Heterogeneous nuclear ribonucleoprotein A1 binds to the transcription-regulatory region of mouse hepatitis virus RNA

Li, Hsin‐Pai; Zhang, Xuming; Duncan, Roger; Comai, Lucio; Lai, Michael M. C.

doi:10.1073/pnas.94.18.9544

Cited by 129 publications

(184 citation statements)

References 42 publications

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“…One notable feature of cellular factors involved in viral RNA synthesis is that they are located predominantly in the nucleus of normal cells, while viral RNA synthesis occurs mostly in the cytoplasm [4]. However, the relocalization of cellular proteins from nucleus to cytoplasm in virus-infected cells has previously been demonstrated for several proteins, including La, hnRNP A1, and PTB [49][50][51]. Here we also showed the relocalization of PTB from nucleus to cytoplasm in HCV replicon cells.…”

Section: E2supporting

confidence: 67%

Polypyrimidine-tract-binding Protein is a Component of the HCV RNA Replication Complex and Necessary for RNA Synthesis

et al. 2006

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The machinery for hepatitis C virus (HCV) RNA replication is still poorly characterized. The relationship between HCV RNA replication and translation is also not clear. We have previously shown that a cellular protein polypyrimidine-tract-binding protein (PTB) binds to HCV RNA at several different sites and modulates HCV translation in several ways. Here we show that PTB also participates in RNA replication. By bromouridine triphosphate (BrUTP) labeling and confocal microscopy of cells harboring an HCV replicon, we showed that the newly synthesized HCV RNA was localized to distinct structures in the cytoplasm, which also contain PTB. Membrane flotation analysis demonstrated that a fraction of cytoplasmic PTB was associated with a detergent-resistant membrane (DRM) structure consisting of lipid rafts, which also contained HCV nonstructural proteins and the human vesicle-associated membrane proteinassociated protein (hVAP-33). PTB in the DRM was resistant to protease digestion, but became sensitive after treatment with the raft-disrupting agents. PTB in the DRM consisted of multiple isoforms and the brain-specific paralog. By using small interfering RNA (siRNA) of PTB, we showed that silencing of the endogenous PTB reduced the replication of HCV RNA replicon. In a cell-free, de novo HCV RNA synthesis system, HCV RNA synthesis was inhibited by anti-PTB antibody. These studies together indicated that PTB is a part of the HCV RNA replication complex and participates in viral RNA synthesis. Thus, PTB has dual functions in HCV life cycle, including translation and RNA replication.

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Section: E2supporting

confidence: 67%

Polypyrimidine-tract-binding Protein is a Component of the HCV RNA Replication Complex and Necessary for RNA Synthesis

et al. 2006

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“…In each case, the complement of the winner 20-mer (above) was incorporated into the positive-strand genome (Fig. 2) such that winner would be present in the negative strand on genome replication, in accord with the reported binding of hnRNP A1 to the negative strand of the IGS in vitro (11,12). The IGS4 region was chosen for these constructs because gene 4 is absolutely nonessential for MHV growth (22), and neither mutant had any aberration in growth phenotype detectable on the basis of plaque size or infectious titer.…”

Section: Generation and Characterization Of Mhv Win And Igs4-win Mutamentioning

confidence: 54%

“…For MHV, hnRNP A1 has been put forward as a candidate factor mediating the leader-body fusion event that is the hallmark of nidovirus sgRNA synthesis (1,12). Evidence supporting this supposition was originally derived from in vitro studies.…”

Section: Discussionmentioning

confidence: 99%

“…As yet, two transcriptionally nonfunctional mutant IGS sequences have been shown to bind hnRNP A1 less well than the wild-type sequence (11,12). More generally, it has been difficult to define the sequence specificity of hnRNP A1 binding to biologically relevant RNA substrates (18)(19)(20).…”

mentioning

confidence: 99%

“…Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 has recently been identified as a host cellular component that binds in vitro to probe RNAs containing the negative-sense strand of the MHV IGS (11,12). This protein is one member of a large set of proteins that bind to cellular pre-mRNA shortly after transcription and function in mRNA maturation and transport.…”

mentioning

confidence: 99%

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Evaluation of the role of heterogeneous nuclear ribonucleoprotein A1 as a host factor in murine coronavirus discontinuous transcription and genome replication

Shen¹,

Masters²

2001

Proc. Natl. Acad. Sci. U.S.A.

