558 results on '"Weitzman, Matthew"'
Search Results
202. DRUMMER—rapid detection of RNA modifications through comparative nanopore sequencing.
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Abebe, Jonathan S, Price, Alexander M, Hayer, Katharina E, Mohr, Ian, Weitzman, Matthew D, Wilson, Angus C, and Depledge, Daniel P
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RNA modification & restriction , *RNA , *PROTEIN-protein interactions , *CURRENT fluctuations , *ERROR rates - Abstract
Motivation The chemical modification of ribonucleotides regulates the structure, stability and interactions of RNAs. Profiling of these modifications using short-read (Illumina) sequencing techniques provides high sensitivity but low-to-medium resolution i.e. modifications cannot be assigned to specific transcript isoforms in regions of sequence overlap. An alternative strategy uses current fluctuations in nanopore-based long read direct RNA sequencing (DRS) to infer the location and identity of nucleotides that differ between two experimental conditions. While highly sensitive, these signal-level analyses require high-quality transcriptome annotations and thus are best suited to the study of model organisms. By contrast, the detection of RNA modifications in microbial organisms which typically have no or low-quality annotations requires an alternative strategy. Here, we demonstrate that signal fluctuations directly influence error rates during base-calling and thus provides an alternative approach for identifying modified nucleotides. Results DRUMMER (Detection of Ribonucleic acid Modifications Manifested in Error Rates) (i) utilizes a range of statistical tests and background noise correction to identify modified nucleotides with high confidence, (ii) operates with similar sensitivity to signal-level analysis approaches and (iii) correlates very well with orthogonal approaches. Using well-characterized DRS datasets supported by independent meRIP-Seq and miCLIP-Seq datasets we demonstrate that DRUMMER operates with high sensitivity and specificity. Availability and implementation DRUMMER is written in Python 3 and is available as open source in the GitHub repository: https://github.com/DepledgeLab/DRUMMER. Supplementary information Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
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203. SAMHD1 Restricts Herpes Simplex Virus 1 in Macrophages by Limiting DNA Replication.
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Eui Tae Kim, White, Tommy E., Brandariz-Núñez, Alberto, Diaz-Griffero, Felipe, and Weitzman, Matthew D.
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HERPES simplex virus , *MACROPHAGES , *DNA replication , *ANTIVIRAL agents , *VIRUS inhibitors , *VIRAL genomes - Abstract
Macrophages play important roles in host immune defense against virus infection. During infection by herpes simplex virus 1 (HSV-1), macrophages acquire enhanced antiviral potential. Restriction of HSV-1 replication and progeny production is important to prevent viral spread, but the cellular mechanisms that inhibit the DNA virus in macrophages are unknown. SAMHD1 was recently identified as a retrovirus restriction factor highly expressed in macrophages. The SAMHD1 protein is expressed in both undifferentiated monocytes and differentiated macrophages, but retroviral restriction is limited to differentiated cells by modulation of SAMHD1 phosphorylation. It is proposed to block reverse transcription of retroviral RNA into DNA by depleting cellular deoxynucleotide triphosphates (dNTPs). Viruses with DNA genomes do not employ reverse transcription during infection, but replication of their viral genomes is also dependent on intracellular dNTP concentrations. Here, we demonstrate that SAMHD1 restricts replication of the HSV-1 DNA genome in differentiated macrophage cell lines. Depleting SAMHD1 in THP-1 cells enhanced HSV-1 replication, while ectopic overexpression of SAMHD1 in U937 cells repressed HSV-1 replication. SAMHD1 did not impact viral gene expression from incoming HSV-1 viral genomes. HSV-1 restriction involved the dNTP triphosphohydrolase activity of SAMHD1 and was partially overcome by addition of exogenous deoxynucleosides. Unlike retroviruses, restriction of HSV-1 was not affected by SAMHD1 phosphorylation status. Our results suggest that SAMHD1 functions broadly to inhibit replication of DNA viruses in nondividing macrophages. [ABSTRACT FROM AUTHOR]
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- 2013
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204. Differential L1 regulation in pluripotent stem cells of humans and apes.
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Marchetto, Maria C. N., Narvaiza, Iñigo, Denli, Ahmet M., Benner, Christopher, Lazzarini, Thomas A., Nathanson, Jason L., Paquola, Apuã C. M., Desai, Keval N., Herai, Roberto H., Weitzman, Matthew D., Yeo, Gene W., Muotri, Alysson R., and Gage, Fred H.
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PLURIPOTENT stem cells , *CHIMPANZEES as laboratory animals , *BONOBO , *INDUCED pluripotent stem cells , *GENE expression , *GENETIC regulation , *THERAPEUTICS - Abstract
Identifying cellular and molecular differences between human and non-human primates (NHPs) is essential to the basic understanding of the evolution and diversity of our own species. Until now, preserved tissues have been the main source for most comparative studies between humans, chimpanzees (Pan troglodytes) and bonobos (Pan paniscus). However, these tissue samples do not fairly represent the distinctive traits of live cell behaviour and are not amenable to genetic manipulation. We propose that induced pluripotent stem (iPS) cells could be a unique biological resource to determine relevant phenotypical differences between human and NHPs, and that those differences could have potential adaptation and speciation value. Here we describe the generation and initial characterization of iPS cells from chimpanzees and bonobos as new tools to explore factors that may have contributed to great ape evolution. Comparative gene expression analysis of human and NHP iPS cells revealed differences in the regulation of long interspersed element-1 (L1, also known as LINE-1) transposons. A force of change in mammalian evolution, L1 elements are retrotransposons that have remained active during primate evolution. Decreased levels of L1-restricting factors APOBEC3B (also known as A3B) and PIWIL2 (ref. 7) in NHP iPS cells correlated with increased L1 mobility and endogenous L1 messenger RNA levels. Moreover, results from the manipulation of A3B and PIWIL2 levels in iPS cells supported a causal inverse relationship between levels of these proteins and L1 retrotransposition. Finally, we found increased copy numbers of species-specific L1 elements in the genome of chimpanzees compared to humans, supporting the idea that increased L1 mobility in NHPs is not limited to iPS cells in culture and may have also occurred in the germ line or embryonic cells developmentally upstream to germline specification during primate evolution. We propose that differences in L1 mobility may have differentially shaped the genomes of humans and NHPs and could have continuing adaptive significance. [ABSTRACT FROM AUTHOR]
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- 2013
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205. Characterization of a nuclear localization signal in the C-terminus of the adeno-associated virus Rep68/78 proteins
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Weitzman, Matthew [Laboratory of Genetics, Salk Institute for Biological Studies, La Jolla, CA 92037 (United States)]
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- 2004
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206. Codon-usage-based inhibition of HIV protein synthesis by human schlafen 11.
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Li, Manqing, Kao, Elaine, Gao, Xia, Sandig, Hilary, Limmer, Kirsten, Pavon-Eternod, Mariana, Jones, Thomas E., Landry, Sebastien, Pan, Tao, Weitzman, Matthew D., and David, Michael
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GENETIC code , *HIV , *PROTEIN synthesis , *INTERFERON regulatory factors , *INTERFERONS , *VIRAL proteins , *PHYSIOLOGY - Abstract
In mammals, one of the most pronounced consequences of viral infection is the induction of type I interferons, cytokines with potent antiviral activity. Schlafen (Slfn) genes are a subset of interferon-stimulated early response genes (ISGs) that are also induced directly by pathogens via the interferon regulatory factor 3 (IRF3) pathway. However, many ISGs are of unknown or incompletely understood function. Here we show that human SLFN11 potently and specifically abrogates the production of retroviruses such as human immunodeficiency virus 1 (HIV-1). Our study revealed that SLFN11 has no effect on the early steps of the retroviral infection cycle, including reverse transcription, integration and transcription. Rather, SLFN11 acts at the late stage of virus production by selectively inhibiting the expression of viral proteins in a codon-usage-dependent manner. We further find that SLFN11 binds transfer RNA, and counteracts changes in the tRNA pool elicited by the presence of HIV. Our studies identified a novel antiviral mechanism within the innate immune response, in which SLFN11 selectively inhibits viral protein synthesis in HIV-infected cells by means of codon-bias discrimination. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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207. Adeno-Associated Virus Type 2 Modulates the Host DNA Damage Response Induced by Herpes Simplex Virus 1 during Coinfection.
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Vogel, Rebecca, Seyffert, Michael, Strasser, Regina, de Oliveira, Anna P., Dresch, Christiane, Glauser, Daniel L., Jolinon, Nelly, Salvetti, Anna, Weitzman, Matthew D., Ackermann, Mathias, and Fraefel, Cornel
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GENETICS of virus diseases , *HERPES simplex virus , *HERPESVIRUS diseases , *VIRAL replication , *ADENOVIRUSES , *DNA damage , *DNA replication - Abstract
Adeno-associated virus type 2 (AAV2) is a human parvovirus that relies on a helper virus for efficient replication. Herpes simplex virus 1 (HSV-1) supplies helper functions and changes the environment of the cell to promote AAV2 replication. In this study, we examined the accumulation of cellular replication and repair proteins at viral replication compartments (RCs) and the influence of replicating AAV2 on HSV-1-induced DNA damage responses (DDR). We observed that the ATM kinase was activated in cells coinfected with AAV2 and HSV-1. We also found that phosphorylated ATR kinase and its cofactor ATR-interacting protein were recruited into AAV2 RCs, but ATR signaling was not activated. DNA-PKcs, another main kinase in the DDR, was degraded during HSV-1 infection in an ICP0-dependent manner, and this degradation was markedly delayed during AAV2 coinfection. Furthermore, we detected phosphorylation of DNA-PKcs during AAV2 but not HSV-1 replication. The AAV2-mediated delay in DNA-PKcs degradation affected signaling through downstream substrates. Overall, our results demonstrate that coinfection with HSV-1 and AAV2 provokes a cellular DDR which is distinct from that induced by HSV-1 alone. [ABSTRACT FROM AUTHOR]
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- 2012
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208. The Adenovirus E1b55K/E4orf6 Complex Induces Degradation of the Bloom Helicase during Infection.
