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Peter D Nagy - One of the best experts on this subject based on the ideXlab platform.
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interviral Recombination between plant insect and fungal RNA viruses role of the intracellular ca 2 mn 2 pump
Journal of Virology, 2019Co-Authors: Nikolay Kovalev, Judit Pogany, Peter D NagyAbstract:Recombination is one of the driving forces of viral evolution. RNA Recombination events among similar RNA viruses are frequent, although RNA Recombination could also take place among unrelated viruses. In this paper, we have established efficient interviral Recombination systems based on yeast and plants. We show that diverse RNA viruses, including the plant viruses tomato bushy stunt virus, caRNAtion Italian ringspot virus, and turnip crinkle virus-associated RNA; the insect plus-strand RNA [(+)RNA] viruses Flock House virus and Nodamura virus; and the double-stranded L-A virus of yeast, are involved in interviral Recombination events. Most interviral recombinants are minus-strand recombinant RNAs, and the junction sites are not randomly distributed, but there are certain hot spot regions. Formation of interviral recombinants in yeast and plants is accelerated by depletion of the cellular SERCA-like Pmr1 ATPase-driven Ca(2+)/Mn(2+) pump, regulating intracellular Ca(2+) and Mn(2+) influx into the Golgi apparatus from the cytosol. The interviral recombinants are generated by a template-switching mechanism during RNA replication by the viral replicase. Replication studies revealed that a group of interviral recombinants is replication competent in cell-free extracts, in yeast, and in the plant Nicotiana benthamiana We propose that there are major differences among the viral replicases to generate and maintain interviral recombinants. Altogether, the obtained data promote the model that host factors greatly contribute to the formation of recombinants among related and unrelated viruses. This is the first time that a host factor's role in affecting interviral Recombination is established.IMPORTANCE Viruses with RNA genomes are abundant, and their genomic sequences show astonishing variation. Genetic Recombination in RNA viruses is a major force behind their rapid evolution, enhanced pathogenesis, and adaptation to their hosts. We utilized a previously identified intracellular Ca(2+)/Mn(2+) pump-deficient yeast to search for interviral recombinants. Noninfectious viral replication systems were used to avoid generating unwanted infectious interviral recombinants. Altogether, interviral RNA recombinants were observed between plant and insect viruses, and between a fungal double-stranded RNA (dsRNA) virus and an insect virus, in the yeast host. In addition, interviral recombinants between two plant virus replicon RNAs were identified in N. benthamiana plants, in which the intracellular Ca(2+)/Mn(2+) pump was depleted. These findings underline the crucial role of the host in promoting RNA Recombination among unrelated viruses.
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interviral Recombination between plant insect and fungal RNA viruses role of the intracellular ca2 mn2 pump
Journal of Virology, 2019Co-Authors: Nikolay Kovalev, Judit Pogany, Peter D NagyAbstract:Recombination is one of the driving forces of viral evolution. RNA Recombination events among similar RNA viruses are frequent, although RNA Recombination could also take place among unrelated viruses. In this paper, we have established efficient interviral Recombination systems based on yeast and plants. We show that diverse RNA viruses, including the plant viruses tomato bushy stunt virus, caRNAtion Italian ringspot virus, and turnip crinkle virus-associated RNA; the insect plus-strand RNA [(+)RNA] viruses Flock House virus and Nodamura virus; and the double-stranded L-A virus of yeast, are involved in interviral Recombination events. Most interviral recombinants are minus-strand recombinant RNAs, and the junction sites are not randomly distributed, but there are certain hot spot regions. Formation of interviral recombinants in yeast and plants is accelerated by depletion of the cellular SERCA-like Pmr1 ATPase-driven Ca2+/Mn2+ pump, regulating intracellular Ca2+ and Mn2+ influx into the Golgi apparatus from the cytosol. The interviral recombinants are generated by a template-switching mechanism during RNA replication by the viral replicase. Replication studies revealed that a group of interviral recombinants is replication competent in cell-free extracts, in yeast, and in the plant Nicotiana benthamiana We propose that there are major differences among the viral replicases to generate and maintain interviral recombinants. Altogether, the obtained data promote the model that host factors greatly contribute to the formation of recombinants among related and unrelated viruses. This is the first time that a host factor's role in affecting interviral Recombination is established.IMPORTANCE Viruses with RNA genomes are abundant, and their genomic sequences show astonishing variation. Genetic Recombination in RNA viruses is a major force behind their rapid evolution, enhanced pathogenesis, and adaptation to their hosts. We utilized a previously identified intracellular Ca2+/Mn2+ pump-deficient yeast to search for interviral recombinants. Noninfectious viral replication systems were used to avoid generating unwanted infectious interviral recombinants. Altogether, interviral RNA recombinants were observed between plant and insect viruses, and between a fungal double-stranded RNA (dsRNA) virus and an insect virus, in the yeast host. In addition, interviral recombinants between two plant virus replicon RNAs were identified in N. benthamiana plants, in which the intracellular Ca2+/Mn2+ pump was depleted. These findings underline the crucial role of the host in promoting RNA Recombination among unrelated viruses.
