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C. Fritsch - One of the best experts on this subject based on the ideXlab platform.
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Intracellular Localization of the Peanut Clump Virus Replication Complex in Tobacco BY-2 Protoplasts Containing Green Fluorescent Protein-Labeled Endoplasmic Reticulum or Golgi Apparatus
Journal of Virology, 2002Co-Authors: Patrice Dunoyer, Odile Hemmer, Christophe Ritzenthaler, Pierre Michler, C. FritschAbstract:Replication is a key event of the viral multiplication cycle that ultimately determines the success of the viral infection. For positive-stranded viruses, replication is thought to occur in complexes containing viral RNA template, viral RNA-dependent RNA polymerase (RdRp), cellular factors, and, in some cases, virus-encoded accessory Proteins (27, 57). All results obtained so far show that RNA replication occurs in close association with intracellular membranes, which generally undergo extensive reorganization in the virus-infected cell (9). Depending upon the virus, the membrane component of the replication complex may be of different origins: membranes of the endoplasmic reticulum (ER) are implicated in viral replication of picorna-like viruses such as comoviruses (11) and potyviruses (50) and of alpha-like viruses such as tobamoviruses (42) and bromoviruses (44, 45). Cytoplasmic invaginations of chloroplast membranes are associated with Tymovirus replication complexes (18, 41), and multivesicular bodies derived from peroxisomes, mitochondria, or vacuoles are the sites of replication for other viruses: cucumber mosaic and tomato aspermy viruses (21), Tomato bushy stunt virus (52), Cymbidium ringspot virus and Carnation Italian ringspot virus (47), and Alfalfa mosaic virus (AMV) (55). In animal cells, the replication of viruses of the Togaviridae and Coronaviridae families occurs in modified endosomes or lysosomes (17, 30, 40, 56). Flavivirus RNA synthesis is thought to occur in vesicle packets derived from the trans-Golgi membranes (29, 58), whereas membranes of the ER, of the Golgi apparatus, and of lysosomes have been detected in the vesicles generated by poliovirus replication (51). In a number of cases (e.g., poliovirus, alphaviruses, and Brome mosaic virus), all of the viral replication Proteins are localized within the replication complex (2, 3, 5, 6, 17, 39, 45). On the other hand, for Flavivirus Kunjin, the replicative Proteins and RNA viral synthesis sites colocalize in vesicle packets, while other nonstructural Proteins are associated with modified membranes from the intermediate compartment (29). For Tobacco etch virus, only the 6-kDa Protein and the viral RNA replication complex were associated with vesicles derived from the ER, whereas NIa and NIb, which are also required for replication, predominantly accumulated in the nucleus (43, 50). To gain insight into the interactions between viral and host factors during Peanut clump virus (PCV) RNA replication, we have investigated the in situ localization of the replicase Proteins and the RNA synthesis sites. PCV, a type member of the Pecluvirus genus, is a positive-strand RNA virus of the alphavirus-like family. The PCV genome is composed of two molecules of RNA. RNA-1 is able to replicate independently of RNA-2 in protoplasts, but both RNAs are indispensable for plant infection (25). Two N-terminally overlapping Proteins encoded by RNA-1 (P131 and P191) are essential replication Proteins. P131 contains sequence motifs common to methyltransferases and helicases, while P191 contains the signature motifs of RNA-dependent RNA polymerases in its C-terminal extension (20, 24). The RNA-1-encoded Protein P15, on the other hand, is not an essential replication factor but is required for efficient viral RNA accumulation. This Protein was not detected near the sites of viral RNA synthesis (15) and therefore is probably not a component of the replication complex. In the present study, we have further investigated the localization of the viral RNA replication complex and provide evidence that both P131 and P191 are present at the replication sites. Using protoplasts from transgenic lines of tobacco BY-2 cells, we demonstrate that the ER, but not the Golgi apparatus, undergoes extensive reorganization upon PCV infection, and we show that the viral RNA replication complex is associated with the modified ER membranes.
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Identification of Genes Involved in Replication and Movement of Peanut Clump Virus
Virology, 1998Co-Authors: Etienne Herzog, Odile Hemmer, S. Bouzoubaa, S. Hauser, G. Meyer, C. FritschAbstract:Abstract The genome of peanut clump pecluvirus (PCV) consists of two messenger RNA components which contain, respectively, three and five open reading frames (ORFs). Inoculation of transcripts from full-length cDNA clones derived from the PCV RNAs showed that RNA-1 is able to replicate in the absence of RNA-2 in protoplasts, but both RNAs are necessary for plant infection. To investigate the role of different gene products in viral RNA replication and movement, transcripts from mutant cDNA clones were inoculated to protoplasts and to Chenopodium quinoa or Nicotiana benthamiana plants, and progeny RNA was detected by Northern blot analysis. The Protein P15, encoded by the third ORF of RNA-1, is essential for efficient replication of the viral genome. The three Proteins, P51, P14, and P17, of the triple gene block contained in RNA-2 are involved in localized movement of the viral genome, whereas the coat Protein (P23) is also required for vascular movement. Insertion of the β-glucuronidase reporter gene (GUS) in place of the P23 or P39 genes (the first and the second genes of RNA-2) allows visualization of the virus infection in inoculated leaves. Although the presence of the GUS gene resulted in a lower accumulation of progeny RNA and, despite instability of the construct in planta, histochemical detection of PCV multiplication was more sensitive than Northern blot detection.
