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David P. Millar - One of the best experts on this subject based on the ideXlab platform.
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A Survey of DDX21 Activity During Rev/RRE Complex Formation
Journal of Molecular Biology, 2018Co-Authors: John A. Hammond, Li Zhou, Rajan Lamichhane, Hui-yi Chu, David P. Millar, Larry Gerace, James R. WilliamsonAbstract:HIV-1 requires a specialized nuclear export pathway to transport unspliced and partially spliced viral transcripts to the cytoplasm. Central to this pathway is the viral protein Rev, which binds to the Rev response element in stem IIB located on unspliced viral transcripts and subsequently oligomerizes in a cooperative manner. Previous work identified a number of cellular DEAD-box helicases as in vivo binding partners of Rev, and siRNA experiments indicated a functional role for many in the HIV replication cycle. Two DEAD-box proteins, DDX1 and DDX3, had previously been shown to play a role in HIV pathogenesis. In this study, another protein identified in that screen, DDX21, is tested for protein and RNA binding and subsequent enzymatic activities in the context of the Rev/RRE pathway. We found that DDX21 can bind to the RRE with high affinity, and this binding stimulates ATPase activity with an enzymatic efficiency similar to DDX1. Furthermore, DDX21 is both an ATP-dependent and ATP-independent helicase, and both ATPase and ATP-dependent helicase activities are inhibited by Rev in a dose-dependent manner, although ATP-independent helicase activity is not. A conserved binding interaction between DDX protein's DEAD domain and Rev was identified, with Rev's nuclear diffusion inhibitory signal motif playing a significant role in binding. Finally, DDX21 was shown to enhance Rev binding to the RRE in a manner similar to that previously described for DDX1, although DDX3 does not. These data indicate that DDX1 and DDX21 have similar biochemical activities with regard to the Rev/RRE system, while DDX3 differs.
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a survey of ddx21 activity during rev rre complex formation
Journal of Molecular Biology, 2017Co-Authors: John A. Hammond, Li Zhou, Rajan Lamichhane, Hui-yi Chu, David P. Millar, Larry Gerace, James R. WilliamsonAbstract:HIV-1 requires a specialized nuclear export pathway to transport unspliced and partially spliced viral transcripts to the cytoplasm. Central to this pathway is the viral protein Rev, which binds to the Rev response element in stem IIB located on unspliced viral transcripts and subsequently oligomerizes in a cooperative manner. Previous work identified a number of cellular DEAD-box helicases as in vivo binding partners of Rev, and siRNA experiments indicated a functional role for many in the HIV replication cycle. Two DEAD-box proteins, DDX1 and DDX3, had previously been shown to play a role in HIV pathogenesis. In this study, another protein identified in that screen, DDX21, is tested for protein and RNA binding and subsequent enzymatic activities in the context of the Rev/RRE pathway. We found that DDX21 can bind to the RRE with high affinity, and this binding stimulates ATPase activity with an enzymatic efficiency similar to DDX1. Furthermore, DDX21 is both an ATP-dependent and ATP-independent helicase, and both ATPase and ATP-dependent helicase activities are inhibited by Rev in a dose-dependent manner, although ATP-independent helicase activity is not. A conserved binding interaction between DDX protein's DEAD domain and Rev was identified, with Rev's nuclear diffusion inhibitory signal motif playing a significant role in binding. Finally, DDX21 was shown to enhance Rev binding to the RRE in a manner similar to that previously described for DDX1, although DDX3 does not. These data indicate that DDX1 and DDX21 have similar biochemical activities with regard to the Rev/RRE system, while DDX3 differs.
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A DEAD-box protein acts through RNA to promote HIV-1 Rev-RRE assembly.
Nucleic Acids Research, 2017Co-Authors: Rajan Lamichhane, John A. Hammond, James R. Williamson, Edwin J. C. Van Der Schans, Raymond F Pauszek, Rae M Anderson, Ingemar Pedron, David P. MillarAbstract:The HIV-1 Rev protein activates nuclear export of unspliced and partially spliced viral RNA transcripts, which encode the viral genome and the genes encoding viral structural proteins, by binding to and oligomerizing on the Rev Response Element (RRE). The human DEAD-box protein 1 (DDX1) enhances the RNA export activity of Rev through an unknown mechanism. Using a single-molecule assembly assay and various DDX1 mutants, we show that DDX1 acts through the RRE RNA to specifically accelerate the nucleation step of the Rev-RRE assembly process. Single-molecule Förster resonance energy transfer (smFRET) experiments using donor-labeled Rev and acceptor-labeled DDX1 show that both proteins can associate with a single RRE molecule. However, simultaneous interaction is only observed in a subset of binding events and does not explain the extent to which DDX1 promotes the nucleation step of Rev-RRE assembly. Together, these results are consistent with a model wherein DDX1 acts as an RNA chaperone, remodeling the RRE into a conformation that is pre-organized to bind the first Rev monomer, thereby promoting the overall Rev-RRE assembly process.