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the proteasomal rpn11 metalloprotease suppresses tombusvirus RNA Recombination and promotes viral replication via facilitating assembly of the viral replicase complex
Journal of Virology, 2015Co-Authors: Reddisiva K Prasanth, Daniel Barajas, Peter D NagyAbstract:RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic Recombination. RNA Recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA Recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and Recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a “matchmaker” that brings the viral p92pol replication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV Recombination. This suggests that Rpn11p can suppress tombusvirus Recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral Recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs. IMPORTANCE RNA viruses evolve rapidly due to genetic changes based on mutations and RNA Recombination. Viral genetic Recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA Recombination, although the role of the host in virus evolution is still understudied. In this study, we used a plant RNA virus, tombusvirus, to examine the role of a cellular proteasomal protein, called Rpn11, in tombusvirus Recombination in a yeast model host, in plants, and in vitro. We found that the cellular Rpn11 is subverted for tombusvirus replication and Rpn11 has a proteasome-independent function in facilitating viral replication. When the Rpn11 level is knocked down or a mutated Rpn11 is expressed, then tombusvirus RNA goes through rapid viral Recombination and evolution. Taken together, the results show that the co-opted cellular Rpn11 is a critical host factor for tombusviruses by regulating viral replication and genetic Recombination.
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coordinated function of cellular dead box helicases in suppression of viral RNA Recombination and maintenance of viral genome integrity
PLOS Pathogens, 2015Co-Authors: Chingkai Chuang, Reddisiva K Prasanth, Peter D NagyAbstract:The intricate interactions between viruses and hosts include an evolutionary arms race and adaptation that is facilitated by the ability of RNA viruses to evolve rapidly due to high frequency mutations and genetic RNA Recombination. In this paper, we show evidence that the co-opted cellular DDX3-like Ded1 DEAD-box helicase suppresses tombusviral RNA Recombination in yeast model host, and the orthologous RH20 helicase functions in a similar way in plants. In vitro replication and Recombination assays confirm the direct role of the ATPase function of Ded1p in suppression of viral Recombination. We also present data supporting a role for Ded1 in facilitating the switch from minus- to plus-strand synthesis. Interestingly, another co-opted cellular helicase, the eIF4AIII-like AtRH2, enhances TBSV Recombination in the absence of Ded1/RH20, suggesting that the coordinated actions of these helicases control viral RNA Recombination events. Altogether, these helicases are the first co-opted cellular factors in the viral replicase complex that directly affect viral RNA Recombination. Ded1 helicase seems to be a key factor maintaining viral genome integrity by promoting the replication of viral RNAs with correct termini, but inhibiting the replication of defective RNAs lacking correct 5’ end sequences. Altogether, a co-opted cellular DEAD-box helicase facilitates the maintenance of full-length viral genome and suppresses viral Recombination, thus limiting the appearance of defective viral RNAs during replication.
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The DDX3-like Ded1p DEAD-box helicase is a suppressor of TBSV RNA Recombination.