David Gil-carton - One of the best experts on this subject based on the ideXlab platform.
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Structure of P15(PAF)-PCNA complex and implications for clamp sliding during DNA replication and repair.
Nature communications, 2015Co-Authors: Alfredo De Biasio, Alain Ibáñez De Opakua, Gulnahar B. Mortuza, Rafael Molina, Tiago N. Cordeiro, Francisco Javier Castillo, Maider Villate, Nekane Merino, Sandra Delgado, David Gil-cartonAbstract:The intrinsically disordered Protein P15(PAF) regulates DNA replication and repair by binding to the proliferating cell nuclear antigen (PCNA) sliding clamp. We present the structure of the human P15(PAF)-PCNA complex. Crystallography and NMR show the central PCNA-interacting Protein motif (PIP-box) of P15(PAF) tightly bound to the front-face of PCNA. In contrast to other PCNA-interacting Proteins, P15(PAF) also contacts the inside of, and passes through, the PCNA ring. The disordered P15(PAF) termini emerge at opposite faces of the ring, but remain protected from 20S proteasomal degradation. Both free and PCNA-bound P15(PAF) binds DNA mainly through its histone-like N-terminal tail, while PCNA does not, and a model of the ternary complex with DNA inside the PCNA ring is consistent with electron micrographs. We propose that P15(PAF) acts as a flexible drag that regulates PCNA sliding along the DNA and facilitates the switch from replicative to translesion synthesis polymerase binding.
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Structure of P15^PAF–PCNA complex and implications for clamp sliding during DNA replication and repair
Nature Communications, 2015Co-Authors: Alfredo De Biasio, Alain Ibáñez De Opakua, Gulnahar B. Mortuza, Rafael Molina, Tiago N. Cordeiro, Maider Villate, Nekane Merino, Sandra Delgado, Francisco Castillo, David Gil-cartonAbstract:The intrinsically disordered Protein P15^PAF regulates DNA replication and repair by binding to the proliferating cell nuclear antigen (PCNA) sliding clamp. We present the structure of the human P15^PAF–PCNA complex. Crystallography and NMR show the central PCNA-interacting Protein motif (PIP-box) of P15^PAF tightly bound to the front-face of PCNA. In contrast to other PCNA-interacting Proteins, P15^PAF also contacts the inside of, and passes through, the PCNA ring. The disordered P15^PAF termini emerge at opposite faces of the ring, but remain protected from 20S proteasomal degradation. Both free and PCNA-bound P15^PAF binds DNA mainly through its histone-like N-terminal tail, while PCNA does not, and a model of the ternary complex with DNA inside the PCNA ring is consistent with electron micrographs. We propose that P15^PAF acts as a flexible drag that regulates PCNA sliding along the DNA and facilitates the switch from replicative to translesion synthesis polymerase binding. P15PAF regulates DNA replication and repair via interactions with the Proliferating Cell Nuclear Antigen (PCNA) sliding clamp. Here the authors present multi-faceted structural analyses of the P15-PCNA-DNA complex that suggests P15 regulates the sliding of PCNA along DNA during replication.
Eric O. Freed - One of the best experts on this subject based on the ideXlab platform.