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Role of Dead Box Helicases in HIV-1 Rev Function: a Single-Molecule Approach
Biophysical Journal, 2014Co-Authors: Rajan Lamichhane, David P. MillarAbstract:The HIV-1 Rev (Regulator of Expression of Virion) protein activates nuclear export of unspliced and partially spliced viral mRNAs, which encode the viral genome and the genes encoding viral structural proteins. Rev interacts with a highly conserved region, the Rev Response Element (RRE), located within the viral mRNA. Initially, a single Rev monomer binds to stem loop IIB of the RRE, whereupon additional Rev monomers are recruited to the RRE through a combination of RNA-protein and protein-protein interactions, resulting in the formation of a functional nuclear export complex. In addition, several cellular host proteins, such as the DEAD box helicases DDX1 and DDX21 are known to be required for efficient Rev function in vivo, although their precise role is unknown. In this study, a variety of single-molecule fluorescence spectroscopic methods were used to dissect the role of DDX1 and DDX21 during assembly of Rev-RRE complexes. To facilitate these studies, the large DDX1 and DDX21 proteins were enzymatically labeled with bright and photostable dyes, while Rev was labeled at a single cysteine by maleimide chemistry. Single-color TIRF measurements with labeled Rev were used to monitor individual Rev monomer binding steps during oligomeric Rev-RRE assembly. Moreover, single-color experiments with labeled DDX1 or DDX21 reveal that each helicase is capable of binding directly to the RRE. Two-color colocalization and FRET measurements were used to monitor the simultaneous binding of both Rev and DDX1 to the RRE, revealing the temporal correlation between individual protein binding steps. Together, these studies are revealing how DEAD box helicases are able to promote the oligomeric assembly of Rev on the RRE, thereby acting as cellular cofactors of HIV-1. Supported by NIH P50 grant GM082545.
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Oligomeric Assembly of HIV-1 Rev on the Rev Response Element: Role of Cellular Cofactors
Biophysical Journal, 2012Co-Authors: Rajan Lamichhane, Rae M. Robertson-anderson, Svitlana Y. Berezhna, Edwin J. C. Van Der Schans, David P. MillarAbstract:Rev, a key regulatory protein of HIV-1, activates nuclear export of unspliced and partially spliced viral mRNAs, which encode the viral genome and the genes encoding viral structural proteins, respectively. Initially, a single Rev monomer binds to a highly conserved region, stem IIB located on the Rev Response Element (RRE) of viral mRNA. Following this nucleation step, additional Rev monomers are recruited to the RRE through a combination of RNA-protein and protein-protein interactions, resulting in the formation of a functional nuclear export complex. In addition, several cellular proteins, such as the DEAD box helicases DDX1 and DDX3 are known to be required for efficient Rev function in vivo, although their precise role is unknown. In this study, single-molecule total internal reflection fluorescence (smTIRF) microscopy was used to visualize oligomeric assembly of Rev on the RRE with single monomer resolution. Binding of up to eight fluorescently labeled Rev monomers to a single immobilized RRE molecule was observed in real-time as discrete jumps in fluorescence intensity, and the event frequencies and corresponding binding and dissociation rates for the different Rev-RRE stoichiometries were determined. The smTIRF assay was used to study Rev-RRE assembly in the presence of DDX1, DDX3 and other cellular proteins. The presence of DDX1 promotes oligomeric assembly by accelerating the first few Rev monomer binding steps, suggesting that DDX1 acts as a chaperone of Rev. The smTIRF measurements are being extended to a multi-color format, in order to directly visualize the colocalization of Rev and selected cellular proteins on the same immobilized RRE molecules. These measurements are revealing the precise timing of various protein binding events during ribonucleoprotein assembly. Supported by NIH grant P50 GM082545.
Roseline Godbout - One of the best experts on this subject based on the ideXlab platform.
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Understanding the Role of DDX1 Expression in Neuroblastoma Response to Treatment
2020Co-Authors: Jessica Bennett, Roseline GodboutAbstract:Introduction: Neuroblastoma is a pediatric cancer originating from neural crest cells and is the most lethal extracranial solid tumour in children. If the neuroblastoma is classified as “high-risk”, over 60% of children diagnosed do not survive past age five. High-risk neuroblastoma is often associated with amplification (increased copies) of the MYCN oncogene. DEAD box proteins are involved in modification of RNA secondary structure and are involved in cellular stress response. Previous research undertaken by the Godbout lab indicates that the DEAD Box 1 (DDX1) gene is co-amplified with the MYCN gene in about 50% of high-risk neuroblastomas, and it may be possible that amplification or knockdown of the DDX1 gene in neuroblastoma cell lines could improve their survival following treatment with DNA damaging agents (ionizing radiation and chemotherapy drugs). Hypothesis: Based on the lab’s prior knowledge of the role of DDX1, my hypothesis is that DDX1 knockdown will result in an improved cellular response to treatment, as DDX1 is known to play roles in cellular survival as well as in DNA double-strand break repair. Methods: Neuroblastoma resistance to treatment was measured indirectly using the soft agar colony formation assay. This assay selects for cancer cells and colony counts can be performed to determine the efficacy of treatment. I am examining the effect of DDX1 knockdown on rate of colony formation in DDX1-amplified and non-amplified cell lines, both prior to and one hour following treatment with ionizing radiation, and the chemotherapy drugs doxorubicin, vincristine, etoposide, bleomycin, and sodium arsenite. Results: Knockdown of DDX1 has opposite effects in DDX1-amplified and non-amplified cells. While knockdown of DDX1 decreases colony formation in DDX1-amplified cell lines, it results in increased colony formation in non-amplified cell lines. While knockdown of DDX1 had no effect on colony formation in cell lines treated with the microtubule-inhibiting chemotherapy drug vincristine, DDX1 knockdown resulted in an improved response to DNA-damaging chemotherapy drugs doxorubicin and etoposide in non-amplified cell lines. There is not yet sufficient data to determine the effect of DNA damaging agents on DDX1-amplified cell lines. Conclusion: Based on my results, DDX1 likely plays a role in the DNA damage response. At basal levels, DDX1 may repair DNA damage induced by ionizing radiation and chemotherapy agents, whereas when DDX1 is overexpressed or knocked down, it is no longer able to perform this function to the same level, thus decreasing neuroblastoma resistance to treatment. Altering the levels of DDX1 could potentially increase the efficacy of radiation and certain chemotherapy drugs in neuroblastoma treatment. leading to a reduction in the mortality of infants and children diagnosed with high-risk neuroblastoma.