2015Co-Authors: Chingkai Chuang, Reddisiva K Prasanth, Peter D NagyAbstract:(A) The previously defined viral RNA Recombination pathway during TBSV replication. The replication-competent TBSV repRNA is cleaved by cellular endoribonucleases, such as the RNAse MRP complex, followed by limited 5’ truncations by the cellular Xrn1p exoribonuclease. These processes lead to the generation of a pool of replication-competent degRNAs that serve as recombinogenic templates in template-switching events driven by the viral replicase. The sequences in recRNAs are shown schematically. (B) Depletion of Ded1p level in yeast leads to the rapid emergence of TBSV recRNAs and degRNAs. Note that doxycycline (+dox samples) leads to depletion of Ded1p expressed from the regulatable TET promoter. Replication of the TBSV DI-AU-FP repRNA (see panel A) in wt and TET::DED1 yeasts co-expressing the tombusvirus p33 and p92 replication proteins was measured by Northern blotting 24 h after initiation of TBSV replication. Note the emergence of different species of recRNAs and degRNA (see panel A) in samples with depleted Ded1p. The accumulation level of repRNA was normalized based on the ribosomal (r)RNA (bottom panel). The bottom images show the results with semi-quantitative RT-PCR, which was used to demonstrate knock-down of Ded1 mRNA levels in TET::Ded1 yeast in the presence of doxycycline. Each sample is obtained from independent yeast colonies. The experiments were repeated two-to-three times. Throughout the paper, +/- means standard deviation. (C) Measuring recRNA levels in yeast expressing wt Ded1p, ded1–95ts or ded1–199ts mutants at 23°C (permissive temperature for yeast growth) or 29°C (semi-permissive temperature). Top panel: The accumulation of TBSV DI-AU-FP repRNA, recRNAs and degRNA was measured by Northern blotting at the 24 h time point. Middle panel: The accumulation level of repRNA was normalized based on the ribosomal (r)RNA. Bottom panel: The accumulation levels of His6-p92 and His6-p33 were tested by Western blotting. Each experiment was repeated. Asterisk marks the SDS-resistant p33 homodimer. (D) Accumulation levels of degRNA and recRNAs in Ded1ts yeasts were measured by Northern blotting. The expressed TBSV template RNA was DI-RIIΔ70 degRNA, which represents a frequently isolated degRNA species lacking RI and part of RII (Panel A). See further details in panel B.
Jozef J Bujarski - One of the best experts on this subject based on the ideXlab platform.
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genetic Recombination in plant infecting messenger sense RNA viruses overview and research perspectives
Frontiers in Plant Science, 2013Co-Authors: Jozef J BujarskiAbstract:RNA Recombination is one of the driving forces of genetic variability in (+)-strand RNA viruses. Various types of RNA-RNA crossovers were described including crosses between the same or different viral RNAs or between viral and cellular RNAs. Likewise, a variety of molecular mechanisms are known to support RNA Recombination, such as replicative events (based on inteRNAl or end-to-end replicase switchings) along with nonreplicative joining among RNA fragments of viral and/or cellular origin. Such mechanisms as RNA decay or RNA interference are responsible for RNA fragmentation and trans-esterification reactions which are likely accountable for ligation of RNA fragments. Numerous host factors were found to affect the profiles of viral RNA recombinants and significant differences in Recombination frequency were observed among various RNA viruses. Comparative analyses of viral sequences allowed for the development of evolutionary models in order to explain adaptive phenotypic changes and co-evolving sites. Many questions remain to be answered by forthcoming RNA Recombination research. (i) How various factors modulate the ability of viral replicase to switch templates, (ii) What is the intracellular location of RNA-RNA template switchings, (iii) Mechanisms and factors responsible for non-replicative RNA Recombination, (iv) Mechanisms of integration of RNA viral sequences with cellular genomic DNA, and (v) What is the role of RNA splicing and ribozyme activity. From an evolutionary stand point, it is not known how RNA viruses parasitize new host species via Recombination, nor is it obvious what the contribution of RNA Recombination is among other RNA modification pathways. We do not understand why the frequency of RNA Recombination varies so much among RNA viruses and the status of RNA Recombination as a form of sex is not well documented.
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RNA RNA Recombination in plant virus replication and evolution
Annual Review of Phytopathology, 2011Co-Authors: Joanna Sztubasolinska, Anna Urbanowicz, Marek Figlerowicz, Jozef J BujarskiAbstract:RNA-RNA Recombination is one of the strongest forces shaping the genomes of plant RNA viruses. The detection of Recombination is a challenging task that prompted the development of both in vitro and in vivo experimental systems. In the divided genome of Brome mosaic virus system, both inter- and intrasegmental crossovers are described. Other systems utilize satellite or defective interfering RNAs (DI-RNAs) of Turnip crinkle virus, Tomato bushy stunt virus, Cucumber necrosis virus, and Potato virus X. These assays identified the mechanistic details of the Recombination process, revealing the role of RNA structure and proteins in the replicase-mediated copy-choice mechanism. In copy choice, the polymerase and the nascent RNA chain from which it is synthesized switch from one RNA template to another. RNA Recombination was found to mediate the rearrangement of viral genes, the repair of deleterious mutations, and the acquisition of nonself sequences influencing the phylogenetics of viral taxa. The evidence fo...