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Orazio, Nicole I., Naeger, Colleen M., Karlseder, Jan, and Weitzman, Matthew D.
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ADENOVIRUSES , *UBIQUITIN , *VIRUS diseases , *VIRAL replication , *VIRAL genomes , *VIROLOGY - Abstract
The adenovirus (Ad) E1b55K and E4orf6 gene products assemble an E3 ubiquitin ligase complex that promotes degradation of cellular proteins. Among the known substrates are p53 and the Mre11-Rad50-Nbs1 (MRN) complex. Since members of the RecQ helicase family function together with MRN in genome maintenance, we investigated whether adenovirus affects RecQ proteins. We show that Bloom helicase (BLM) is degraded during adenovirus type 5 (Ad5) infection. BLM degradation is mediated by E1b55K/E4orf6 but is independent of MRN. We detected BLM localized at discrete foci around viral replication centers. These studies identify BLM as a new substrate for degradation by the adenovirus E1b55K/E4orf6 complex. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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209. Adenovirus Type 5 E4orf3 Protein Targets the Mre11 Complex to Cytoplasmic Aggresomes.
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raujo, Felipe D., Stracker, Tavis H., Carson, Christian T., Lee, Darwin V., and Weitzman, Matthew D.
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ADENOVIRUSES , *VIRUS diseases , *VIRAL replication , *PROTEIN synthesis , *VIRAL genomes , *DNA viruses - Abstract
Virus infections have dramatic effects on structural and morphological characteristics of the host cell. The gene product of open reading frame 3 in the early region 4 (EAorf3) of adenovirus serotype 5 (Ad5) is involved in efficient replication and late protein synthesis. During infection with adenovirus mutants lacking the E4 region, the viral genomic DNA is joined into concatemers by cellular DNA repair factors, and this requires the Mre11/Rad50/Nbs1 complex. Concatemer formation can be prevented by the E4orf3 protein, which causes the cellular redistribution of the Mre11 complex. Here we show that E4orf3 colocalizes with components of the Mre11 complex in nuclear tracks and also in large cytoplasmic accumulations. Rearrangement of Mre11 and Rad50 by Ad5 E4orf3 is not dependent on interactions with Nbs1 or promyelocytic leukemia protein nuclear bodies. Late in infection the cytoplasmic inclusions appear as a distinct juxtanuclear accumulation at the centrosome and this requires an intact microtubule cytoskeleton. The large cytoplasmic accumulations meet the criteria defined for aggresomes, including γ-tubulin colocalization and formation of a surrounding vimentin cage. E4orf3 also appears to alter the solubility of the cellular Mre11 complex. These data suggest that E4orf3 can target the Mre11 complex to an aggresome and may explain how the cellular repair complex is inactivated during adenovirus infection. [ABSTRACT FROM AUTHOR]
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- 2005
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210. DNA repair proteins affect the lifecycle of herpes simplex virus 1.
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Lilley, Caroline E., Carson, Christian T., Muotri, Alysson R., Gage, Fred H., and Weitzman, Matthew D.
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DNA repair , *HERPES simplex virus , *VIRAL replication , *DNA damage , *NEURONS , *GENETIC mutation - Abstract
We report that herpes simplex virus 1 (HSV-1) infection can activate and exploit a cellular DNA damage response that aids viral replication in nonneuronal cells Early in HSV-1 infection, several members of the cellular DNA damage-sensing machinery are activated and accumulate at sites of viral DNA replication. When this cellular response is abrogated, formation of HSV-1 replication centers is retarded, and viral production is compromised. In neurons, HSV-1 replication centers fail to mature, and the DNA damage response is not initiated. These data suggest that the failure of neurons to mount a DNA damage response to HSV-1 may contribute to the establishment of latency. [ABSTRACT FROM AUTHOR]
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- 2005
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211. A genetic screen identifies a cellular regulator of adeno-associated virus.
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Cathomen, Toni, Stracker, Travis H., Gilbert, Luz Beatriz, and Weitzman, Matthew D.
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HUMAN chromosome abnormality diagnosis , *CARRIER proteins , *VIRUSES - Abstract
Describes a design of a genetic screen to identify cellular Rep recognition sequence (RRS)-binding proteins. Presence of RRS proteins in the cellular adeno-associated virus type 1 (AAV1) integration locus; Reactivity of human zinc finger protein with RRS motifs; Contribution of ZF5 to AAV2 repression.
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- 2001
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212. The HSV-1 ubiquitin ligase ICP0: Modifying the cellular proteome to promote infection.
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Rodríguez, Milagros Collados, Dybas, Joseph M., Hughes, Joseph, Weitzman, Matthew D., and Boutell, Chris
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UBIQUITINATION , *HERPES simplex virus , *VIRAL genomes , *VIRUS reactivation , *UBIQUITIN , *VIRAL replication - Abstract
• ICP0 is a viral E3 ubiquitin ligase that promotes HSV-1 infection. • ICP0 interacts with multiple component proteins of the ubiquitin pathway. • ICP0 disrupts multiple cellular processes activated in response to infection • ICP0 remodels the SUMO proteome to counteract host immune defences to infection. • ICP0 is an attractive drug target for the development of antiviral HSV-1 therapeutics. Herpes simplex virus 1 (HSV-1) hijacks ubiquitination machinery to modify the cellular proteome to create an environment permissive for virus replication. HSV-1 encodes its own RING-finger E3 ubiquitin (Ub) ligase, Infected Cell Protein 0 (ICP0), that directly interfaces with component proteins of the Ub pathway to inactivate host immune defences and cellular processes that restrict the progression of HSV-1 infection. Consequently, ICP0 plays a critical role in the infectious cycle of HSV-1 that is required to promote the efficient onset of lytic infection and productive reactivation of viral genomes from latency. This review will describe the current knowledge regarding the biochemical properties and known substrates of ICP0 during HSV-1 infection. We will highlight the gaps in the characterization of ICP0 function and propose future areas of research required to understand fully the biological properties of this important HSV-1 regulatory protein. [ABSTRACT FROM AUTHOR]
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- 2020
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213. 371. Optimizing Custom Zinc-Finger Nucleases for Use in Human Cells
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Alwin, Stephen, Gere, Maja B., Guhl, Eva, Effertz, Karin, Barbas, Carlos F., Segal, David J., Weitzman, Matthew D., and Cathomen, Toni
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NUCLEASES , *CELLS - Abstract
An abstract of the article "Optimizing Custom Zinc-Finger Nucleases for Use in Human Cells," by Stephen Alwin, Maja B. Gere, Eva Guhl, Karin Effertz, Carlos F. Barbas III, David J. Segal, Matthew D. Weitzman, and Toni Cathomen is presented.
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- 2005
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214. The SMC5/6 complex prevents genotoxicity upon APOBEC3A-mediated replication stress.
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Fingerman DF, O'Leary DR, Hansen AR, Tran T, Harris BR, DeWeerd RA, Hayer KE, Fan J, Chen E, Tennakoon M, Meroni A, Szeto JH, Devenport J, LaVigne D, Weitzman MD, Shalem O, Bednarski J, Vindigni A, Zhao X, and Green AM
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- Humans, Genomic Instability, Cell Line, Tumor, Proteins, Cytidine Deaminase metabolism, Cytidine Deaminase genetics, DNA Replication, Cell Cycle Proteins metabolism, Cell Cycle Proteins genetics, DNA Damage, Chromosomal Proteins, Non-Histone metabolism, Chromosomal Proteins, Non-Histone genetics
- Abstract
Mutational patterns caused by APOBEC3 cytidine deaminase activity are evident throughout human cancer genomes. In particular, the APOBEC3A family member is a potent genotoxin that causes substantial DNA damage in experimental systems and human tumors. However, the mechanisms that ensure genome stability in cells with active APOBEC3A are unknown. Through an unbiased genome-wide screen, we define the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex as essential for cell viability when APOBEC3A is active. We observe an absence of APOBEC3A mutagenesis in human tumors with SMC5/6 dysfunction, consistent with synthetic lethality. Cancer cells depleted of SMC5/6 incur substantial genome damage from APOBEC3A activity during DNA replication. Further, APOBEC3A activity results in replication tract lengthening which is dependent on PrimPol, consistent with re-initiation of DNA synthesis downstream of APOBEC3A-induced lesions. Loss of SMC5/6 abrogates elongated replication tracts and increases DNA breaks upon APOBEC3A activity. Our findings indicate that replication fork lengthening reflects a DNA damage response to APOBEC3A activity that promotes genome stability in an SMC5/6-dependent manner. Therefore, SMC5/6 presents a potential therapeutic vulnerability in tumors with active APOBEC3A., (© 2024. The Author(s).)
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- 2024
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215. Identifying Protein Interactions with Viral DNA Genomes during Virus Infection.