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Cytoplasmic aggregation of DDX1 in developing embryos: Early embryonic lethality associated with DDX1 knockout.
Developmental Biology, 2019Co-Authors: Matthew R. Hildebrandt, Yixiong Wang, Lubna Yasmin, Darryl D. Glubrecht, Roseline GodboutAbstract:Abstract Temporally-regulated maternal RNA translation is essential for embryonic development, with defective degradation resulting in stalled 2-cell embryos. We show that DDX1, a DEAD box protein implicated in RNA transport, may be a key regulator of maternal RNA utilization. DDX1 protein localizes exclusively to cytoplasmic granules in both oocytes and early stage mouse embryos, with DDX1 requiring RNA for retention at these sites. Homozygous knockout of DDX1 causes stalling of mouse embryos at the 2–4 cell stages. These results suggest a maternal RNA-dependent role for DDX1 in the progression of embryos past the 2–4 cell stage. The change in appearance of DDX1-containing granules in developing embryos further supports a role in temporally-regulated degradation of RNAs. We carried out RNA-immunoprecipitations (RNA-IPs) to identify mRNAs bound to DDX1 in 2-cell embryos, focusing on 16 maternal genes previously shown to be essential for embryonic development past the 1- to 2-cell stages. Five of these RNAs were preferentially bound by DDX1: Ago2, Zar1, Tle6, Floped and Tif1α. We propose that DDX1 controls access to subsets of key maternal RNAs required for early embryonic development.
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Role for RIF1-interacting partner DDX1 in BLM recruitment to DNA double-strand breaks.
DNA Repair, 2017Co-Authors: Ho-yin Poon, Devon R. Germain, Matthew R. Hildebrandt, Elizabeth A. Monckton, Richard P. Fahlman, Roseline GodboutAbstract:Human Rap1-interacting factor 1 (RIF1) is an important player in the repair of DNA double strand breaks (DSBs). RIF1 acts downstream of 53BP1, with well-documented roles in class switch recombination in B-cells and inhibition of end resection initiation in BRCA1-defective cells. Here, we report that DEAD Box 1 (DDX1), a RNA helicase also implicated in DSB repair, interacts with RIF1, with co-localization of DDX1 and RIF1 observed throughout interphase. Recruitment of DDX1 to DSBs is dependent on RIF1, with RIF1 depletion abolishing DDX1-mediated facilitation of homologous recombination at DSBs. As previously demonstrated for RIF1, DDX1 is also required for chromatin loading of Bloom syndrome helicase (BLM) to ionizing radiation-induced DSBs, a RIF1-related activity that is independent of 53BP1. We show that DDX1 and RIF1 have different nucleic acid requirements for accumulation at DSBs, with RNA-DNA hybrids required for DDX1 accrual at DSBs, and single-strand RNA required for accumulation of RIF1 at these sites. Our data suggest both convergent and divergent roles for DDX1 and RIF1 in DSB repair, and may help explain why RIF1 depletion does not fully mimic 53BP1 ablation in the restoration of homologous recombination defects in BRCA1-deficient cells.
