IFN) response, and it interacts with the Med8 component of Mediator. A spontaneous mutant of a recombinant NSs-deleted Bunyamwera virus (rBUNdelNSs2) was identified and characterized. This mutant virus, termed mBUNNSs22, expresses a 21 aa N-terminally truncated form of NSs. Like rBUNdelNSs2, mBUNNSs22 is attenuated in IFN-deficient cells, and to a greater extent in IFN-competent cells. Both rBUNdelNSs2 and mBUNNSs22 are potent IFN inducers and their growth can be rescued by depleting cellular IRF3. Strikingly, despite encoding an NSs protein that contains the Med8 interaction domain, mBUNNSs22 fails to block RNA polymerase II activity during infection. Overall, our data suggest that both the interaction of NSs with Med8 and a novel unidentified function of the NSs N-terminus, seem necessary for Bunyamwera virus to counteract host antiviral responses.


Assuntos
Vírus Bunyamwera/imunologia , Interferons/antagonistas & inibidores , Proteínas não Estruturais Virais/imunologia , Fatores de Virulência/imunologia , Sequência de Aminoácidos , Sequência de Bases , Vírus Bunyamwera/genética , Vírus Bunyamwera/patogenicidade , Linhagem Celular , Humanos , Complexo Mediador/metabolismo , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Deleção de Sequência , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/fisiologia , Ensaio de Placa Viral , Fatores de Virulência/genética , Fatores de Virulência/fisiologia , Replicação Viral
6.
Mol Cell ; 36(4): 654-66, 2009 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-19941825

RESUMO

Nucleoprotein complexes present challenges to genome stability by acting as potent blocks to replication. One attractive model of how such conflicts are resolved is direct targeting of blocked forks by helicases with the ability to displace the blocking protein-DNA complex. We show that Rep and UvrD each promote movement of E. coli replisomes blocked by nucleoprotein complexes in vitro, that such an activity is required to clear protein blocks (primarily transcription complexes) in vivo, and that a polarity of translocation opposite that of the replicative helicase is critical for this activity. However, these two helicases are not equivalent. Rep but not UvrD interacts physically and functionally with the replicative helicase. In contrast, UvrD likely provides a general means of protein-DNA complex turnover during replication, repair, and recombination. Rep and UvrD therefore provide two contrasting solutions as to how organisms may promote replication of protein-bound DNA.


Assuntos
DNA Helicases/metabolismo , DNA Bacteriano/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Motores Moleculares/metabolismo , Complexos Multienzimáticos/metabolismo , Meios de Cultura , Replicação do DNA , DnaB Helicases/metabolismo , Escherichia coli/citologia , Escherichia coli/enzimologia , Escherichia coli/crescimento & desenvolvimento , Teste de Complementação Genética , Mutação/genética , Nucleoproteínas/metabolismo , Ligação Proteica , Supressão Genética , Transcrição Gênica
7.
J Virol ; 83(8): 3637-46, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19193790

RESUMO

The mRNAs of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family, possess a 5' cap structure but lack a 3' poly(A) tail, a common feature of eukaryotic mRNAs that greatly enhances translation efficiency. Viral mRNAs also contain untranslated regions (UTRs) that flank the coding sequence. Using model virus-like mRNAs that harbor the Renilla luciferase reporter gene, we found that the 3' UTR of the BUNV small-segment mRNA mediated efficient translation in the absence of a poly(A) tail. Viral UTRs did not increase RNA stability, and polyadenylation did not significantly enhance reporter activity. Translation of virus-like mRNAs in transfected cells was unaffected by knockdown of poly(A)-binding protein (PABP) but was markedly reduced by depletion of eukaryotic initiation factor 4G, suggesting a PABP-independent process for translation initiation. In BUNV-infected cells, translation of polyadenylated but not virus-like mRNAs was inhibited. Furthermore, we demonstrate that the viral nucleocapsid protein binds to, and colocalizes with, PABP in the cytoplasm early in infection, followed by nuclear retention of PABP. Our results suggest that BUNV corrupts PABP function in order to inhibit translation of polyadenylated cellular mRNAs while its own mRNAs are translated in a PABP-independent process.


Assuntos
Regiões 3' não Traduzidas/fisiologia , Vírus Bunyamwera/fisiologia , Regulação Viral da Expressão Gênica/fisiologia , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Animais , Linhagem Celular , Núcleo Celular/química , Cricetinae , Citoplasma/química , Fator de Iniciação Eucariótico 4G/genética , Fator de Iniciação Eucariótico 4G/metabolismo , Técnicas de Silenciamento de Genes , Genes Reporter , Humanos , Luciferases/biossíntese , Proteínas do Nucleocapsídeo/metabolismo , Proteínas de Ligação a Poli(A)/genética , Proteínas de Ligação a Poli(A)/metabolismo , Ligação Proteica , Estabilidade de RNA
8.
Nucleic Acids Res ; 34(18): 5194-202, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17000639

RESUMO

All organisms require mechanisms that resuscitate replication forks when they break down, reflecting the complex intracellular environments within which DNA replication occurs. Here we show that as few as three lac repressor-operator complexes block Escherichia coli replication forks in vitro regardless of the topological state of the DNA. Blockage with tandem repressor-operator complexes was also observed in vivo, demonstrating that replisomes have a limited ability to translocate through high affinity protein-DNA complexes. However, cells could tolerate tandem repressor-bound operators within the chromosome that were sufficient to block all forks in vitro. This discrepancy between in vitro and in vivo observations was at least partly explained by the ability of RecA, RecBCD and RecG to abrogate the effects of repressor-operator complexes on cell viability. However, neither RuvABC nor RecF were needed for normal cell growth in the face of such complexes. Holliday junction resolution by RuvABC and facilitated loading of RecA by RecF were not therefore critical for tolerance of protein-DNA blocks. We conclude that there is a trade-off between efficient genome duplication and other aspects of DNA metabolism such as transcriptional control, and that recombination enzymes, either directly or indirectly, provide the means to tolerate such conflicts.