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Efficient In Vitro System of Homologous Recombination in Brome Mosaic Bromovirus
Journal of Virology, 2006Co-Authors: Rafal Wierzchoslawski, Jozef J BujarskiAbstract:Recent in vivo studies have revealed that the subgenomic promoter (sgp) in brome mosaic bromovirus (BMV) RNA3 supports frequent homologous Recombination events (R. Wierzchoslawski, A. Dzianott, and J. Bujarski, J. Virol. 78:8552-8564, 2004). In this paper, we describe an sgp-driven in vitro system that supports efficient RNA3 crossovers. A 1:1 mixture of two (-)-sense RNA3 templates was copied with either a BMV replicase (RdRp) preparation or recombinant BMV protein 2a. The BMV replicase enzyme supported a lower Recombination frequency than 2a, demonstrating a role of other viral and/or host factors. The described in vitro system will allow us to study the mechanism of homologous RNA Recombination.
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Infection and RNA Recombination of Brome mosaic virus in Arabidopsis thaliana.
Virology, 2004Co-Authors: Aleksandra Dzianott, Jozef J BujarskiAbstract:Ecotypes of Arabidopsis thaliana supported the replication and systemic spread of Brome mosaic virus (BMV) RNAs. Infection was induced either by manual inoculation with viral RNA or by BMV virions, demonstrating that virus disassembly did not prevent infection. When in vitro-transcribed BMV RNAs 1-3 were used, production of subgenomic RNA4 was observed, showing that BMV RNA replication and transcription had occurred. Furthermore, inoculations of the transgenic Arabidopsis line that expressed a suppressor of RNA interference (RNAi) pathway markedly increased the BMV RNA concentrations. Inoculations with designed BMV RNA3 Recombination vectors generated both homologous and nonhomologous BMV RNA-RNA recombinants. Thus, all cellular factors essential for BMV RNA replication, transcription, and RNA Recombination were shown to be present in Arabidopsis. The current scope of understanding of the model Arabidopsis plant system should facilitate the identification of these factors governing the BMV life cycle.
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A Transcriptionally Active Subgenomic Promoter Supports Homologous Crossovers in a Plus-Strand RNA Virus
Journal of Virology, 2003Co-Authors: Rafal Wierzchoslawski, Aleksandra Dzianott, Selvi Kunimalayan, Jozef J BujarskiAbstract:Genetic RNA Recombination plays an important role in viral evolution, but its molecular mechanism is not well understood. In this work we describe homologous RNA Recombination activity that is supported by a subgenomic promoter (sgp) region in the RNA3 segment of brome mosaic bromovirus (BMV), a tripartite plus-strand RNA virus. The crossover frequencies were determined by coinoculations with pairs of BMV RNA3 variants that carried a duplicated sgp region flanked by marker restriction sites. A region composed of the sgp core, a poly(A) tract, and an upstream enhancer supported homologous exchanges in 25% of the analyzed RNA3 progeny. However, mutations in the sgp core stopped both the transcription of the sgp RNA and homologous Recombination. These data provide evidence for an association of RNA Recombination with transcription.
Chi-ping Cheng - One of the best experts on this subject based on the ideXlab platform.
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suppression of viral RNA Recombination by a host exoribonuclease
Journal of Virology, 2006Co-Authors: Chi-ping Cheng, Elena Serviene, Peter D NagyAbstract:RNA viruses of humans, animals, and plants evolve rapidly due to mutations and RNA Recombination. A previous genome-wide screen in Saccharomyces cerevisiae, a model host, identified five host genes, including XRN1, encoding a 5'-3' exoribonuclease, whose absence led to an approximately 10- to 50-fold enhancement of RNA Recombination in Tomato bushy stunt virus (E. Serviene, N. Shapka, C. P. Cheng, T. Panavas, B. Phuangrat, J. Baker, and P. D. Nagy, Proc. Natl. Acad. Sci. USA 102:10545-10550, 2005). In this study, we found abundant 5'-truncated viral RNAs in xrn1delta mutant strains but not in the parental yeast strains, suggesting that these RNAs might serve as Recombination substrates promoting RNA Recombination in xrn1delta mutant yeast. This model is supported by data showing that an enhanced level of viral recombinant accumulation occurred when two different 5'-truncated viral RNAs were expressed in the parental and xrn1delta mutant yeast strains or electroporated into plant protoplasts. Moreover, we demonstrate that purified Xrn1p can degrade the 5'-truncated viral RNAs in vitro. Based on these findings, we propose that Xrn1p can suppress viral RNA Recombination by rapidly removing the 5'-truncated RNAs, the substrates of Recombination, and thus reducing the chance for Recombination to occur in the parental yeast strain. In addition, we show that the 5'-truncated viral RNAs are generated by host endoribonucleases. Accordingly, overexpression of the Ngl2p endoribonuclease led to an increased accumulation of cleaved viral RNAs in vivo and in vitro. Altogether, this paper establishes that host ribonucleases and host-mediated viral RNA turnover play major roles in RNA virus Recombination and evolution.