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DEAD Box 1 Facilitates Removal of RNA and Homologous Recombination at DNA Double-Strand Breaks
Molecular and Cellular Biology, 2016Co-Authors: Devon R. Germain, Matthew R. Hildebrandt, Elizabeth A. Monckton, Ho-yin Poon, Michael J. Hendzel, Darin Mcdonald, Roseline GodboutAbstract:Although RNA and RNA-binding proteins have been linked to double-strand breaks (DSBs), little is known regarding their roles in the cellular response to DSBs and, if any, in the repair process. Here, we provide direct evidence for the presence of RNA-DNA hybrids at DSBs and suggest that binding of RNA to DNA at DSBs may impact repair efficiency. Our data indicate that the RNA-unwinding protein DEAD box 1 (DDX1) is required for efficient DSB repair and cell survival after ionizing radiation (IR), with depletion of DDX1 resulting in reduced DSB repair by homologous recombination (HR). While DDX1 is not essential for end resection, a key step in homology-directed DSB repair, DDX1 is required for maintenance of the single-stranded DNA once generated by end resection. We show that transcription deregulation has a significant effect on DSB repair by HR in DDX1-depleted cells and that RNA-DNA duplexes are elevated at DSBs in DDX1-depleted cells. Based on our combined data, we propose a role for DDX1 in resolving RNA-DNA structures that accumulate at DSBs located at sites of active transcription. Our findings point to a previously uncharacterized requirement for clearing RNA at DSBs for efficient repair by HR.
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DDX1 knockout results in transgenerational wild-type lethality in mice
Scientific Reports, 2015Co-Authors: Matthew R. Hildebrandt, Devon R. Germain, Elizabeth A. Monckton, Miranda Brun, Roseline GodboutAbstract:DEAD box 1 (DDX1) is a member of the DEAD box family of RNA helicases which are involved in all aspects of RNA metabolism. DDX1 has been implicated in a variety of biological processes, including 3’-end processing of mRNA, DNA repair, microRNA processing, tRNA maturation and mRNA transport. To study the role of DDX1 during development, we have generated mice carrying a constitutive DDX1 knock-out allele. DDX1+/− mice have no obvious phenotype and express similar levels of DDX1 as wild-type mice indicating compensation from the intact DDX1 allele. Heterozygote matings produce no viable DDX1−/− progeny, with DDX1−/− embryos dying prior to embryonic day (E) 3.5. Intriguingly, the number of wild-type progeny is significantly decreased in heterozygote crosses, with two different heterozygote populations identified based on parental genotype: (i) normal DDX1+/− mice which generate the expected number of wild-type progeny and (ii) DDX1*/− mice (with * signifying a non-genetically altered allele) which generate a significantly reduced number of wild-type mice. The transgenerational inheritance of wild-type lethality observed upon crossing DDX1*/− mice is independent of parental sex and occurs in cis through a mechanism that is different from other types of previously reported transgenerational epigenetic inheritance.
James R. Williamson - One of the best experts on this subject based on the ideXlab platform.
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A Survey of DDX21 Activity During Rev/RRE Complex Formation
Journal of Molecular Biology, 2018Co-Authors: John A. Hammond, Li Zhou, Rajan Lamichhane, Hui-yi Chu, David P. Millar, Larry Gerace, James R. WilliamsonAbstract:HIV-1 requires a specialized nuclear export pathway to transport unspliced and partially spliced viral transcripts to the cytoplasm. Central to this pathway is the viral protein Rev, which binds to the Rev response element in stem IIB located on unspliced viral transcripts and subsequently oligomerizes in a cooperative manner. Previous work identified a number of cellular DEAD-box helicases as in vivo binding partners of Rev, and siRNA experiments indicated a functional role for many in the HIV replication cycle. Two DEAD-box proteins, DDX1 and DDX3, had previously been shown to play a role in HIV pathogenesis. In this study, another protein identified in that screen, DDX21, is tested for protein and RNA binding and subsequent enzymatic activities in the context of the Rev/RRE pathway. We found that DDX21 can bind to the RRE with high affinity, and this binding stimulates ATPase activity with an enzymatic efficiency similar to DDX1. Furthermore, DDX21 is both an ATP-dependent and ATP-independent helicase, and both ATPase and ATP-dependent helicase activities are inhibited by Rev in a dose-dependent manner, although ATP-independent helicase activity is not. A conserved binding interaction between DDX protein's DEAD domain and Rev was identified, with Rev's nuclear diffusion inhibitory signal motif playing a significant role in binding. Finally, DDX21 was shown to enhance Rev binding to the RRE in a manner similar to that previously described for DDX1, although DDX3 does not. These data indicate that DDX1 and DDX21 have similar biochemical activities with regard to the Rev/RRE system, while DDX3 differs.
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a survey of ddx21 activity during rev rre complex formation
Journal of Molecular Biology, 2017Co-Authors: John A. Hammond, Li Zhou, Rajan Lamichhane, Hui-yi Chu, David P. Millar, Larry Gerace, James R. WilliamsonAbstract:HIV-1 requires a specialized nuclear export pathway to transport unspliced and partially spliced viral transcripts to the cytoplasm. Central to this pathway is the viral protein Rev, which binds to the Rev response element in stem IIB located on unspliced viral transcripts and subsequently oligomerizes in a cooperative manner. Previous work identified a number of cellular DEAD-box helicases as in vivo binding partners of Rev, and siRNA experiments indicated a functional role for many in the HIV replication cycle. Two DEAD-box proteins, DDX1 and DDX3, had previously been shown to play a role in HIV pathogenesis. In this study, another protein identified in that screen, DDX21, is tested for protein and RNA binding and subsequent enzymatic activities in the context of the Rev/RRE pathway. We found that DDX21 can bind to the RRE with high affinity, and this binding stimulates ATPase activity with an enzymatic efficiency similar to DDX1. Furthermore, DDX21 is both an ATP-dependent and ATP-independent helicase, and both ATPase and ATP-dependent helicase activities are inhibited by Rev in a dose-dependent manner, although ATP-independent helicase activity is not. A conserved binding interaction between DDX protein's DEAD domain and Rev was identified, with Rev's nuclear diffusion inhibitory signal motif playing a significant role in binding. Finally, DDX21 was shown to enhance Rev binding to the RRE in a manner similar to that previously described for DDX1, although DDX3 does not. These data indicate that DDX1 and DDX21 have similar biochemical activities with regard to the Rev/RRE system, while DDX3 differs.
