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1.
Elife ; 62017 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-28653620

RESUMO

Positive-strand RNA viruses, the largest genetic class of viruses, include numerous important pathogens such as Zika virus. These viruses replicate their RNA genomes in novel, membrane-bounded mini-organelles, but the organization of viral proteins and RNAs in these compartments has been largely unknown. We used cryo-electron tomography to reveal many previously unrecognized features of Flock house nodavirus (FHV) RNA replication compartments. These spherular invaginations of outer mitochondrial membranes are packed with electron-dense RNA fibrils and their volumes are closely correlated with RNA replication template length. Each spherule's necked aperture is crowned by a striking cupped ring structure containing multifunctional FHV RNA replication protein A. Subtomogram averaging of these crowns revealed twelve-fold symmetry, concentric flanking protrusions, and a central electron density. Many crowns were associated with long cytoplasmic fibrils, likely to be exported progeny RNA. These results provide new mechanistic insights into positive-strand RNA virus replication compartment structure, assembly, function and control.


Assuntos
Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Nodaviridae/fisiologia , RNA Viral/metabolismo , Replicação Viral , Animais , Linhagem Celular , Drosophila , Membranas Mitocondriais/ultraestrutura , Membranas Mitocondriais/virologia
2.
Virology ; 400(2): 240-7, 2010 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-20189623

RESUMO

The hnRNP C heterotetramer [(C1(3))C2] binds RNA polymerase II transcripts in the nucleus, along with other proteins of the core hnRNP complex, and plays an important role in mRNA biogenesis and transport. Infection of HeLa cells with poliovirus causes hnRNP C to re-localize from the nucleus, where it is normally retained during interphase, to the cytoplasm. We have proposed that in the cytoplasm, the protein isoforms of hnRNP C participate in the recognition of viral specific RNAs by the poliovirus replication proteins and/or in the assembly of membrane-bound RNA replication complexes. In SK-OV-3 cells, which express reduced levels of hnRNP C compared to HeLa cells or 293 cells, the kinetics of poliovirus replication are delayed. hnRNP C is also re-localized from the nucleus to the cytoplasm in SK-OV-3 cells infected with poliovirus. Increased expression of hnRNP C in SK-OV-3 cells by transient transfection increases the rate of virus production and overall yield over that seen in mock-transfected cells. We propose that hnRNP C interacts with poliovirus RNA and replication proteins to increase the efficiency of viral genomic RNA synthesis.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo C/metabolismo , Interações Hospedeiro-Patógeno , Poliovirus/fisiologia , RNA Viral/biossíntese , Replicação Viral , Linhagem Celular Tumoral , Núcleo Celular/química , Citoplasma/química , Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/genética , Humanos , Transfecção
3.
J Virol ; 84(9): 4229-42, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20164237

RESUMO

The poliovirus 3' noncoding region (3' NCR) is necessary for efficient virus replication. A poliovirus mutant, PVDelta3'NCR, with a deletion of the entire 3' NCR, yielded a virus that was capable of synthesizing viral RNA, albeit with a replication defect caused by deficient positive-strand RNA synthesis compared to wild-type virus. We detected multiple ribonucleoprotein (RNP) complexes in extracts from poliovirus-infected HeLa cells formed with a probe corresponding to the 5' end of poliovirus negative-strand RNA (the complement of the genomic 3' NCR), and the levels of these RNP complexes increased during the course of viral infection. Previous studies have identified RNP complexes formed with the 3' end of poliovirus negative-strand RNA, including one that contains a 36-kDa protein later identified as heterogeneous nuclear ribonucleoprotein C (hnRNP C). We report here that the 5' end of poliovirus negative-strand RNA is capable of interacting with endogenous hnRNP C, as well as with poliovirus nonstructural proteins. Further, we demonstrate that the addition of recombinant purified hnRNP C proteins can stimulate virus RNA synthesis in vitro and that depletion of hnRNP C proteins in cultured cells results in decreased virus yields and a correspondingly diminished accumulation of positive-strand RNAs. We propose that the association of hnRNP C with poliovirus negative-strand termini acts to stabilize or otherwise promote efficient positive-strand RNA synthesis.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo C/metabolismo , Interações Hospedeiro-Patógeno , Poliovirus/fisiologia , RNA Viral/metabolismo , Replicação Viral , Regiões 3' não Traduzidas , Células HeLa , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , RNA Viral/genética , Deleção de Sequência , Proteínas não Estruturais Virais/metabolismo
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