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screening of the yeast ythc collection identifies essential host factors affecting tombusvirus RNA Recombination
Journal of Virology, 2006Co-Authors: Elena Serviene, Chi-ping Cheng, Jannine Baker, Yi Jiang, Peter D NagyAbstract:RNA Recombination is a major process in promoting rapid virus evolution in an infected host. A previous genome-wide screen with the yeast single-gene deletion library of 4,848 strains, representing ∼80% of all genes of yeast, led to the identification of 11 host genes affecting RNA Recombination in Tomato bushy stunt virus (TBSV), a small model plant virus (E. Serviene, N. Shapka, C. P. Cheng, T. Panavas, B. Phuangrat, J. Baker, and P. D. Nagy, Proc. Natl. Acad. Sci. USA 102:10545-10550, 2005). To further test the role of host genes in viral RNA Recombination, in this paper, we extended the screening to 800 essential yeast genes present in the yeast Tet-promoters Hughes Collection (yTHC). In total, we identified 16 new host genes that either increased or decreased the ratio of TBSV recombinants to the nonrecombined TBSV RNA. The identified essential yeast genes are involved in RNA transcription/metabolism, in protein metabolism/transport, or unknown cellular processes. Detailed analysis of the effect of the identified yeast genes revealed that they might affect RNA Recombination by altering (i) the ratio of the two viral replication proteins, (ii) the stability of the viral RNA, and/or (iii) the replicability of the recombinant RNAs. Overall, this and previous works firmly establish that a set of essential and nonessential host genes could affect TBSV Recombination and evolution.
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screening of the yeast ythc collection identifies essential host factors affecting tombusvirus RNA Recombination
Journal of Virology, 2006Co-Authors: Elena Serviene, Chi-ping Cheng, Jannine Baker, Yi Jiang, Peter D NagyAbstract:RNA Recombination is a major process in promoting rapid virus evolution in an infected host. A previous genome-wide screen with the yeast single-gene deletion library of 4,848 strains, representing approximately 80% of all genes of yeast, led to the identification of 11 host genes affecting RNA Recombination in Tomato bushy stunt virus (TBSV), a small model plant virus (E. Serviene, N. Shapka, C. P. Cheng, T. Panavas, B. Phuangrat, J. Baker, and P. D. Nagy, Proc. Natl. Acad. Sci. USA 102:10545-10550, 2005). To further test the role of host genes in viral RNA Recombination, in this paper, we extended the screening to 800 essential yeast genes present in the yeast Tet-promoters Hughes Collection (yTHC). In total, we identified 16 new host genes that either increased or decreased the ratio of TBSV recombinants to the nonrecombined TBSV RNA. The identified essential yeast genes are involved in RNA transcription/metabolism, in protein metabolism/transport, or unknown cellular processes. Detailed analysis of the effect of the identified yeast genes revealed that they might affect RNA Recombination by altering (i) the ratio of the two viral replication proteins, (ii) the stability of the viral RNA, and/or (iii) the replicability of the recombinant RNAs. Overall, this and previous works firmly establish that a set of essential and nonessential host genes could affect TBSV Recombination and evolution.
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Genome-wide screen identifies host genes affecting viral RNA Recombination.
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Elena Servienė, Chi-ping Cheng, Bencharong Phuangrat, Jannine Baker, Natalia Shapka, Tadas Panavas, Peter D NagyAbstract:Rapid evolution of RNA viruses with mRNA-sense genomes is a major concern to health and economic welfare because of the devastating diseases these viruses inflict on humans, animals, and plants. To test whether host genes can affect the evolution of RNA viruses, we used a Saccharomyces cerevisiae single-gene deletion library, which includes ≈80% of yeast genes, in RNA Recombination studies based on a small viral replicon RNA derived from tomato bushy stunt virus. The genome-wide screen led to the identification of five host genes whose absence resulted in the rapid generation of new viral RNA recombinants. Thus, these genes normally suppress viral RNA Recombination, but in their absence, hosts become viral Recombination “hotbeds.” Four of the five suppressor genes are likely involved in RNA degradation, suggesting that RNA degradation could play a role in viral RNA Recombination. In contrast, deletion of four other host genes inhibited virus Recombination, indicating that these genes normally accelerate the RNA Recombination process. A comparison of deletion strains with the lowest and the highest Recombination rate revealed that host genes could affect recombinant accumulation by up to 80-fold. Overall, our results demonstrate that a set of host genes have a major effect on RNA virus Recombination and evolution.