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Packard JE, Kumar N, Weitzman MD, and Dembowski JA
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- Humans, DNA Viruses genetics, Animals, Viral Proteins metabolism, Viral Proteins genetics, Herpesviridae genetics, Herpesviridae metabolism, Herpesviridae physiology, Vaccinia virus genetics, Genome, Viral, Virus Replication, DNA, Viral genetics, DNA, Viral metabolism, Host-Pathogen Interactions
- Abstract
Viruses exploit the host cell machinery to enable infection and propagation. This review discusses the complex landscape of DNA virus-host interactions, focusing primarily on herpesviruses and adenoviruses, which replicate in the nucleus of infected cells, and vaccinia virus, which replicates in the cytoplasm. We discuss experimental approaches used to discover and validate interactions of host proteins with viral genomes and how these interactions impact processes that occur during infection, including the host DNA damage response and viral genome replication, repair, and transcription. We highlight the current state of knowledge regarding virus-host protein interactions and also outline emerging areas and future directions for research.
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- 2024
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216. The SMC5/6 complex prevents genotoxicity upon APOBEC3A-mediated replication stress.
- Author
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O'Leary DR, Hansen AR, Fingerman DF, Tran T, Harris BR, Hayer KE, Fan J, Chen E, Tennakoon M, DeWeerd RA, Meroni A, Szeto JH, Weitzman MD, Shalem O, Bednarski J, Vindigni A, Zhao X, and Green AM
- Abstract
Mutational patterns caused by APOBEC3 cytidine deaminase activity are evident throughout human cancer genomes. In particular, the APOBEC3A family member is a potent genotoxin that causes substantial DNA damage in experimental systems and human tumors. However, the mechanisms that ensure genome stability in cells with active APOBEC3A are unknown. Through an unbiased genome-wide screen, we define the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex as essential for cell viability when APOBEC3A is active. We observe an absence of APOBEC3A mutagenesis in human tumors with SMC5/6 dysfunction, consistent with synthetic lethality. Cancer cells depleted of SMC5/6 incur substantial genome damage from APOBEC3A activity during DNA replication. Further, APOBEC3A activity results in replication tract lengthening which is dependent on PrimPol, consistent with re-initiation of DNA synthesis downstream of APOBEC3A-induced lesions. Loss of SMC5/6 abrogates elongated replication tracts and increases DNA breaks upon APOBEC3A activity. Our findings indicate that replication fork lengthening reflects a DNA damage response to APOBEC3A activity that promotes genome stability in an SMC5/6-dependent manner. Therefore, SMC5/6 presents a potential therapeutic vulnerability in tumors with active APOBEC3A.
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- 2024
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217. Feasibility and Usability of EnergyPoints: A Mobile Health App to Guide Acupressure Use for Cancer Symptom Management.
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Beck SL, Smith R, Mindes J, Beck K, Leah Kim J, Weitzman M, Stone JAM, Veleber S, and Dudley WN
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- Adult, Humans, Female, Adolescent, Male, Feasibility Studies, Fatigue, Acupressure, Mobile Applications, Cancer Survivors, Neoplasms therapy
- Abstract
Objective: To examine the feasibility and usability of EnergyPoints™, an innovative mobile health app that teaches and guides people with cancer to implement daily acupressure to self-manage their fatigue and sleep disturbances., Methods and Intervention: The study used an integrated agile, human-centered approach. Adults (age 18 years and over) with cancer experiencing at least moderate fatigue, and living in the Greater New York City community, were recruited from social media, patient advocacy groups, and referrals. Twenty participants (in 3 sprints of 3, 5, and 12) were video-recorded thinking aloud while using the app for the first time. They then used the app at home to self-administer acupressure (twice daily for 1 week) while continuously wearing a fitness tracker. Each participant completed an exit interview and modified Computer System Usability Questionnaire post-participation., Results: Participants were ages 40 to 76 years and 65% female; 65% were non-Hispanic white. Mean pass rates per ritual exceeded 80%. Users completed (totally or partially) greater than 90% of stimulating acupressure and 70% of relaxing acupressure rituals. Sprint 3 SPs totally completed at least 1 ritual 87% of the time. The majority agreed or strongly agreed the app was easy to use (90%), easy to learn (85%), easy to understand (75%), and effective in helping perform self-acupressure (85%). In an analysis of ease of completing 5 key tasks, all successfully completed the tasks; 3 users required some assistance. Of 654 usability statements, those coded as personal experience/context (197), content related to acupressure learning (105), and content related to the onboarding/profile (71) were most frequent. The design team integrated recommendations into the app before the next sprint., Conclusions: Findings supported feasibility and usability, as well as acceptability, and led to significant alterations and improvements. EnergyPoints™ offers an opportunity to mainstream acupressure and help cancer survivors self-manage their symptoms., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Authors have no conflicts to declare. When it is commercialized, EnergyPoints will be a for-profit product of 5 Point App Inc. All funding for the research was supported by a grant from the National Cancer Institute.
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- 2024
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218. Characteristics and Challenges of Providing Acupuncture and Chinese Herbal Medicine in Oncology Treatment: Report of Survey Data and Experience of Five Unique Clinical Settings.
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Veleber S, Cohen MR, Weitzman M, Maimon Y, Adamo CA, Siman J, Lu W, Sajdyk T, and Stone JAM
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- Humans, Medical Oncology, Surveys and Questionnaires, Drugs, Chinese Herbal therapeutic use, Acupuncture Therapy, Acupuncture
- Abstract
Acupuncture is an integrative therapy with strong evidence to support its use in the oncology setting, yet barriers exist for implementation into conventional medical clinics. Though acupuncture is recommended in clinical practice guidelines for oncology, there is little data in the literature showing how acupuncture and other related therapies, including herbal medicine are successfully implemented in some oncology clinics, while others experience barriers to care. To characterize the current use of acupuncture (ACU) and herbal medicine (HM) in oncology clinics, we collected general demographic and usage data from 5 example clinics. In addition, to better understand the barriers faced by ACU and HM clinics in implementing acupuncture as a treatment modality, a survey was deployed to 2320 members of the Society for Integrative Oncology. This article examines the characteristics of oncology settings around the world, and shares data from the survey on the use of these therapies in the field of oncology. The primary barrier to acupuncture care, as reported by providers, was cost. With just under 70% of the oncologists reporting it as the most important obstacle. Additional barriers to implementation included concerns about competency and training, accessibility and safety of herbal medicine during treatment. Though acupuncture is being incorporated into more conventional oncology settings, organized strategies for implementation involving payers and policymakers is needed., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Susan Veleber is an employee of Fred Hutchinson Cancer Center. Jonathan Siman is an employee of Fred Hutchinson Cancer Center. Matthew Weizman is an employee of Memorial Sloan Kettering. Misha Cohen is the owner of Chicken Soup Chinese Medicine. Yair Maimon is the owner of Refuot Integrative Medical Center. Christine Adamo is a contracted provider for cCARE. Jennifer Stone, Weidong Lu and Tammy Sajdyk have no conflicts to declare.
- Published
- 2024
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219. Intestinal transit-amplifying cells require METTL3 for growth factor signaling and cell survival.
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Danan CH, Naughton KE, Hayer KE, Vellappan S, McMillan EA, Zhou Y, Matsuda R, Nettleford SK, Katada K, Parham LR, Ma X, Chowdhury A, Wilkins BJ, Shah P, Weitzman MD, and Hamilton KE
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- Animals, Mice, Cell Proliferation physiology, Cell Survival genetics, Intercellular Signaling Peptides and Proteins metabolism, Intestines, RNA metabolism, Signal Transduction physiology, Proto-Oncogene Proteins p21(ras) metabolism, Stem Cells metabolism
- Abstract
Intestinal epithelial transit-amplifying cells are essential stem progenitors required for intestinal homeostasis, but their rapid proliferation renders them vulnerable to DNA damage from radiation and chemotherapy. Despite these cells' critical roles in intestinal homeostasis and disease, few studies have described genes that are essential to transit-amplifying cell function. We report that RNA methyltransferase-like 3 (METTL3) is required for survival of transit-amplifying cells in the murine small intestine. Transit-amplifying cell death after METTL3 deletion was associated with crypt and villus atrophy, loss of absorptive enterocytes, and uniform wasting and death in METTL3-depleted mice. Sequencing of polysome-bound and methylated RNAs in enteroids and in vivo demonstrated decreased translation of hundreds of methylated transcripts after METTL3 deletion, particularly transcripts involved in growth factor signal transduction such as Kras. Further investigation verified a relationship between METTL3 and Kras methylation and protein levels in vivo. Our study identifies METTL3 as an essential factor supporting the homeostasis of small intestinal tissue via direct maintenance of transit-amplifying cell survival. We highlight the crucial role of RNA modifications in regulating growth factor signaling in the intestine with important implications for both homeostatic tissue renewal and epithelial regeneration.
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- 2023
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220. Battlefield acupuncture for chronic musculoskeletal pain in cancer survivors: a novel care delivery model for oncology acupuncture.