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A DEAD-box protein acts through RNA to promote HIV-1 Rev-RRE assembly.
Nucleic Acids Research, 2017Co-Authors: Rajan Lamichhane, John A. Hammond, James R. Williamson, Edwin J. C. Van Der Schans, Raymond F Pauszek, Rae M Anderson, Ingemar Pedron, David P. MillarAbstract:The HIV-1 Rev protein activates nuclear export of unspliced and partially spliced viral RNA transcripts, which encode the viral genome and the genes encoding viral structural proteins, by binding to and oligomerizing on the Rev Response Element (RRE). The human DEAD-box protein 1 (DDX1) enhances the RNA export activity of Rev through an unknown mechanism. Using a single-molecule assembly assay and various DDX1 mutants, we show that DDX1 acts through the RRE RNA to specifically accelerate the nucleation step of the Rev-RRE assembly process. Single-molecule Förster resonance energy transfer (smFRET) experiments using donor-labeled Rev and acceptor-labeled DDX1 show that both proteins can associate with a single RRE molecule. However, simultaneous interaction is only observed in a subset of binding events and does not explain the extent to which DDX1 promotes the nucleation step of Rev-RRE assembly. Together, these results are consistent with a model wherein DDX1 acts as an RNA chaperone, remodeling the RRE into a conformation that is pre-organized to bind the first Rev monomer, thereby promoting the overall Rev-RRE assembly process.
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DDX1 Is an RNA-Dependent ATPase Involved in HIV-1 Rev Function and Virus Replication
Journal of Molecular Biology, 2012Co-Authors: Stephen P. Edgcomb, Larry Gerace, Andrew B. Carmel, Souad Naji, Geza Ambrus-aikelin, Jason R. Reyes, Andrew C. S. Saphire, James R. WilliamsonAbstract:The human immunodeficiency virus type 1 (HIV-1) Rev protein is essential for the virus because it promotes nuclear export of alternatively processed mRNAs, and Rev is also linked to translation of viral mRNAs and genome encapsidation. Previously, the human DEAD-box helicase DDX1 was suggested to be involved in Rev functions, but this relationship is not well understood. Biochemical studies of DDX1 and its interactions with Rev and model RNA oligonucleotides were carried out to investigate the molecular basis for association of these components. A combination of gel-filtration chromatography and circular dichroism spectroscopy demonstrated that recombinant DDX1 expressed in Escherichia coli is a well-behaved folded protein. Binding assays using fluorescently labeled Rev and cell-based immunoprecipitation analysis confirmed a specific RNA-independent DDX1–Rev interaction. Additionally, DDX1 was shown to be an RNA-activated ATPase, wherein Rev-bound RNA was equally effective at stimulating ATPase activity as protein-free RNA. Gel mobility shift assays further demonstrated that DDX1 forms complexes with Rev-bound RNA. RNA silencing of DDX1 provided strong evidence that DDX1 is required for both Rev activity and HIV production from infected cells. Collectively, these studies demonstrate a clear link between DDX1 and HIV-1 Rev in cell-based assays of HIV-1 production and provide the first demonstration that recombinant DDX1 binds Rev and RNA and has RNA-dependent catalytic activity.
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Single-Molecule Studies Reveal that DEAD-Box Protein DDX1 Promotes Oligomerization of HIV-1 Rev on the Rev Response Element
Journal of Molecular Biology, 2011Co-Authors: Rae M. Robertson-anderson, James R. Williamson, Stephen P. Edgcomb, Andrew B. Carmel, Jun Wang, David P. MillarAbstract:Oligomeric assembly of Rev on the Rev response element (RRE) is essential for the nuclear export of unspliced and singly spliced human immunodeficiency virus type 1 viral mRNA transcripts. Several host factors, including the human DEAD box protein DDX1, are also known to be required for efficient Rev function. In this study, spontaneous assembly and dissociation of individual Rev–RRE complexes in the presence or absence of DDX1 were observed in real time via single-molecule total internal reflection fluorescence microscopy. Binding of up to eight fluorescently labeled Rev monomers to a single RRE molecule was visualized, and the event frequencies and corresponding binding and dissociation rates for the different Rev–RRE stoichiometries were determined. The presence of DDX1 eliminated a second kinetic phase present during the initial Rev binding step, attributed to nonproductive nucleation events, resulting in increased occurrence of higher-order Rev–RRE stoichiometries. This effect was further enhanced upon the addition of a non-hydrolyzable ATP analog (adenylyl-imidophosphate), whereas ADP had no effect beyond that of DDX1 alone. Notably, the first three Rev monomer binding events were accelerated in the presence of DDX1 and adenylyl-imidophosphate, while the dissociation rates remained unchanged. Measurements performed across a range of DDX1 concentrations suggest that DDX1 targets Rev rather than the RRE to promote oligomeric assembly. Moreover, DDX1 is able to restore the oligomerization activity of a Rev mutant that is otherwise unable to assemble on the RRE beyond a monomeric complex. Taken together, these results suggest that DDX1 acts as a cellular cofactor by promoting oligomerization of Rev on the RRE.