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mechanism of RNA Recombination in carmo and tombusviruses evidence for template switching by the RNA dependent RNA polymerase in vitro
Journal of Virology, 2003Co-Authors: Chi-ping Cheng, Peter D NagyAbstract:RNA Recombination occurs frequently during replication of tombusviruses and carmoviruses, which are related small plus-sense RNA viruses of plants. The most common recombinants generated by these viruses are either defective interfering (DI) RNAs or chimeric satellite RNAs, which are thought to be generated by template switching of the viral RNA-dependent RNA polymerase (RdRp) during the viral replication process. To test if RNA Recombination is mediated by the viral RdRp, we used either a purified recombinant RdRp of Turnip crinkle carmovirus or a partially purified RdRp preparation of Cucumber necrosis tombusvirus. We demonstrated that these RdRp preparations generated RNA recombinants in vitro. The RdRp-driven template switching events occurred between either identical templates or two different RNA templates. The template containing a replication enhancer recombined more efficiently than templates containing artificial sequences. We also observed that AU-rich sequences promote Recombination more efficiently than GC-rich sequences. Cloning and sequencing of the generated recombinants revealed that the junction sites were located frequently at the ends of the templates (end-to-end template switching). We also found several recombinants that were generated by template switching involving inteRNAl positions in the RNA templates. In contrast, RNA ligation-based RNA Recombination was not detected in vitro. Demonstration of the ability of carmo- and tombusvirus RdRps to switch RNA templates in vitro supports the copy-choice models of RNA Recombination and DI RNA formation for these viruses.
Elena Serviene - One of the best experts on this subject based on the ideXlab platform.
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suppression of viral RNA Recombination by a host exoribonuclease
Journal of Virology, 2006Co-Authors: Chi-ping Cheng, Elena Serviene, Peter D NagyAbstract:RNA viruses of humans, animals, and plants evolve rapidly due to mutations and RNA Recombination. A previous genome-wide screen in Saccharomyces cerevisiae, a model host, identified five host genes, including XRN1, encoding a 5'-3' exoribonuclease, whose absence led to an approximately 10- to 50-fold enhancement of RNA Recombination in Tomato bushy stunt virus (E. Serviene, N. Shapka, C. P. Cheng, T. Panavas, B. Phuangrat, J. Baker, and P. D. Nagy, Proc. Natl. Acad. Sci. USA 102:10545-10550, 2005). In this study, we found abundant 5'-truncated viral RNAs in xrn1delta mutant strains but not in the parental yeast strains, suggesting that these RNAs might serve as Recombination substrates promoting RNA Recombination in xrn1delta mutant yeast. This model is supported by data showing that an enhanced level of viral recombinant accumulation occurred when two different 5'-truncated viral RNAs were expressed in the parental and xrn1delta mutant yeast strains or electroporated into plant protoplasts. Moreover, we demonstrate that purified Xrn1p can degrade the 5'-truncated viral RNAs in vitro. Based on these findings, we propose that Xrn1p can suppress viral RNA Recombination by rapidly removing the 5'-truncated RNAs, the substrates of Recombination, and thus reducing the chance for Recombination to occur in the parental yeast strain. In addition, we show that the 5'-truncated viral RNAs are generated by host endoribonucleases. Accordingly, overexpression of the Ngl2p endoribonuclease led to an increased accumulation of cleaved viral RNAs in vivo and in vitro. Altogether, this paper establishes that host ribonucleases and host-mediated viral RNA turnover play major roles in RNA virus Recombination and evolution.