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Zhang YL, Mao JJ, Li QS, Weitzman M, and Liou KT
- Abstract
Introduction: Battlefield Acupuncture (BFA), a standardized auricular acupuncture protocol, is widely used for pain in the military but is not well-studied in oncology. This study examined cancer survivors who received BFA for pain., Methods: This is a secondary analysis of a randomized trial that compared the effectiveness of BFA and electroacupuncture vs. usual care for chronic musculoskeletal pain in cancer survivors. This study focused on participants randomized to BFA. Participants received 10 weekly treatments. Needles were placed until one of these stop conditions were satisfied: ten needles were administered; pain severity decreased to ≤1 out of 10; patient declined further needling, or vasovagal reaction was observed. Pain severity was assessed using Brief Pain Inventory. Responders were those with ≥30% pain severity reduction. We examined pain location, BFA stop reason, and pain reduction of participants during the first session. We also examined which factors predicted responder status after the first session (week 1) or the full treatment (week 12)., Results: Among 143 randomized to BFA, most common pain locations were lower back (30.8%) and knee/leg (18.2%). Of 138 who initiated treatment, 41 (30.0%) received ten needles; 81 (59.1%) achieved pain ≤1; 14 (10.2%) declined further needling; and 1 (0.7%) had vasovagal reaction. BFA reduced pain severity by 2.9 points (95% CI 2.6 to 3.2) after the first session ( P < 0.001). After adjusting for baseline pain severity, responders at week 1 were 2.5 times more likely to be responders at week 12, compared to those who were non-responders at week 1 (AOR 2.5, 95% CI 1.02 to 6.11, P = 0.04). Among those who achieved pain ≤1, 74% were responders at week 12, a higher proportion compared to the proportion of responders among those who received ten needles (39.5%), those who declined further needling (50%), and those with vasovagal reaction (0.0%) ( P = 0.001). Those with pain in proximal joints had a higher proportion of responders at week 12, compared to those with pain in distal joints (64.2% vs. 20%, P = 0.008)., Conclusion: Specific factors may predict the likelihood of achieving meaningful pain reduction from BFA. Understanding these predictors could inform precision pain management and acupuncture delivery models., Competing Interests: JM reports grants from Tibet CheeZheng Tibetan Medicine Co Ltd outside the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Zhang, Mao, Li, Weitzman and Liou.)
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- 2023
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221. Electro-acupuncture versus battle field auricular acupuncture in breast cancer survivors with chronic musculoskeletal pain: subgroup analysis of a randomized clinical trial.
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Bao T, Zhi WI, Baser RE, Li QS, Weitzman M, Gillespie EF, Robson M, and Mao JJ
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- Humans, Female, Breast Neoplasms complications, Breast Neoplasms therapy, Cancer Survivors, Musculoskeletal Pain therapy, Acupuncture, Ear, Acupuncture Therapy
- Abstract
Purpose: Chronic musculoskeletal pain is common and debilitating among breast cancer survivors. The PEACE trial demonstrated that electro-acupuncture (EA) and battle field auricular acupuncture (BFAA) both reduced pain more than usual care (UC) in cancer survivors. However, the comparative effectiveness between EA and BFAA among breast cancer survivors is unknown., Methods: EA and BFAA received ten weekly treatments. UC was offered ten EA treatments after week 12. The primary endpoint was change in mean Brief Pain Inventory (BPI) pain severity from baseline to week 12. We analyzed the subset of 165 (46%) trial participants with a breast cancer primary diagnosis. We conducted constrained linear mixed model analyses, which constrained all arms to a common pre-randomization baseline mean. Model-based mean estimates at weeks 12 and 24 were compared between arms using model contrasts., Results: Among 165 breast cancer survivors, common pre-randomization mean pain severity was 5.35 [95% Confidence Interval (CI) 5.04, 5.66]. At week 12, BPI pain severity score was 2.69 (2.26. 3.13) in EA, 3.60 (3.17, 4.02) in BFAA, and 5.06 (4.47, 5.65) in UC. EA reduced pain severity significantly more than BFAA at weeks 12 [- 0.90 (- 1.45, - 0.36), p = 0.001] and 24 [- 0.82, (- 1.38, - 0.27), p = 0.004]. EA and BFAA significantly improved both Patient-Reported Outcomes Measurement Information System (PROMIS) - Global Health physical health and mental health component scores at week 12 compared to UC. Mild toxicities were reported., Conclusion: EA was more effective than BFAA at reducing pain severity, but both similarly improved physical and mental health scores. Breast cancer survivors with chronic musculoskeletal pain may consider EA before BFAA., Trial Registration: ClinicalTrials.gov Identifier: NCT02979574. https://clinicaltrials.gov/ct2/show/NCT02979574., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2023
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222. Double-Strand Break Repair Pathways Differentially Affect Processing and Transduction by Dual AAV Vectors.
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Maurer AC, Benyamini B, Fan VB, Whitney ON, Dailey GM, Darzacq X, Weitzman MD, and Tjian R
- Abstract
Recombinant adeno-associated viral vectors (rAAV) are a powerful tool for gene delivery but have a limited DNA carrying capacity. Efforts to expand this genetic payload have focused on engineering the vector components, such as dual trans-splicing vectors which double the delivery size by exploiting the natural concatenation of rAAV genomes in host nuclei. We hypothesized that inefficient dual vector transduction could be improved by modulating host factors which affect concatenation. Since factors mediating concatenation are not well defined, we performed a genome-wide screen to identify host cell regulators. We discovered that Homologous Recombination (HR) is inhibitory to dual vector transduction. We demonstrate that depletion or inhibition of HR factors BRCA1 and Rad51 significantly increase reconstitution of a large split transgene by increasing both concatenation and expression from rAAVs. Our results define new roles for DNA damage repair in rAAV transduction and highlight the potential for pharmacological intervention to increase genetic payload of rAAV vectors.
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- 2023
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223. Author Correction: A viral biomolecular condensate coordinates assembly of progeny particles.
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Charman M, Grams N, Kumar N, Halko E, Dybas JM, Abbott A, Lum KK, Blumenthal D, Tsopurashvili E, and Weitzman MD
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- 2023
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224. Intestinal transit amplifying cells require METTL3 for growth factor signaling, KRAS expression, and cell survival.
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Danan CH, Naughton KE, Hayer KE, Vellappan S, McMillan EA, Zhou Y, Matsuda R, Nettleford SK, Katada K, Parham LR, Ma X, Chowdhury A, Wilkins BJ, Shah P, Weitzman MD, and Hamilton KE
- Abstract
Intestinal epithelial transit amplifying cells are essential stem progenitors required for intestinal homeostasis, but their rapid proliferation renders them vulnerable to DNA damage from radiation and chemotherapy. Despite their critical roles in intestinal homeostasis and disease, few studies have described genes that are essential to transit amplifying cell function. We report that the RNA methyltransferase, METTL3, is required for survival of transit amplifying cells in the murine small intestine. Transit amplifying cell death after METTL3 deletion was associated with crypt and villus atrophy, loss of absorptive enterocytes, and uniform wasting and death in METTL3-depleted mice. Ribosome profiling and sequencing of methylated RNAs in enteroids and in vivo demonstrated decreased translation of hundreds of unique methylated transcripts after METTL3 deletion, particularly transcripts involved in growth factor signal transduction such as Kras . Further investigation confirmed a novel relationship between METTL3 and Kras methylation and protein levels in vivo . Our study identifies METTL3 as an essential factor supporting the homeostasis of small intestinal tissue via direct maintenance of transit amplifying cell survival. We highlight the crucial role of RNA modifications in regulating growth factor signaling in the intestine, with important implications for both homeostatic tissue renewal and epithelial regeneration., Competing Interests: Conflict-of-interest statement Premal Shah is a member of the Scientific Advisory Board of Trestle Biosciences and is Director at Ananke Therapeutics. All other authors declare they have no competing interests.
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- 2023
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225. Virology under the Microscope-a Call for Rational Discourse.
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Goodrum F, Lowen AC, Lakdawala S, Alwine J, Casadevall A, Imperiale MJ, Atwood W, Avgousti D, Baines J, Banfield B, Banks L, Bhaduri-McIntosh S, Bhattacharya D, Blanco-Melo D, Bloom D, Boon A, Boulant S, Brandt C, Broadbent A, Brooke C, Cameron C, Campos S, Caposio P, Chan G, Cliffe A, Coffin J, Collins K, Damania B, Daugherty M, Debbink K, DeCaprio J, Dermody T, Dikeakos J, DiMaio D, Dinglasan R, Duprex WP, Dutch R, Elde N, Emerman M, Enquist L, Fane B, Fernandez-Sesma A, Flenniken M, Frappier L, Frieman M, Frueh K, Gack M, Gaglia M, Gallagher T, Galloway D, García-Sastre A, Geballe A, Glaunsinger B, Goff S, Greninger A, Hancock M, Harris E, Heaton N, Heise M, Heldwein E, Hogue B, Horner S, Hutchinson E, Hyser J, Jackson W, Kalejta R, Kamil J, Karst S, Kirchhoff F, Knipe D, Kowalik T, Lagunoff M, Laimins L, Langlois R, Lauring A, Lee B, Leib D, Liu SL, Longnecker R, Lopez C, Luftig M, Lund J, Manicassamy B, McFadden G, McIntosh M, Mehle A, Miller WA, Mohr I, Moody C, Moorman N, Moscona A, Mounce B, Munger J, Münger K, Murphy E, Naghavi M, Nelson J, Neufeldt C, Nikolich J, O'Connor C, Ono A, Orenstein W, Ornelles D, Ou JH, Parker J, Parrish C, Pekosz A, Pellett P, Pfeiffer J, Plemper R, Polyak S, Purdy J, Pyeon D, Quinones-Mateu M, Renne R, Rice C, Schoggins J, Roller R, Russell C, Sandri-Goldin R, Sapp M, Schang L, Schmid S, Schultz-Cherry S, Semler B, Shenk T, Silvestri G, Simon V, Smith G, Smith J, Spindler K, Stanifer M, Subbarao K, Sundquist W, Suthar M, Sutton T, Tai A, Tarakanova V, tenOever B, Tibbetts S, Tompkins S, Toth Z, van Doorslaer K, Vignuzzi M, Wallace N, Walsh D, Weekes M, Weinberg J, Weitzman M, Weller S, Whelan S, White E, Williams B, Wobus C, Wong S, and Yurochko A
- Subjects
- Humans, SARS-CoV-2, Pandemics prevention & control, Antiviral Agents, COVID-19 prevention & control, Viruses
- Abstract
Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.
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- 2023
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226. A viral biomolecular condensate coordinates assembly of progeny particles.