Rajan Lamichhane - One of the best experts on this subject based on the ideXlab platform.
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A Survey of DDX21 Activity During Rev/RRE Complex Formation
Journal of Molecular Biology, 2018Co-Authors: John A. Hammond, Li Zhou, Rajan Lamichhane, Hui-yi Chu, David P. Millar, Larry Gerace, James R. WilliamsonAbstract:HIV-1 requires a specialized nuclear export pathway to transport unspliced and partially spliced viral transcripts to the cytoplasm. Central to this pathway is the viral protein Rev, which binds to the Rev response element in stem IIB located on unspliced viral transcripts and subsequently oligomerizes in a cooperative manner. Previous work identified a number of cellular DEAD-box helicases as in vivo binding partners of Rev, and siRNA experiments indicated a functional role for many in the HIV replication cycle. Two DEAD-box proteins, DDX1 and DDX3, had previously been shown to play a role in HIV pathogenesis. In this study, another protein identified in that screen, DDX21, is tested for protein and RNA binding and subsequent enzymatic activities in the context of the Rev/RRE pathway. We found that DDX21 can bind to the RRE with high affinity, and this binding stimulates ATPase activity with an enzymatic efficiency similar to DDX1. Furthermore, DDX21 is both an ATP-dependent and ATP-independent helicase, and both ATPase and ATP-dependent helicase activities are inhibited by Rev in a dose-dependent manner, although ATP-independent helicase activity is not. A conserved binding interaction between DDX protein's DEAD domain and Rev was identified, with Rev's nuclear diffusion inhibitory signal motif playing a significant role in binding. Finally, DDX21 was shown to enhance Rev binding to the RRE in a manner similar to that previously described for DDX1, although DDX3 does not. These data indicate that DDX1 and DDX21 have similar biochemical activities with regard to the Rev/RRE system, while DDX3 differs.
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a survey of ddx21 activity during rev rre complex formation
Journal of Molecular Biology, 2017Co-Authors: John A. Hammond, Li Zhou, Rajan Lamichhane, Hui-yi Chu, David P. Millar, Larry Gerace, James R. WilliamsonAbstract:HIV-1 requires a specialized nuclear export pathway to transport unspliced and partially spliced viral transcripts to the cytoplasm. Central to this pathway is the viral protein Rev, which binds to the Rev response element in stem IIB located on unspliced viral transcripts and subsequently oligomerizes in a cooperative manner. Previous work identified a number of cellular DEAD-box helicases as in vivo binding partners of Rev, and siRNA experiments indicated a functional role for many in the HIV replication cycle. Two DEAD-box proteins, DDX1 and DDX3, had previously been shown to play a role in HIV pathogenesis. In this study, another protein identified in that screen, DDX21, is tested for protein and RNA binding and subsequent enzymatic activities in the context of the Rev/RRE pathway. We found that DDX21 can bind to the RRE with high affinity, and this binding stimulates ATPase activity with an enzymatic efficiency similar to DDX1. Furthermore, DDX21 is both an ATP-dependent and ATP-independent helicase, and both ATPase and ATP-dependent helicase activities are inhibited by Rev in a dose-dependent manner, although ATP-independent helicase activity is not. A conserved binding interaction between DDX protein's DEAD domain and Rev was identified, with Rev's nuclear diffusion inhibitory signal motif playing a significant role in binding. Finally, DDX21 was shown to enhance Rev binding to the RRE in a manner similar to that previously described for DDX1, although DDX3 does not. These data indicate that DDX1 and DDX21 have similar biochemical activities with regard to the Rev/RRE system, while DDX3 differs.
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A DEAD-box protein acts through RNA to promote HIV-1 Rev-RRE assembly.