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screening of the yeast ythc collection identifies essential host factors affecting tombusvirus RNA Recombination
Journal of Virology, 2006Co-Authors: Elena Serviene, Chi-ping Cheng, Jannine Baker, Yi Jiang, Peter D NagyAbstract:RNA Recombination is a major process in promoting rapid virus evolution in an infected host. A previous genome-wide screen with the yeast single-gene deletion library of 4,848 strains, representing ∼80% of all genes of yeast, led to the identification of 11 host genes affecting RNA Recombination in Tomato bushy stunt virus (TBSV), a small model plant virus (E. Serviene, N. Shapka, C. P. Cheng, T. Panavas, B. Phuangrat, J. Baker, and P. D. Nagy, Proc. Natl. Acad. Sci. USA 102:10545-10550, 2005). To further test the role of host genes in viral RNA Recombination, in this paper, we extended the screening to 800 essential yeast genes present in the yeast Tet-promoters Hughes Collection (yTHC). In total, we identified 16 new host genes that either increased or decreased the ratio of TBSV recombinants to the nonrecombined TBSV RNA. The identified essential yeast genes are involved in RNA transcription/metabolism, in protein metabolism/transport, or unknown cellular processes. Detailed analysis of the effect of the identified yeast genes revealed that they might affect RNA Recombination by altering (i) the ratio of the two viral replication proteins, (ii) the stability of the viral RNA, and/or (iii) the replicability of the recombinant RNAs. Overall, this and previous works firmly establish that a set of essential and nonessential host genes could affect TBSV Recombination and evolution.
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screening of the yeast ythc collection identifies essential host factors affecting tombusvirus RNA Recombination
Journal of Virology, 2006Co-Authors: Elena Serviene, Chi-ping Cheng, Jannine Baker, Yi Jiang, Peter D NagyAbstract:RNA Recombination is a major process in promoting rapid virus evolution in an infected host. A previous genome-wide screen with the yeast single-gene deletion library of 4,848 strains, representing approximately 80% of all genes of yeast, led to the identification of 11 host genes affecting RNA Recombination in Tomato bushy stunt virus (TBSV), a small model plant virus (E. Serviene, N. Shapka, C. P. Cheng, T. Panavas, B. Phuangrat, J. Baker, and P. D. Nagy, Proc. Natl. Acad. Sci. USA 102:10545-10550, 2005). To further test the role of host genes in viral RNA Recombination, in this paper, we extended the screening to 800 essential yeast genes present in the yeast Tet-promoters Hughes Collection (yTHC). In total, we identified 16 new host genes that either increased or decreased the ratio of TBSV recombinants to the nonrecombined TBSV RNA. The identified essential yeast genes are involved in RNA transcription/metabolism, in protein metabolism/transport, or unknown cellular processes. Detailed analysis of the effect of the identified yeast genes revealed that they might affect RNA Recombination by altering (i) the ratio of the two viral replication proteins, (ii) the stability of the viral RNA, and/or (iii) the replicability of the recombinant RNAs. Overall, this and previous works firmly establish that a set of essential and nonessential host genes could affect TBSV Recombination and evolution.
Paul Becher - One of the best experts on this subject based on the ideXlab platform.
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Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins.
Genome Biology and Evolution, 2017Co-Authors: Maximiliane Kleine Büning, Denise Meyer, Sophia Austermann-busch, Gleyder Roman-sosa, Tillmann H. Rümenapf, Paul BecherAbstract:RNA Recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of Recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA Recombination depends on translation of viral proteins, a nonreplicative Recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the inteRNAl ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient Recombination and suggests that RNA Recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5' terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA Recombination. Taken together, this study highlights that nonreplicative RNA Recombination does not require translation of viral proteins.
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RNA Recombination in pestiviruses cellular RNA sequences in viral genomes highlight the role of host factors for viral persistence and lethal disease
RNA Biology, 2011Co-Authors: Paul Becher, Norbert TautzAbstract:Persistence of the positive strand RNA virus bovine viral diarrhea virus (BVDV) in its host may last for years. However, it frequently ends in lethal disease triggered by emerging virus mutants created by RNA Recombination. Those mutant genomes often encompass cellular mRNA fragments. Persistence of BVDV depends on a mechanism limiting viral RNA replication efficiency. This restriction is based on the dependency of a viral protease on a cellular cofactor available only in limiting amounts. Virus mutants leading to progression from persistence to lethal disease elude this regulatory mechanism by various genomic changes achieved by RNA Recombination. Cell culture based studies on the underlying mechanisms demonstrated that RNA Recombination occurs even in the absence of an active viral RNA-dependent RNA polymerase. This implicates that mechanisms besides the commonly accepted replicative template switching model are involved in viral RNA Recombination.