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Charman M, Grams N, Kumar N, Halko E, Dybas JM, Abbott A, Lum KK, Blumenthal D, Tsopurashvili E, and Weitzman MD
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- Humans, Capsid chemistry, Capsid metabolism, Capsid Proteins chemistry, Capsid Proteins metabolism, Intrinsically Disordered Proteins chemistry, Intrinsically Disordered Proteins metabolism, Biomolecular Condensates chemistry, Biomolecular Condensates metabolism, Viral Proteins chemistry, Viral Proteins metabolism, Adenoviruses, Human chemistry, Adenoviruses, Human growth & development, Adenoviruses, Human metabolism
- Abstract
Biomolecular condensates formed by phase separation can compartmentalize and regulate cellular processes
1,2 . Emerging evidence has suggested that membraneless subcellular compartments in virus-infected cells form by phase separation3-8 . Although linked to several viral processes3-5,9,10 , evidence that phase separation contributes functionally to the assembly of progeny particles in infected cells is lacking. Here we show that phase separation of the human adenovirus 52-kDa protein has a critical role in the coordinated assembly of infectious progeny particles. We demonstrate that the 52-kDa protein is essential for the organization of viral structural proteins into biomolecular condensates. This organization regulates viral assembly such that capsid assembly is coordinated with the provision of viral genomes needed to produce complete packaged particles. We show that this function is governed by the molecular grammar of an intrinsically disordered region of the 52-kDa protein, and that failure to form condensates or to recruit viral factors that are critical for assembly results in failed packaging and assembly of only non-infectious particles. Our findings identify essential requirements for coordinated assembly of progeny particles and demonstrate that phase separation of a viral protein is critical for production of infectious progeny during adenovirus infection., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)- Published
- 2023
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227. Addressing the benefits of inhibiting APOBEC3-dependent mutagenesis in cancer.
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Petljak M, Green AM, Maciejowski J, and Weitzman MD
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- Humans, Mutagenesis genetics, APOBEC-1 Deaminase genetics, Mutation, Cytidine Deaminase genetics, APOBEC Deaminases genetics, Neoplasms genetics, Neoplasms pathology
- Abstract
Mutational signatures associated with apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC)3 cytosine deaminase activity have been found in over half of cancer types, including some therapy-resistant and metastatic tumors. Driver mutations can occur in APOBEC3-favored sequence contexts, suggesting that mutagenesis by APOBEC3 enzymes may drive cancer evolution. The APOBEC3-mediated signatures are often detected in subclonal branches of tumor phylogenies and are acquired in cancer cell lines over long periods of time, indicating that APOBEC3 mutagenesis can be ongoing in cancer. Collectively, these and other observations have led to the proposal that APOBEC3 mutagenesis represents a disease-modifying process that could be inhibited to limit tumor heterogeneity, metastasis and drug resistance. However, critical aspects of APOBEC3 biology in cancer and in healthy tissues have not been clearly defined, limiting well-grounded predictions regarding the benefits of inhibiting APOBEC3 mutagenesis in different settings in cancer. We discuss the relevant mechanistic gaps and strategies to address them to investigate whether inhibiting APOBEC3 mutagenesis may confer clinical benefits in cancer., (© 2022. Springer Nature America, Inc.)
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- 2022
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228. Antigen glycosylation regulates efficacy of CAR T cells targeting CD19.
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Heard A, Landmann JH, Hansen AR, Papadopolou A, Hsu YS, Selli ME, Warrington JM, Lattin J, Chang J, Ha H, Haug-Kroeper M, Doray B, Gill S, Ruella M, Hayer KE, Weitzman MD, Green AM, Fluhrer R, and Singh N
- Subjects
- B-Lymphocytes, Glycosylation, Humans, Receptors, Antigen, T-Cell metabolism, T-Lymphocytes, Antigens, CD19 metabolism, Immunotherapy, Adoptive methods
- Abstract
While chimeric antigen receptor (CAR) T cells targeting CD19 can cure a subset of patients with B cell malignancies, most patients treated will not achieve durable remission. Identification of the mechanisms leading to failure is essential to broadening the efficacy of this promising platform. Several studies have demonstrated that disruption of CD19 genes and transcripts can lead to disease relapse after initial response; however, few other tumor-intrinsic drivers of CAR T cell failure have been reported. Here we identify expression of the Golgi-resident intramembrane protease Signal peptide peptidase-like 3 (SPPL3) in malignant B cells as a potent regulator of resistance to CAR therapy. Loss of SPPL3 results in hyperglycosylation of CD19, an alteration that directly inhibits CAR T cell effector function and suppresses anti-tumor cytotoxicity. Alternatively, over-expression of SPPL3 drives loss of CD19 protein, also enabling resistance. In this pre-clinical model these findings identify post-translational modification of CD19 as a mechanism of antigen escape from CAR T cell therapy., (© 2022. The Author(s).)
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- 2022
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229. Adenovirus prevents dsRNA formation by promoting efficient splicing of viral RNA.
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Price AM, Steinbock RT, Di C, Hayer KE, Li Y, Herrmann C, Parenti NA, Whelan JN, Weiss SR, and Weitzman MD
- Subjects
- RNA, Double-Stranded genetics, RNA, Double-Stranded metabolism, RNA, Messenger metabolism, Adenoviridae genetics, Adenoviridae metabolism, RNA Splicing, RNA, Viral genetics, RNA, Viral metabolism
- Abstract
Eukaryotic cells recognize intracellular pathogens through pattern recognition receptors, including sensors of aberrant nucleic acid structures. Sensors of double-stranded RNA (dsRNA) are known to detect replication intermediates of RNA viruses. It has long been suggested that annealing of mRNA from symmetrical transcription of both top and bottom strands of DNA virus genomes can produce dsRNA during infection. Supporting this hypothesis, nearly all DNA viruses encode inhibitors of dsRNA-recognition pathways. However, direct evidence that DNA viruses produce dsRNA is lacking. Contrary to dogma, we show that the nuclear-replicating DNA virus adenovirus (AdV) does not produce detectable levels of dsRNA during infection. In contrast, abundant dsRNA is detected within the nucleus of cells infected with AdV mutants defective for viral RNA processing. In the presence of nuclear dsRNA, the cytoplasmic dsRNA sensor PKR is relocalized and activated within the nucleus. Accumulation of viral dsRNA occurs in the late phase of infection, when unspliced viral transcripts form intron/exon base pairs between top and bottom strand transcripts. We propose that DNA viruses actively limit dsRNA formation by promoting efficient splicing and mRNA processing, thus avoiding detection and restriction by host innate immune sensors of pathogenic nucleic acids., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2022
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230. Schlafens Can Put Viruses to Sleep.
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Kim ET and Weitzman MD
- Subjects
- Animals, Cell Cycle Proteins immunology, Humans, Immunity, Innate, Mice, Endoribonucleases immunology, Host-Pathogen Interactions immunology, Immune Evasion, Virus Diseases immunology
- Abstract
The Schlafen gene family encodes for proteins involved in various biological tasks, including cell proliferation, differentiation, and T cell development. Schlafens were initially discovered in mice, and have been studied in the context of cancer biology, as well as their role in protecting cells during viral infection. This protein family provides antiviral barriers via direct and indirect effects on virus infection. Schlafens can inhibit the replication of viruses with both RNA and DNA genomes. In this review, we summarize the cellular functions and the emerging relationship between Schlafens and innate immunity. We also discuss the functions and distinctions of this emerging family of proteins as host restriction factors against viral infection. Further research into Schlafen protein function will provide insight into their mechanisms that contribute to intrinsic and innate host immunity.
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- 2022
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231. Health-Related Quality of Life in Cancer Survivors with Chemotherapy-Induced Peripheral Neuropathy: A Randomized Clinical Trial.
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Bao T, Baser R, Chen C, Weitzman M, Zhang YL, Seluzicki C, Li QS, Piulson L, and Zhi WI
- Subjects
- Female, Humans, Quality of Life, Acupuncture Therapy, Antineoplastic Agents adverse effects, Cancer Survivors, Neoplasms drug therapy, Peripheral Nervous System Diseases chemically induced
- Abstract
Background: Chemotherapy-induced peripheral neuropathy (CIPN) is a common, debilitating adverse effect of neurotoxic chemotherapy that significantly worsens the quality of life of cancer survivors., Materials and Methods: Survivors of solid tumors with persistent moderate-to-severe CIPN defined as numbness, tingling, or pain rated ≥4 on an 11-point numeric rating scale (NRS) were randomized in a 1:1:1 ratio to 8 weeks of real acupuncture (RA) versus sham acupuncture (SA) versus usual care (UC). We previously reported the primary endpoint (NRS); here we report the following health-related quality of life endpoints: Functional Assessment of Cancer Therapy/Gynecologic Oncology Group-Neurotoxicity (FACT/GOG-Ntx), Hospital Anxiety and Depression Scale (HADS), Insomnia Severity Index (ISI), and Brief Fatigue Inventory (BFI). For each endpoint, the mean changes from baseline and 95% confidence intervals were estimated within each arm and compared between arms using linear mixed models., Results: We enrolled 75 survivors of solid tumors with moderate-to-severe CIPN into the study. Compared with baseline, at week 8, FACT/GOG-Ntx, HADS anxiety, and ISI scores significantly improved in RA and SA, but not in UC. Compared with UC, at week 8, FACT/GOG-Ntx scores significantly increased in RA and SA arms indicating improved CIPN-related symptoms and quality of life (p = .001 and p = .01). There was no statistically significant difference between RA and SA. There was no difference in HADS depression or BFI among RA, SA, and UC at weeks 8 and 12., Conclusion: Acupuncture may improve CIPN-related symptoms and quality of life in cancer survivors with persistent CIPN. Further large sample size studies are needed to delineate placebo effects., Implications for Practice: The authors conducted a randomized sham acupuncture- and usual care-controlled clinical trial to evaluate the impact of acupuncture on health-related quality of life outcomes in patients with solid tumors with chemotherapy-induced peripheral neuropathy (CIPN). Statistically significant improvements in quality of life, anxiety, insomnia, and fatigue were achieved with 8 weeks of real acupuncture when compared with baseline, without statistically significant differences between real and sham acupuncture. These findings suggest that acupuncture may be effective for improving CIPN-related symptoms and quality of life and reducing anxiety and insomnia in cancer survivors with persistent CIPN, with further study needed to delineate placebo effects., (© 2021 AlphaMed Press.)