Assuntos
Proteínas de Bactérias/metabolismo , Replicação do DNA , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regiões Operadoras Genéticas , Proteínas Repressoras/metabolismo , DNA Super-Helicoidal/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Repressores Lac , Complexos Multienzimáticos/metabolismo , Recombinação Genética
9.
Virology ; 335(1): 122-30, 2005 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-15823611

RESUMO

Transcription of segmented negative-strand RNA viruses is initiated by cap snatching: a host mRNA is cleaved generally at 10-20 nt from its 5' capped end and the resulting capped leader used to prime viral transcription. For Tomato spotted wilt virus (TSWV), type species of the plant-infecting Tospovirus genus within the Bunyaviridae, cap donors were previously shown to require a single base complementarity to the ultimate or penultimate viral template sequence. More recently, the occurrence in vitro of "re-snatching" of viral mRNAs, i.e., the use of viral mRNAs as cap donors, has been demonstrated for TSWV. To estimate the relative occurrence of re-snatching compared to snatching of host mRNAs, the use of cap donors with either single, double, or multiple complementarity to the viral template was analyzed in pair-wise competition in TSWV in vitro transcription assays. A strong preference was observed for multiple-basepairing donors.


Assuntos
Pareamento de Bases , RNA Polimerases Dirigidas por DNA/metabolismo , Capuzes de RNA/metabolismo , RNA Mensageiro/metabolismo , Tospovirus/enzimologia , Transcrição Gênica , Animais , Sequência de Bases , Primers do DNA , Solanum lycopersicum , Dados de Sequência Molecular , RNA Mensageiro/química , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Coelhos , Moldes Genéticos , Tospovirus/genética , Tospovirus/metabolismo
10.
Virus Res ; 110(1-2): 125-31, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15845263

RESUMO

The Tomato spotted wilt virus ambisense M- and S-RNA segments contain an A/U-rich intergenic region predicted to form a stable hairpin structure. The site of transcription termination of S-segment encoded N and NSs mRNAs synthesised in an in vitro transcription system was roughly mapped to the 3'-end of the intergenic hairpin, i.e. position 1568-1574 for N and position 1852-1839 for NSs, as determined by RT-PCR cloning and size estimation on Northern blots. This suggests that these viral transcripts contain a predicted stem-loop structure at their 3'-end. The potential involvement of the 3'-end structure in transcription termination is discussed.


Assuntos
Regiões 3' não Traduzidas/genética , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Viral/química , Tospovirus/genética , Sequência de Bases , Northern Blotting , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
11.
J Gen Virol ; 85(Pt 5): 1335-1338, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15105551

RESUMO

Ongoing transcription in vitro of Tomato spotted wilt virus (TSWV) has previously been demonstrated to require the presence of reticulocyte lysate. This dependence was further investigated by testing the occurrence of transcription in the presence of two translation inhibitors: edeine, an inhibitor that still allows scanning of nascent mRNAs by the 40S ribosomal subunit, and cycloheximide, an inhibitor that completely blocks translation including ribosome scanning. Neither of these inhibitors blocked TSWV transcription initiation or elongation in vitro, as demonstrated by de novo-synthesized viral mRNAs with globin mRNA-derived leader sequences, suggesting that TSWV transcription in vitro requires the presence of (a component within) reticulocyte lysate, rather than a viral protein resulting from translation.


Assuntos
Tospovirus/genética , Transcrição Gênica , Cicloeximida/farmacologia , Edeína/farmacologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Biossíntese de Proteínas , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/biossíntese , RNA Viral/biossíntese
12.
Virology ; 303(2): 278-86, 2002 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-12490389

RESUMO

Purified Tomato spotted wilt virus particles were shown to support either genome replication or transcription in vitro, depending on the conditions chosen. Transcriptional activity was observed only upon addition of rabbit reticulocyte lysate, indicating a dependence on translation. Under these conditions RNA molecules of subgenomic length were synthesized that hybridized to strand-specific probes for the N and NSs genes. Cloning of these transcripts demonstrated the presence of nonviral leader sequences at their 5' ends, confirming the occurrence of genuine viral transcription initiation known as "cap snatching." Sequence analyses revealed that both alpha- and beta-globin mRNA, present in the reticulocyte lysate, as well as added Alfalfa mosaic virus (AMV) RNA sequences, were utilized as cap donors. Moreover, an artificially produced N mRNA containing an AMV-derived leader was shown to be used as cap donor, indicating that resnatching of viral mRNAs takes place in vitro.


Assuntos
RNA Viral/biossíntese , Tospovirus/fisiologia , Transcrição Gênica , Vírion/fisiologia , Replicação Viral , Animais , Capuzes de RNA/metabolismo , RNA Mensageiro/metabolismo , Coelhos , Reticulócitos/metabolismo , Tospovirus/genética
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