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noncytopathogenic pestivirus strains generated by nonhomologous RNA Recombination alterations in the ns4a ns4b coding region
Journal of Virology, 2005Co-Authors: Andreas Gallei, Michaela Orlich, H J Thiel, Paul BecherAbstract:Several studies have demonstrated that cytopathogenic (cp) pestivirus strains evolve from noncytopathogenic (noncp) viruses by nonhomologous RNA Recombination. In addition, two recent reports showed the rapid emergence of noncp Bovine viral diarrhea virus (BVDV) after a few cell culture passages of cp BVDV strains by homologous Recombination between identical duplicated viral sequences. To allow the identification of Recombination sites from noncp BVDV strains that evolve from cp viruses, we constructed the cp BVDV strains CP442 and CP552. Both harbor duplicated viral sequences of different origin flanking the cellular insertion Nedd8*; the latter is a prerequisite for their cytopathogenicity. In contrast to the previous studies, isolation of noncp strains was possible only after extensive cell culture passages of CP442 and CP552. Sequence analysis of 15 isolated noncp BVDVs confirmed that all recombinant strains lack at least most of Nedd8*. Interestingly, only one strain resulted from homologous Recombination while the other 14 strains were generated by nonhomologous Recombination. Accordingly, our data suggest that the extent of sequence identity between participating sequences influences both frequency and mode (homologous versus nonhomologous) of RNA Recombination in pestiviruses. Further analyses of the noncp recombinant strains revealed that a duplication of 14 codons in the BVDV nonstructural protein 4B (NS4B) gene does not interfere with efficient viral replication. Moreover, an insertion of viral sequences between the NS4A and NS4B genes was well tolerated. These findings thus led to the identification of two genomic loci which appear to be suited for the insertion of heterologous sequences into the genomes of pestiviruses and related viruses.
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Noncytopathogenic pestivirus strains generated by nonhomologous RNA Recombination: alterations in the NS4A/NS4B coding region.
Journal of Virology, 2005Co-Authors: Andreas Gallei, Michaela Orlich, H J Thiel, Paul BecherAbstract:Several studies have demonstrated that cytopathogenic (cp) pestivirus strains evolve from noncytopathogenic (noncp) viruses by nonhomologous RNA Recombination. In addition, two recent reports showed the rapid emergence of noncp Bovine viral diarrhea virus (BVDV) after a few cell culture passages of cp BVDV strains by homologous Recombination between identical duplicated viral sequences. To allow the identification of Recombination sites from noncp BVDV strains that evolve from cp viruses, we constructed the cp BVDV strains CP442 and CP552. Both harbor duplicated viral sequences of different origin flanking the cellular insertion Nedd8*; the latter is a prerequisite for their cytopathogenicity. In contrast to the previous studies, isolation of noncp strains was possible only after extensive cell culture passages of CP442 and CP552. Sequence analysis of 15 isolated noncp BVDVs confirmed that all recombinant strains lack at least most of Nedd8*. Interestingly, only one strain resulted from homologous Recombination while the other 14 strains were generated by nonhomologous Recombination. Accordingly, our data suggest that the extent of sequence identity between participating sequences influences both frequency and mode (homologous versus nonhomologous) of RNA Recombination in pestiviruses. Further analyses of the noncp recombinant strains revealed that a duplication of 14 codons in the BVDV nonstructural protein 4B (NS4B) gene does not interfere with efficient viral replication. Moreover, an insertion of viral sequences between the NS4A and NS4B genes was well tolerated. These findings thus led to the identification of two genomic loci which appear to be suited for the insertion of heterologous sequences into the genomes of pestiviruses and related viruses.
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RNA Recombination in vivo in the absence of viral replication
Journal of Virology, 2004Co-Authors: Andreas Gallei, Heinzjurgen Thiel, Alexander Pankraz, Paul BecherAbstract:To study fundamental aspects of RNA Recombination, an in vivo RNA Recombination system was established. This system allowed the efficient generation of recombinant cytopathogenic pestiviruses after transfection of synthetic, nonreplicatable, subgenomic transcripts in cells infected with a replicating noncytopathogenic virus. Studies addressing the interplay between RNA Recombination and replication revealed that cotransfection of noninfected cells with various pairs of nonreplicatable RNA derivatives also led to the emergence of recombinant viral genomes. Remarkably, homologous and nonhomologous Recombination occurred between two overlapping transcripts, each lacking different essential parts of the viral RNA-dependent RNA polymerase (RdRp) gene. Apart from the generally accepted viral replicative copy choice Recombination, our results prove the existence of a viral RdRp-independent mechanism of RNA Recombination that occurs in vivo. It appears likely that such a mechanism not only contributes to the evolution of RNA viruses but also leads to the generation of recombinant cellular RNAs.