- Published
- 2021
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232. Interaction with the CCT chaperonin complex limits APOBEC3A cytidine deaminase cytotoxicity.
- Author
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Green AM, DeWeerd RA, O'Leary DR, Hansen AR, Hayer KE, Kulej K, Dineen AS, Szeto JH, Garcia BA, and Weitzman MD
- Subjects
- Mutagenesis, Proteins genetics, Chaperonin Containing TCP-1 genetics, Cytidine Deaminase genetics
- Abstract
The APOBEC3 cytidine deaminases are implicated as the cause of a prevalent somatic mutation pattern found in cancer genomes. The APOBEC3 enzymes act as viral restriction factors by mutating viral genomes. Mutation of the cellular genome is presumed to be an off-target activity of the enzymes, although the regulatory measures for APOBEC3 expression and activity remain undefined. It is therefore difficult to predict circumstances that enable APOBEC3 interaction with cellular DNA that leads to mutagenesis. The APOBEC3A (A3A) enzyme is the most potent deaminase of the family. Using proteomics, we evaluate protein interactors of A3A to identify potential regulators. We find that A3A interacts with the chaperonin-containing TCP-1 (CCT) complex, a cellular machine that assists in protein folding and function. Importantly, depletion of CCT results in A3A-induced DNA damage and cytotoxicity. Evaluation of cancer genomes demonstrates an enrichment of A3A mutational signatures in cancers with silencing mutations in CCT subunit genes. Together, these data suggest that the CCT complex interacts with A3A, and that disruption of CCT function results in increased A3A mutational activity., (© 2021 The Authors.)
- Published
- 2021
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233. Adenovirus Remodeling of the Host Proteome and Host Factors Associated with Viral Genomes.
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Dybas JM, Lum KK, Kulej K, Reyes ED, Lauman R, Charman M, Purman CE, Steinbock RT, Grams N, Price AM, Mendoza L, Garcia BA, and Weitzman MD
- Abstract
Viral infections are associated with extensive remodeling of the cellular proteome. Viruses encode gene products that manipulate host proteins to redirect cellular processes or subvert antiviral immune responses. Adenovirus (AdV) encodes proteins from the early E4 region which are necessary for productive infection. Some cellular antiviral proteins are known to be targeted by AdV E4 gene products, resulting in their degradation or mislocalization. However, the full repertoire of host proteome changes induced by viral E4 proteins has not been defined. To identify cellular proteins and processes manipulated by viral products, we developed a global, unbiased proteomics approach to analyze changes to the host proteome during infection with adenovirus serotype 5 (Ad5) virus. We used whole-cell proteomics to measure total protein abundances in the proteome during Ad5 infection. Since host antiviral proteins can antagonize viral infection by associating with viral genomes and inhibiting essential viral processes, we used Isolation of Proteins on Nascent DNA (iPOND) proteomics to identify proteins associated with viral genomes during infection with wild-type Ad5 or an E4 mutant virus. By integrating these proteomics data sets, we identified cellular factors that are degraded in an E4-dependent manner or are associated with the viral genome in the absence of E4 proteins. We further show that some identified proteins exert inhibitory effects on Ad5 infection. Our systems-level analysis reveals cellular processes that are manipulated during Ad5 infection and points to host factors counteracted by early viral proteins as they remodel the host proteome to promote efficient infection. IMPORTANCE Viral infections induce myriad changes to the host cell proteome. As viruses harness cellular processes and counteract host defenses, they impact abundance, post-translational modifications, interactions, or localization of cellular proteins. Elucidating the dynamic changes to the cellular proteome during viral replication is integral to understanding how virus-host interactions influence the outcome of infection. Adenovirus encodes early gene products from the E4 genomic region that are known to alter host response pathways and promote replication, but the full extent of proteome modifications they mediate is not known. We used an integrated proteomics approach to quantitate protein abundance and protein associations with viral DNA during virus infection. Systems-level analysis identifies cellular proteins and processes impacted in an E4-dependent manner, suggesting ways that adenovirus counteracts potentially inhibitory host defenses. This study provides a global view of adenovirus-mediated proteome remodeling, which can serve as a model to investigate virus-host interactions of DNA viruses.
- Published
- 2021
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234. Comparative proteomics identifies Schlafen 5 (SLFN5) as a herpes simplex virus restriction factor that suppresses viral transcription.
- Author
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Kim ET, Dybas JM, Kulej K, Reyes ED, Price AM, Akhtar LN, Orr A, Garcia BA, Boutell C, and Weitzman MD
- Subjects
- Animals, Cell Cycle Proteins genetics, Chlorocebus aethiops, DNA, Viral metabolism, HEK293 Cells, HeLa Cells, Herpes Simplex metabolism, Humans, Immediate-Early Proteins genetics, Immediate-Early Proteins metabolism, Promoter Regions, Genetic, Proteomics, RNA Polymerase II metabolism, Ubiquitin-Protein Ligases genetics, Ubiquitin-Protein Ligases metabolism, Ubiquitination, Vero Cells, Cell Cycle Proteins metabolism, Gene Expression Regulation, Viral, Herpes Simplex virology, Host-Pathogen Interactions, Simplexvirus genetics, Transcription, Genetic
- Abstract
Intrinsic antiviral host factors confer cellular defence by limiting virus replication and are often counteracted by viral countermeasures. We reasoned that host factors that inhibit viral gene expression could be identified by determining proteins bound to viral DNA (vDNA) in the absence of key viral antagonists. Herpes simplex virus 1 (HSV-1) expresses E3 ubiquitin-protein ligase ICP0 (ICP0), which functions as an E3 ubiquitin ligase required to promote infection. Cellular substrates of ICP0 have been discovered as host barriers to infection but the mechanisms for inhibition of viral gene expression are not fully understood. To identify restriction factors antagonized by ICP0, we compared proteomes associated with vDNA during HSV-1 infection with wild-type virus and a mutant lacking functional ICP0 (ΔICP0). We identified the cellular protein Schlafen family member 5 (SLFN5) as an ICP0 target that binds vDNA during HSV-1 ΔICP0 infection. We demonstrated that ICP0 mediates ubiquitination of SLFN5, which leads to its proteasomal degradation. In the absence of ICP0, SLFN5 binds vDNA to repress HSV-1 transcription by limiting accessibility of RNA polymerase II to viral promoters. These results highlight how comparative proteomics of proteins associated with viral genomes can identify host restriction factors and reveal that viral countermeasures can overcome SLFN antiviral activity.
- Published
- 2021
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235. Direct RNA sequencing reveals m 6 A modifications on adenovirus RNA are necessary for efficient splicing.
- Author
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Price AM, Hayer KE, McIntyre ABR, Gokhale NS, Abebe JS, Della Fera AN, Mason CE, Horner SM, Wilson AC, Depledge DP, and Weitzman MD
- Subjects
- A549 Cells, Adenosine metabolism, Adenoviruses, Human pathogenicity, DNA, Viral genetics, Gene Knockdown Techniques, Gene Knockout Techniques, HEK293 Cells, Host-Pathogen Interactions genetics, Humans, Methyltransferases genetics, Methyltransferases metabolism, RNA, Small Interfering metabolism, RNA, Viral genetics, Sequence Analysis, RNA, Virus Replication, Adenosine analogs & derivatives, Adenovirus Infections, Human virology, Adenoviruses, Human genetics, RNA Splicing, RNA, Viral metabolism
- Abstract
Adenovirus is a nuclear replicating DNA virus reliant on host RNA processing machinery. Processing and metabolism of cellular RNAs can be regulated by METTL3, which catalyzes the addition of N6-methyladenosine (m
6 A) to mRNAs. While m6 A-modified adenoviral RNAs have been previously detected, the location and function of this mark within the infectious cycle is unknown. Since the complex adenovirus transcriptome includes overlapping spliced units that would impede accurate m6 A mapping using short-read sequencing, here we profile m6 A within the adenovirus transcriptome using a combination of meRIP-seq and direct RNA long-read sequencing to yield both nucleotide and transcript-resolved m6 A detection. Although both early and late viral transcripts contain m6 A, depletion of m6 A writer METTL3 specifically impacts viral late transcripts by reducing their splicing efficiency. These data showcase a new technique for m6 A discovery within individual transcripts at nucleotide resolution, and highlight the role of m6 A in regulating splicing of a viral pathogen.- Published
- 2020
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236. STAT3-BDNF-TrkB signalling promotes alveolar epithelial regeneration after lung injury.