Nucleic Acids Research, 2017Co-Authors: Rajan Lamichhane, John A. Hammond, James R. Williamson, Edwin J. C. Van Der Schans, Raymond F Pauszek, Rae M Anderson, Ingemar Pedron, David P. MillarAbstract:The HIV-1 Rev protein activates nuclear export of unspliced and partially spliced viral RNA transcripts, which encode the viral genome and the genes encoding viral structural proteins, by binding to and oligomerizing on the Rev Response Element (RRE). The human DEAD-box protein 1 (DDX1) enhances the RNA export activity of Rev through an unknown mechanism. Using a single-molecule assembly assay and various DDX1 mutants, we show that DDX1 acts through the RRE RNA to specifically accelerate the nucleation step of the Rev-RRE assembly process. Single-molecule Förster resonance energy transfer (smFRET) experiments using donor-labeled Rev and acceptor-labeled DDX1 show that both proteins can associate with a single RRE molecule. However, simultaneous interaction is only observed in a subset of binding events and does not explain the extent to which DDX1 promotes the nucleation step of Rev-RRE assembly. Together, these results are consistent with a model wherein DDX1 acts as an RNA chaperone, remodeling the RRE into a conformation that is pre-organized to bind the first Rev monomer, thereby promoting the overall Rev-RRE assembly process.
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Role of Dead Box Helicases in HIV-1 Rev Function: a Single-Molecule Approach
Biophysical Journal, 2014Co-Authors: Rajan Lamichhane, David P. MillarAbstract:The HIV-1 Rev (Regulator of Expression of Virion) protein activates nuclear export of unspliced and partially spliced viral mRNAs, which encode the viral genome and the genes encoding viral structural proteins. Rev interacts with a highly conserved region, the Rev Response Element (RRE), located within the viral mRNA. Initially, a single Rev monomer binds to stem loop IIB of the RRE, whereupon additional Rev monomers are recruited to the RRE through a combination of RNA-protein and protein-protein interactions, resulting in the formation of a functional nuclear export complex. In addition, several cellular host proteins, such as the DEAD box helicases DDX1 and DDX21 are known to be required for efficient Rev function in vivo, although their precise role is unknown. In this study, a variety of single-molecule fluorescence spectroscopic methods were used to dissect the role of DDX1 and DDX21 during assembly of Rev-RRE complexes. To facilitate these studies, the large DDX1 and DDX21 proteins were enzymatically labeled with bright and photostable dyes, while Rev was labeled at a single cysteine by maleimide chemistry. Single-color TIRF measurements with labeled Rev were used to monitor individual Rev monomer binding steps during oligomeric Rev-RRE assembly. Moreover, single-color experiments with labeled DDX1 or DDX21 reveal that each helicase is capable of binding directly to the RRE. Two-color colocalization and FRET measurements were used to monitor the simultaneous binding of both Rev and DDX1 to the RRE, revealing the temporal correlation between individual protein binding steps. Together, these studies are revealing how DEAD box helicases are able to promote the oligomeric assembly of Rev on the RRE, thereby acting as cellular cofactors of HIV-1. Supported by NIH P50 grant GM082545.
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Oligomeric Assembly of HIV-1 Rev on the Rev Response Element: Role of Cellular Cofactors
Biophysical Journal, 2012Co-Authors: Rajan Lamichhane, Rae M. Robertson-anderson, Svitlana Y. Berezhna, Edwin J. C. Van Der Schans, David P. MillarAbstract:Rev, a key regulatory protein of HIV-1, activates nuclear export of unspliced and partially spliced viral mRNAs, which encode the viral genome and the genes encoding viral structural proteins, respectively. Initially, a single Rev monomer binds to a highly conserved region, stem IIB located on the Rev Response Element (RRE) of viral mRNA. Following this nucleation step, additional Rev monomers are recruited to the RRE through a combination of RNA-protein and protein-protein interactions, resulting in the formation of a functional nuclear export complex. In addition, several cellular proteins, such as the DEAD box helicases DDX1 and DDX3 are known to be required for efficient Rev function in vivo, although their precise role is unknown. In this study, single-molecule total internal reflection fluorescence (smTIRF) microscopy was used to visualize oligomeric assembly of Rev on the RRE with single monomer resolution. Binding of up to eight fluorescently labeled Rev monomers to a single immobilized RRE molecule was observed in real-time as discrete jumps in fluorescence intensity, and the event frequencies and corresponding binding and dissociation rates for the different Rev-RRE stoichiometries were determined. The smTIRF assay was used to study Rev-RRE assembly in the presence of DDX1, DDX3 and other cellular proteins. The presence of DDX1 promotes oligomeric assembly by accelerating the first few Rev monomer binding steps, suggesting that DDX1 acts as a chaperone of Rev. The smTIRF measurements are being extended to a multi-color format, in order to directly visualize the colocalization of Rev and selected cellular proteins on the same immobilized RRE molecules. These measurements are revealing the precise timing of various protein binding events during ribonucleoprotein assembly. Supported by NIH grant P50 GM082545.
Krzysztof Rakus - One of the best experts on this subject based on the ideXlab platform.
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Cytosolic Sensors for Pathogenic Viral and Bacterial Nucleic Acids in Fish.