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Viruses with RNA genomes often capture and redirect host cell components to assist in mechanisms particular to RNA-dependent RNA synthesis. The nidoviruses are an order of positive-stranded RNA viruses, comprising coronaviruses and arteriviruses, that employ a unique strategy of discontinuous transcription, producing a series of subgenomic mRNAs linking a 5 leader to distal portions of the genome. For the prototype coronavirus mouse hepatitis virus (MHV), heterogeneous nuclear ribonucleoprotein (hnRNP) A1 has been shown to be able to bind in vitro to the negative strand of the intergenic sequence, a cis-acting element found in the leader RNA and preceding each downstream ORF in the genome. hnRNP A1 thus has been proposed as a host factor in MHV transcription. To test this hypothesis genetically, we initially constructed MHV mutants with a very high-affinity hnRNP A1 binding site inserted in place of, or adjacent to, an intergenic sequence in the MHV genome. This inserted hnRNP A1 binding site was not able to functionally replace, or enhance transcription from, the intergenic sequence. This finding led us to test more directly the role of hnRNP A1 by analysis of MHV replication and RNA synthesis in a murine cell line that does not express this protein. The cellular absence of hnRNP A1 had no detectable effect on the production of infectious virus, the synthesis of genomic RNA, or the quantity or quality of subgenomic mRNAs. These results strongly suggest that hnRNP A1 is not a required host factor for MHV discontinuous transcription or genome replication.

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Heterogeneous nuclear ribonucleoproteins as regulators of gene expression through interactions with the human thymidine kinase promoter

et al. 2000

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In search for nuclear proteins that interact with the human thymidine kinase (htk) promoter, we discovered that p37AUF, a hnRNP C-like protein, and hnRNP A1, both members of the heterogeneous ribonucleoprotein family, can bind with high affinity to an ATTT sequence motif contained within the cell cycle regulatory unit (CCRU). We report here that over-expression of p37AUF stimulates gene expression mediated by the htk promoter in a promoter-sequence specific manner, whereas hnRNP A1 suppresses it. Both recombinant p37AUF and hnRNP A1 can bind the htk CCRU, suggesting that their binding to the DNA target does not require additional cellular components. We further discovered that hnRNP K is a potent suppressor of htk mediated gene activity. However, its mechanism of action is mediated through protein-protein interaction, since hnRNP K itself cannot bind the htk CCRU but can competitively inhibit the binding of other hnRNPs. The binding site for the hnRNPs on the htk CCRU is not required for S-phase induction of the htk promoter. However, in stable but not transient transfectants, the mutation of the hnRNP binding site results in 5- to 10-fold reduction of htk mediated gene activity in synchronized and exponentially growing cells. Collectively, these findings support emerging evidence that hnRNPs, in addition to their traditional role in RNA biogenesis, could be regulators of gene expression through direct DNA binding or interaction with other proteins.

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Heterogeneous nuclear ribonucleoprotein A1 binds to the transcription-regulatory region of mouse hepatitis virus RNA

Cited by 129 publications

References 42 publications

Polypyrimidine-tract-binding Protein is a Component of the HCV RNA Replication Complex and Necessary for RNA Synthesis

Polypyrimidine-tract-binding Protein is a Component of the HCV RNA Replication Complex and Necessary for RNA Synthesis

Evaluation of the role of heterogeneous nuclear ribonucleoprotein A1 as a host factor in murine coronavirus discontinuous transcription and genome replication

Heterogeneous nuclear ribonucleoproteins as regulators of gene expression through interactions with the human thymidine kinase promoter

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