- Author
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Paris AJ, Hayer KE, Oved JH, Avgousti DC, Toulmin SA, Zepp JA, Zacharias WJ, Katzen JB, Basil MC, Kremp MM, Slamowitz AR, Jayachandran S, Sivakumar A, Dai N, Wang P, Frank DB, Eisenlohr LC, Cantu E 3rd, Beers MF, Weitzman MD, Morrisey EE, and Worthen GS
- Subjects
- Alveolar Epithelial Cells metabolism, Animals, Brain-Derived Neurotrophic Factor genetics, Female, Humans, Lung Injury etiology, Lung Injury pathology, Male, Membrane Glycoproteins genetics, Protein-Tyrosine Kinases genetics, Receptor, trkB genetics, STAT3 Transcription Factor genetics, Alveolar Epithelial Cells cytology, Brain-Derived Neurotrophic Factor metabolism, Lung Injury prevention & control, Membrane Glycoproteins metabolism, Protein-Tyrosine Kinases metabolism, Receptor, trkB metabolism, Regeneration, STAT3 Transcription Factor metabolism
- Abstract
Alveolar epithelial regeneration is essential for recovery from devastating lung diseases. This process occurs when type II alveolar pneumocytes (AT2 cells) proliferate and transdifferentiate into type I alveolar pneumocytes (AT1 cells). We used genome-wide analysis of chromatin accessibility and gene expression following acute lung injury to elucidate repair mechanisms. AT2 chromatin accessibility changed substantially following injury to reveal STAT3 binding motifs adjacent to genes that regulate essential regenerative pathways. Single-cell transcriptome analysis identified brain-derived neurotrophic factor (Bdnf) as a STAT3 target gene with newly accessible chromatin in a unique population of regenerating AT2 cells. Furthermore, the BDNF receptor tropomyosin receptor kinase B (TrkB) was enriched on mesenchymal alveolar niche cells (MANCs). Loss or blockade of AT2-specific Stat3, Bdnf or mesenchyme-specific TrkB compromised repair and reduced Fgf7 expression by niche cells. A TrkB agonist improved outcomes in vivo following lung injury. These data highlight the biological and therapeutic importance of the STAT3-BDNF-TrkB axis in orchestrating alveolar epithelial regeneration.
- Published
- 2020
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237. Adenovirus-mediated ubiquitination alters protein-RNA binding and aids viral RNA processing.
- Author
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Herrmann C, Dybas JM, Liddle JC, Price AM, Hayer KE, Lauman R, Purman CE, Charman M, Kim ET, Garcia BA, and Weitzman MD
- Subjects
- Adenoviridae Infections metabolism, Base Sequence, Binding Sites, Computational Biology methods, Humans, Nucleotide Motifs, Protein Binding, Proteome, Proteomics methods, RNA Processing, Post-Transcriptional, RNA Splicing, Ubiquitination, Adenoviridae physiology, Adenoviridae Infections virology, Gene Expression Regulation, Viral, Host-Pathogen Interactions, RNA, Viral genetics, RNA, Viral metabolism, RNA-Binding Proteins metabolism
- Abstract
Viruses promote infection by hijacking the ubiquitin machinery of the host to counteract or redirect cellular processes. Adenovirus encodes two early proteins, E1B55K and E4orf6, that together co-opt a cellular ubiquitin ligase complex to overcome host defences and promote virus production. Adenovirus mutants lacking E1B55K or E4orf6 display defects in viral RNA processing and protein production, but previously identified substrates of the redirected ligase do not explain these phenotypes. Here, we used a quantitative proteomics approach to identify substrates of E1B55K/E4orf6-mediated ubiquitination that facilitate RNA processing. While all currently known cellular substrates of E1B55K and E4orf6 are degraded by the proteasome, we uncovered RNA-binding proteins as high-confidence substrates that are not decreased in overall abundance. We focused on two RNA-binding proteins, RALY and hnRNP-C, which we confirm are ubiquitinated without degradation. Knockdown of RALY and hnRNP-C increased levels of viral RNA splicing, protein abundance and progeny production during infection with E1B55K-deleted virus. Furthermore, infection with E1B55K-deleted virus resulted in an increased interaction of hnRNP-C with viral RNA and attenuation of viral RNA processing. These data suggest that viral-mediated ubiquitination of RALY and hnRNP-C relieves a restriction on viral RNA processing and reveal an unexpected role for non-degradative ubiquitination in the manipulation of cellular processes during virus infection.
- Published
- 2020
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238. Adeno-Associated Virus Genome Interactions Important for Vector Production and Transduction.
- Author
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Maurer AC and Weitzman MD
- Subjects
- Animals, Gene Transfer Techniques, Genome, Viral, Helper Viruses physiology, Humans, Dependovirus genetics, Genetic Therapy, Genetic Vectors, Host Microbial Interactions, Transduction, Genetic, Virus Replication
- Abstract
Recombinant adeno-associated virus has emerged as one of the most promising gene therapy delivery vectors. Development of these vectors took advantage of key features of the wild-type adeno-associated virus (AAV), enabled by basic studies of the underlying biology and requirements for transcription, replication, and packaging of the viral genome. Each step in generating and utilizing viral vectors involves numerous molecular interactions that together determine the efficiency of vector production and gene delivery. Once delivered into the cell, interactions with host proteins will determine the fate of the viral genome, and these will impact the intended goal of gene delivery. Here, we provide an overview of known interactions of the AAV genome with viral and cellular proteins involved in its amplification, packaging, and expression. Further appreciation of how the AAV genome interacts with host factors will enhance how this simple virus can be harnessed for an array of vector purposes that benefit human health.
- Published
- 2020
- Full Text
- View/download PDF
239. A Tribute to Barrie J. Carter.
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Srivastava A, Weitzman M, Chatterjee S, Engelhardt JF, Owens RA, Muzyczka N, and Ali R
- Subjects
- History, 20th Century, History, 21st Century, Humans, United States, Dependovirus, Genetic Therapy history
- Published
- 2020
- Full Text
- View/download PDF
240. Impaired Death Receptor Signaling in Leukemia Causes Antigen-Independent Resistance by Inducing CAR T-cell Dysfunction.
- Author
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Singh N, Lee YG, Shestova O, Ravikumar P, Hayer KE, Hong SJ, Lu XM, Pajarillo R, Agarwal S, Kuramitsu S, Orlando EJ, Mueller KT, Good CR, Berger SL, Shalem O, Weitzman MD, Frey NV, Maude SL, Grupp SA, June CH, Gill S, and Ruella M
- Subjects
- Humans, Precursor Cell Lymphoblastic Leukemia-Lymphoma pathology, Signal Transduction, Precursor Cell Lymphoblastic Leukemia-Lymphoma genetics, Receptors, Chimeric Antigen metabolism, Receptors, Death Domain metabolism
- Abstract
Primary resistance to CD19-directed chimeric antigen receptor T-cell therapy (CART19) occurs in 10% to 20% of patients with acute lymphoblastic leukemia (ALL); however, the mechanisms of this resistance remain elusive. Using a genome-wide loss-of-function screen, we identified that impaired death receptor signaling in ALL led to rapidly progressive disease despite CART19 treatment. This was mediated by an inherent resistance to T-cell cytotoxicity that permitted antigen persistence and was subsequently magnified by the induction of CAR T-cell functional impairment. These findings were validated using samples from two CAR T-cell clinical trials in ALL, where we found that reduced expression of death receptor genes was associated with worse overall survival and reduced T-cell fitness. Our findings suggest that inherent dysregulation of death receptor signaling in ALL directly leads to CAR T-cell failure by impairing T-cell cytotoxicity and promoting progressive CAR T-cell dysfunction. SIGNIFICANCE: Resistance to CART19 is a significant barrier to efficacy in the treatment of B-cell malignancies. This work demonstrates that impaired death receptor signaling in tumor cells causes failed CART19 cytotoxicity and drives CART19 dysfunction, identifying a novel mechanism of antigen-independent resistance to CAR therapy. See related commentary by Green and Neelapu, p. 492 ., (©2020 American Association for Cancer Research.)
- Published
- 2020
- Full Text
- View/download PDF
241. Quantitative live cell imaging reveals influenza virus manipulation of Rab11A transport through reduced dynein association.
- Author
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Bhagwat AR, Le Sage V, Nturibi E, Kulej K, Jones J, Guo M, Tae Kim E, Garcia BA, Weitzman MD, Shroff H, and Lakdawala SS
- Subjects
- Biological Transport, Dyneins genetics, Host-Pathogen Interactions, Humans, Influenza A virus genetics, Influenza, Human genetics, Influenza, Human virology, RNA, Viral genetics, RNA, Viral metabolism, rab GTP-Binding Proteins genetics, Dyneins metabolism, Influenza A virus physiology, Influenza, Human metabolism, rab GTP-Binding Proteins metabolism
- Abstract
Assembly of infectious influenza A viruses (IAV) is a complex process involving transport from the nucleus to the plasma membrane. Rab11A-containing recycling endosomes have been identified as a platform for intracellular transport of viral RNA (vRNA). Here, using high spatiotemporal resolution light-sheet microscopy (~1.4 volumes/second, 330 nm isotropic resolution), we quantify Rab11A and vRNA movement in live cells during IAV infection and report that IAV infection decreases speed and increases arrest of Rab11A. Unexpectedly, infection with respiratory syncytial virus alters Rab11A motion in a manner opposite to IAV, suggesting that Rab11A is a common host component that is differentially manipulated by respiratory RNA viruses. Using two-color imaging we demonstrate co-transport of Rab11A and IAV vRNA in infected cells and provide direct evidence that vRNA-associated Rab11A have altered transport. The mechanism of altered Rab11A movement is likely related to a decrease in dynein motors bound to Rab11A vesicles during IAV infection.