International Journal of Molecular Sciences, 2020Co-Authors: Miriam Mojzesz, Magdalena Chadzinska, Krzysztof Rakus, Kentaro Nakagami, Gouranga Biswas, Masahiro Sakai, Jun-ichi HikimaAbstract:Recognition of the non-self signature of invading pathogens is a crucial step for the initiation of the innate immune mechanisms of the host. The host response to viral and bacterial infection involves sets of pattern recognition receptors (PRRs), which bind evolutionarily conserved pathogen structures, known as pathogen-associated molecular patterns (PAMPs). Recent advances in the identification of different types of PRRs in teleost fish revealed a number of cytosolic sensors for recognition of viral and bacterial nucleic acids. These are DExD/H-box RNA helicases including a group of well-characterized retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) and non-RLR DExD/H-box RNA helicases (e.g., DDX1, DDX3, DHX9, DDX21, DHX36 and DDX41) both involved in recognition of viral RNAs. Another group of PRRs includes cytosolic DNA sensors (CDSs), such as cGAS and LSm14A involved in recognition of viral and intracellular bacterial dsDNAs. Moreover, dsRNA-sensing protein kinase R (PKR), which has a role in antiviral immune responses in higher vertebrates, has been identified in fish. Additionally, fish possess a novel PKR-like protein kinase containing Z-DNA binding domain, known as PKZ. Here, we review the current knowledge concerning cytosolic sensors for recognition of viral and bacterial nucleic acids in teleosts.
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viral infection induced changes in the expression profile of non rlr dexd h box rna helicases DDX1 ddx3 dhx9 ddx21 and dhx36 in zebrafish and common carp
Fish & Shellfish Immunology, 2020Co-Authors: Miriam Mojzesz, Katarzyna Klak, Paulina Wojtal, Mikolaj Adamek, Piotr Podlasz, Marek Matras, Michal Reichert, Magdalena Chadzinska, M Chmielewskakrzesinska, Krzysztof RakusAbstract:In mammals, several non-RLR DExD/H-box RNA helicases are involve in sensing of viral nucleic acids and activation of antiviral immune response, however their role in the immune defense of fish is much less known. In this study, the expression profile of non-RLR DExD/H-box RNA helicase genes: DDX1, ddx3, dhx9, ddx21 and dhx36, was studied in zebrafish (Danio rerio) and common carp (Cyprinus carpio L.) during infection with two RNA viruses: spring viremia of carp virus (SVCV) and Chum salmon reovirus (CSV). Bioinformatic analysis of the amino acid sequences of the core helicase of DDX1, DDX3, DHX9, DDX21 and DHX36 in zebrafish and common carp revealed presence of all conserved motifs found amongst all other species, with the exception of common carp DHX9 which do not possess motif V. The transcripts of studied DExD/H-box RNA helicases were found in zebrafish ZF4 cell line as well as in all studied organs from zebrafish and common carp. The expression study demonstrated the up-regulation of the expression of selected non-RLR DExD/H-box RNA helicases during viral infections in ZF4 cell line (in vitro study) and in zebrafish and common carp organs (in vivo study). DDX1 was the only DExD/H-box RNA helicase which expression was repetitively up-regulated during in vivo infections with SVCV and CSV in zebrafish and SVCV in common carp. In ZF4 cells and kidney of common carp, viral infection-induced up-regulation of DExD/H-box RNA helicases preceded the up-regulation of type I IFN gene. Our results suggest that studied non-RLR DExD/H-box RNA helicases might be involved in antiviral immune response in fish.
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Viral infection-induced changes in the expression profile of non-RLR DExD/H-box RNA helicases (DDX1, DDX3, DHX9, DDX21 and DHX36) in zebrafish and common carp.
Fish & Shellfish Immunology, 2020Co-Authors: Miriam Mojzesz, Katarzyna Klak, Paulina Wojtal, Mikolaj Adamek, Piotr Podlasz, M. Chmielewska-krzesinska, Marek Matras, Michal Reichert, Magdalena Chadzinska, Krzysztof RakusAbstract:In mammals, several non-RLR DExD/H-box RNA helicases are involve in sensing of viral nucleic acids and activation of antiviral immune response, however their role in the immune defense of fish is much less known. In this study, the expression profile of non-RLR DExD/H-box RNA helicase genes: DDX1, ddx3, dhx9, ddx21 and dhx36, was studied in zebrafish (Danio rerio) and common carp (Cyprinus carpio L.) during infection with two RNA viruses: spring viremia of carp virus (SVCV) and Chum salmon reovirus (CSV). Bioinformatic analysis of the amino acid sequences of the core helicase of DDX1, DDX3, DHX9, DDX21 and DHX36 in zebrafish and common carp revealed presence of all conserved motifs found amongst all other species, with the exception of common carp DHX9 which do not possess motif V. The transcripts of studied DExD/H-box RNA helicases were found in zebrafish ZF4 cell line as well as in all studied organs from zebrafish and common carp. The expression study demonstrated the up-regulation of the expression of selected non-RLR DExD/H-box RNA helicases during viral infections in ZF4 cell line (in vitro study) and in zebrafish and common carp organs (in vivo study). DDX1 was the only DExD/H-box RNA helicase which expression was repetitively up-regulated during in vivo infections with SVCV and CSV in zebrafish and SVCV in common carp. In ZF4 cells and kidney of common carp, viral infection-induced up-regulation of DExD/H-box RNA helicases preceded the up-regulation of type I IFN gene. Our results suggest that studied non-RLR DExD/H-box RNA helicases might be involved in antiviral immune response in fish.