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Cleavage of the Murine Leukemia Virus Transmembrane Env Protein by Human Immunodeficiency Virus Type 1 Protease: Transdominant Inhibition by Matrix Mutations
Journal of virology, 1998Co-Authors: Rosemary E. Kiernan, Eric O. FreedAbstract:We have identified mutations in the human immunodeficiency virus type 1 (HIV-1) matrix Protein (MA) which block infectivity of virions pseudotyped with murine leukemia virus (MuLV) envelope (Env) glycoProteins without affecting infectivity conferred by HIV-1 Env or vesicular stomatitis virus G glycoProteins. This inhibition is very potent and displays a strong transdominant effect; infectivity is reduced more than 100-fold when wild-type and mutant molecular clones are cotransfected at a 1:1 ratio. This phenomenon is observed with both ecotropic and amphotropic MuLV Env. The MA mutations do not affect the incorporation of MuLV Env into virions. We demonstrate that in HIV-1 virions pseudotyped with MuLV Env, the HIV-1 protease (PR) efficiently catalyzes the cleavage of the P15(E) transmembrane (TM) Protein to p12(E). Immunoprecipitation analysis of pseudotyped virions reveals that the mutant MA blocks this HIV-1 PR-mediated cleavage of MuLV TM. Furthermore, the transdominant inhibition exerted by the mutant MA on wild-type infectivity correlates with the relative level of P15(E) cleavage. Consistent with the hypothesis that abrogation of infectivity imposed by the mutant MA is due to inhibition of P15(E) cleavage, mutant virions are significantly more infectious when pseudotyped with a truncated p12(E) form of MuLV Env. These results indicate that HIV-1 Gag sequences can influence the viral PR-mediated processing of the MuLV TM Env Protein P15(E). These findings have implications for the development of HIV-1-based retroviral vectors pseudotyped with MuLV Env, since P15(E) cleavage is essential for activating membrane fusion and virus infectivity.
Qi Feng-ying - One of the best experts on this subject based on the ideXlab platform.
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Expression and biological significance of TGF-β_1,P15 in esophageal squamous carcinoma
Shandong Medical Journal, 2006Co-Authors: Qi Feng-yingAbstract:[Objective] To investigate transforming growth factor-β_1(TGF-β_1)and P15 expression in the esophageal squamous carcinoma and the putative roles in carcinogenesis of esophageal squamous carcinoma.[Methods] The expressions of TGF-β_1 Protein,P15 Protein and P15 mRNA were examined by SP immunohistochemistry(IHC) method and in suit hybridyzation(ISH) in 48 cases of esophageal squamous carcinoma,18 cases of esophageal dysplasia and 10 cases of incisal margin normal tissues.[Results] The positive expression rate of TGF-β_1 in esophageal carcinoma(62.5 %) was lower than that of normal tissues(100 %);The positive expression rates of P15 mRNA and P15 Protein in esophageal carcinoma were 56.3 % and 47.9 %,which were respectively lower than those in incisal margin normal tissues and adjacent dysplasia tissues(P0.05).There was marked correlation between the expression of P15 mRNA and P15 Protein in esophageal carcinoma tissues(χ~2=22,P0.05).There had marked relation between the expression of TGF-β_1 and P15 mRNA in esophageal carcinoma tissues(χ~2=23.84,P0.05).[Conclusions] TGF-β_1 may play positive roles in P15 transcription.The low expressions of P15 and TGF-β_1 might involve in esophageal carcinogenesis.
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Expression and biological significance of CyclinD1,P15 in esophageal squamous carcinoma
Journal of Modern Oncology, 2006Co-Authors: Qi Feng-yingAbstract:Objective:To investigate CyclinD1 and P15 expression in the esophageal squamous carcinoma and the putative roles in carcinogenesis of esophageal squamous carcinoma. Methods: The expressions of CyclinD1 Protein,P15 Protein and P15 mRNA were examined by streptavidin-horseradish peroxidase(SP)immunohistochemistry (IHC) method and in suit hybridyzation (ISH) in 48 cases of esophageal squamous carcinoma, 18 cases of esophageal dysplasia and 10 cases of incisal margin normal tissues. Results: The positive expression rates of CyclinD1 in esophageal carcinoma (58.3 %) and dysplasia tissues(66.7 %) were both higher than that in normal tissues (0 ); The positive expression rates of P15 mRNA and P15 Protein in esophageal carcinoma were 56.3 % and 47.9 %, which were respectively lower than those in incisal margin normal tissues and adjacent dysplasia tissues (P0.05). There was marked correlation between the expression of P15 mRNA and P15 Protein in esophageal carcinoma tissues (P0.05). There have negative relation between the expression of CyclinD1 and P15 Protein in esophageal carcinoma tissues (P0.05). Conclusion: The high expressions of CyclinD1 ,which may be the earlier molecular event during the esophageal carcinogenesis and the low expressions of P15 might involve in esophageal carcinogenesis, CyclinD1 and P15 Protein had positive or negative effect on the carcinoma progression respectively, and there may be some interaction between them.
Odile Hemmer - One of the best experts on this subject based on the ideXlab platform.
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Intracellular Localization of the Peanut Clump Virus Replication Complex in Tobacco BY-2 Protoplasts Containing Green Fluorescent Protein-Labeled Endoplasmic Reticulum or Golgi Apparatus
Journal of Virology, 2002Co-Authors: Patrice Dunoyer, Odile Hemmer, Christophe Ritzenthaler, Pierre Michler, C. FritschAbstract:Replication is a key event of the viral multiplication cycle that ultimately determines the success of the viral infection. For positive-stranded viruses, replication is thought to occur in complexes containing viral RNA template, viral RNA-dependent RNA polymerase (RdRp), cellular factors, and, in some cases, virus-encoded accessory Proteins (27, 57). All results obtained so far show that RNA replication occurs in close association with intracellular membranes, which generally undergo extensive reorganization in the virus-infected cell (9). Depending upon the virus, the membrane component of the replication complex may be of different origins: membranes of the endoplasmic reticulum (ER) are implicated in viral replication of picorna-like viruses such as comoviruses (11) and potyviruses (50) and of alpha-like viruses such as tobamoviruses (42) and bromoviruses (44, 45). Cytoplasmic invaginations of chloroplast membranes are associated with Tymovirus replication complexes (18, 41), and multivesicular bodies derived from peroxisomes, mitochondria, or vacuoles are the sites of replication for other viruses: cucumber mosaic and tomato aspermy viruses (21), Tomato bushy stunt virus (52), Cymbidium ringspot virus and Carnation Italian ringspot virus (47), and Alfalfa mosaic virus (AMV) (55). In animal cells, the replication of viruses of the Togaviridae and Coronaviridae families occurs in modified endosomes or lysosomes (17, 30, 40, 56). Flavivirus RNA synthesis is thought to occur in vesicle packets derived from the trans-Golgi membranes (29, 58), whereas membranes of the ER, of the Golgi apparatus, and of lysosomes have been detected in the vesicles generated by poliovirus replication (51). In a number of cases (e.g., poliovirus, alphaviruses, and Brome mosaic virus), all of the viral replication Proteins are localized within the replication complex (2, 3, 5, 6, 17, 39, 45). On the other hand, for Flavivirus Kunjin, the replicative Proteins and RNA viral synthesis sites colocalize in vesicle packets, while other nonstructural Proteins are associated with modified membranes from the intermediate compartment (29). For Tobacco etch virus, only the 6-kDa Protein and the viral RNA replication complex were associated with vesicles derived from the ER, whereas NIa and NIb, which are also required for replication, predominantly accumulated in the nucleus (43, 50). To gain insight into the interactions between viral and host factors during Peanut clump virus (PCV) RNA replication, we have investigated the in situ localization of the replicase Proteins and the RNA synthesis sites. PCV, a type member of the Pecluvirus genus, is a positive-strand RNA virus of the alphavirus-like family. The PCV genome is composed of two molecules of RNA. RNA-1 is able to replicate independently of RNA-2 in protoplasts, but both RNAs are indispensable for plant infection (25). Two N-terminally overlapping Proteins encoded by RNA-1 (P131 and P191) are essential replication Proteins. P131 contains sequence motifs common to methyltransferases and helicases, while P191 contains the signature motifs of RNA-dependent RNA polymerases in its C-terminal extension (20, 24). The RNA-1-encoded Protein P15, on the other hand, is not an essential replication factor but is required for efficient viral RNA accumulation. This Protein was not detected near the sites of viral RNA synthesis (15) and therefore is probably not a component of the replication complex. In the present study, we have further investigated the localization of the viral RNA replication complex and provide evidence that both P131 and P191 are present at the replication sites. Using protoplasts from transgenic lines of tobacco BY-2 cells, we demonstrate that the ER, but not the Golgi apparatus, undergoes extensive reorganization upon PCV infection, and we show that the viral RNA replication complex is associated with the modified ER membranes.
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Identification of Genes Involved in Replication and Movement of Peanut Clump Virus
Virology, 1998Co-Authors: Etienne Herzog, Odile Hemmer, S. Bouzoubaa, S. Hauser, G. Meyer, C. FritschAbstract:Abstract The genome of peanut clump pecluvirus (PCV) consists of two messenger RNA components which contain, respectively, three and five open reading frames (ORFs). Inoculation of transcripts from full-length cDNA clones derived from the PCV RNAs showed that RNA-1 is able to replicate in the absence of RNA-2 in protoplasts, but both RNAs are necessary for plant infection. To investigate the role of different gene products in viral RNA replication and movement, transcripts from mutant cDNA clones were inoculated to protoplasts and to Chenopodium quinoa or Nicotiana benthamiana plants, and progeny RNA was detected by Northern blot analysis. The Protein P15, encoded by the third ORF of RNA-1, is essential for efficient replication of the viral genome. The three Proteins, P51, P14, and P17, of the triple gene block contained in RNA-2 are involved in localized movement of the viral genome, whereas the coat Protein (P23) is also required for vascular movement. Insertion of the β-glucuronidase reporter gene (GUS) in place of the P23 or P39 genes (the first and the second genes of RNA-2) allows visualization of the virus infection in inoculated leaves. Although the presence of the GUS gene resulted in a lower accumulation of progeny RNA and, despite instability of the construct in planta, histochemical detection of PCV multiplication was more sensitive than Northern blot detection.