- Published
- 2020
- Full Text
- View/download PDF
242. A core viral protein binds host nucleosomes to sequester immune danger signals.
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Avgousti DC, Herrmann C, Kulej K, Pancholi NJ, Sekulic N, Petrescu J, Molden RC, Blumenthal D, Paris AJ, Reyes ED, Ostapchuk P, Hearing P, Seeholzer SH, Worthen GS, Black BE, Garcia BA, and Weitzman MD
- Subjects
- Alarmins metabolism, Animals, Bronchoalveolar Lavage Fluid cytology, Bronchoalveolar Lavage Fluid immunology, Cell Line, Chromatin Assembly and Disassembly drug effects, HMGB1 Protein metabolism, Histones metabolism, Humans, Inflammation immunology, Inflammation metabolism, Lung immunology, Lung metabolism, Male, Mice, Neutrophil Infiltration drug effects, Neutrophil Infiltration immunology, Nucleosomes chemistry, Nucleosomes drug effects, Nucleosomes genetics, Protein Binding, Protein Processing, Post-Translational, Proteomics, Viral Core Proteins chemistry, Viral Core Proteins pharmacology, Adenoviridae chemistry, Immunity, Innate drug effects, Nucleosomes metabolism, Viral Core Proteins metabolism
- Abstract
Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses. Small basic proteins compact and regulate both viral and cellular DNA genomes. Nucleosomes are the repeating units of cellular chromatin and play an important part in innate immune responses. Viral-encoded core basic proteins compact viral genomes, but their impact on host chromatin structure and function remains unexplored. Adenoviruses encode a highly basic protein called protein VII that resembles cellular histones. Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles, it is unknown whether protein VII affects cellular chromatin. Here we show that protein VII alters cellular chromatin, leading us to hypothesize that this has an impact on antiviral responses during adenovirus infection in human cells. We find that protein VII forms complexes with nucleosomes and limits DNA accessibility. We identified post-translational modifications on protein VII that are responsible for chromatin localization. Furthermore, proteomic analysis demonstrated that protein VII is sufficient to alter the protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in the chromatin of members of the high-mobility-group protein B family (HMGB1, HMGB2 and HMGB3). HMGB1 is actively released in response to inflammatory stimuli and functions as a danger signal to activate immune responses. We showed that protein VII can directly bind HMGB1 in vitro and further demonstrated that protein VII expression in mouse lungs is sufficient to decrease inflammation-induced HMGB1 content and neutrophil recruitment in the bronchoalveolar lavage fluid. Together, our in vitro and in vivo results show that protein VII sequesters HMGB1 and can prevent its release. This study uncovers a viral strategy in which nucleosome binding is exploited to control extracellular immune signaling.
- Published
- 2016
- Full Text
- View/download PDF
243. Changing the ubiquitin landscape during viral manipulation of the DNA damage response.
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Weitzman MD, Lilley CE, and Chaurushiya MS
- Subjects
- Host-Pathogen Interactions, Humans, Signal Transduction, Viral Proteins metabolism, Viral Proteins physiology, Virus Diseases genetics, Virus Diseases virology, Viruses metabolism, DNA Damage, DNA Repair, Ubiquitin metabolism, Virus Diseases physiopathology
- Abstract
Viruses often induce signaling through the same cellular cascades that are activated by damage to the cellular genome. Signaling triggered by viral proteins or exogenous DNA delivered by viruses can be beneficial or detrimental to viral infection. Viruses have therefore evolved to dissect the cellular DNA damage response pathway during infection, often marking key cellular regulators with ubiquitin to induce their degradation or change their function. Signaling controlled by ubiquitin or ubiquitin-like proteins has recently emerged as key regulator of the cellular DNA damage response. Situated at the interface between DNA damage signaling and the ubiquitin system, viruses can reveal key convergence points in this important cellular pathway. In this review, we examine how viruses harness the diversity of the cellular ubiquitin system to modulate the DNA damage signaling pathway. We discuss the implications of viral infiltration of this pathway for both the transcriptional program of the virus and for the cellular response to DNA damage., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
244. Adeno-associated virus biology.
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Weitzman MD and Linden RM
- Subjects
- Dependovirus classification, Dependovirus growth & development, Dependovirus metabolism, Gene Transfer Techniques, Genetic Vectors metabolism, Serotyping, Virus Replication genetics, Virus Replication physiology, Dependovirus genetics, Genetic Vectors genetics
- Abstract
Adeno-associated virus (AAV) was first discovered as a contaminant of adenovirus stocks in the 1960s. The development of recombinant AAV vectors (rAAV) was facilitated by early studies that generated infectious molecular clones, determined the sequence of the genome, and defined the genetic elements of the virus. The refinement of methods and protocols for the production and application of rAAV vectors has come from years of studies that explored the basic biology of this virus and its interaction with host cells. Interest in improving vector performance has in turn driven studies that have provided tremendous insights into the basic biology of the AAV lifecycle. In this chapter, we review the background on AAV biology and its exploitation for vectors and gene delivery.
- Published
- 2011
- Full Text
- View/download PDF
245. Genomes in conflict: maintaining genome integrity during virus infection.
- Author
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Weitzman MD, Lilley CE, and Chaurushiya MS
- Subjects
- Animals, Humans, Models, Biological, Neoplasms genetics, Neoplasms virology, DNA Damage, DNA Repair, Genomic Instability, Virus Diseases pathology, Viruses pathogenicity
- Abstract
The cellular surveillance network for sensing and repairing damaged DNA prevents an array of human diseases, and when compromised it can lead to genomic instability and cancer. The carefully maintained cellular response to DNA damage is challenged during viral infection, when foreign DNA is introduced into the cell. The battle between virus and host generates a genomic conflict. The host attempts to limit viral infection and protect its genome, while the virus deploys tactics to eliminate, evade, or exploit aspects of the cellular defense. Studying this conflict has revealed that the cellular DNA damage response machinery comprises part of the intrinsic cellular defense against viral infection. In this review we examine recent advances in this emerging field. We identify common themes used by viruses in their attempts to commandeer or circumvent the host cell's DNA repair machinery, and highlight potential outcomes of the conflict for both virus and host.
- Published
- 2010
- Full Text
- View/download PDF
246. Analysis of the RPE transcriptome reveals dynamic changes during the development of the outer blood-retinal barrier.
- Author
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Rizzolo LJ, Chen X, Weitzman M, Sun R, and Zhang H
- Subjects
- Actins genetics, Actins metabolism, Animals, Cell Cycle Proteins genetics, Chick Embryo, Cluster Analysis, Collagen genetics, Collagen metabolism, Extracellular Matrix genetics, Intercellular Junctions genetics, Laminin genetics, Laminin metabolism, Membrane Proteins genetics, Membrane Proteins metabolism, Microtubule-Associated Proteins genetics, Microtubule-Associated Proteins metabolism, Myosins genetics, Myosins metabolism, Oligonucleotide Array Sequence Analysis, Phagocytosis genetics, Protein Subunits genetics, Protein Subunits metabolism, Proteins genetics, Proteins metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, Receptors, Cell Surface genetics, Receptors, Cell Surface metabolism, Reverse Transcriptase Polymerase Chain Reaction, Blood-Retinal Barrier embryology, Blood-Retinal Barrier metabolism, Gene Expression Profiling, Pigment Epithelium of Eye metabolism, Transcription, Genetic
- Abstract
Purpose: The morphology of the RPE shows minimal change as the neural retina and choriocapillaris differentiate. Nonetheless, initial studies of proteins related to the outer blood-retinal barrier suggest extensive remodeling of the retinal pigment epithelium (RPE) in response to this changing environment. A genomic approach was used to investigate the extent of this remodeling., Methods: RPE was isolated from E7, E10, E14, and E18 chick embryos and total RNA extracted for probing the entire genome on Affymetrix microarray chips. Statistical parameters using ANOVA were adjusted to yield a theoretical false discovery rate of 5%. STEM software was used to cluster genes into statistically related patterns of expression. Gene ontology clustering, using Affymetrix software was used for functional clustering. The proteinlounge.com database was used as a source of known biological pathways., Results: Of the 37,694 probesets on the microarray, 17,199 were absent. Of the 20,495 expressed probes, the expression of 8,889 was developmentally regulated. 4,814 of these could be clustered into 12 patterns of expression that were statistically significant. Minimal contamination by surrounding tissues was detected. The developmental patterns of 22 tight and adherens junction proteins were compared using hybridization to the microarray and quantitative PCR. Only two showed small variations from the patterns revealed by the microarray. The data indicate extensive remodeling of the extracellular matrix, cell surface receptors, cell-cell junctions, transcellular ion transport, and signal transduction pathways throughout development. Notably, the appearance of the mRNAs for claudin 20, ZO-3, and cadherins 13 and 20 very late in development suggest barrier properties continue to change after functional junctions are formed., Conclusions: The data reveal a far more dynamic view of the RPE and its interactions with its environment than would be expected from morphological examination. The remodeling of junctional complexes, extracellular matrix interactions and transcellular transport capabilities indicates a continuous remodeling of the blood-retinal barrier as the retina develops. These data provide a standard whereby culture models of RPE function and regulation may be judged.
- Published
- 2007
247. Inactivating intracellular antiviral responses during adenovirus infection.
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Weitzman MD and Ornelles DA
- Subjects
- Adenoviridae Infections genetics, DNA Repair, Protein Biosynthesis, Signal Transduction, Virus Replication, Adenoviridae pathogenicity, Adenoviridae Infections physiopathology, DNA Damage, DNA, Viral genetics
- Abstract
DNA viruses promote cell cycle progression, stimulate unscheduled DNA synthesis, and present the cell with an extraordinary amount of exogenous DNA. These insults elicit vigorous responses mediated by cellular factors that govern cellular homeostasis. To ensure productive infection, adenovirus has developed means to inactivate these intracellular antiviral responses. Among the challenges to the host cell is the viral DNA genome, which is viewed as DNA damage and elicits a cellular response to inhibit replication. Adenovirus therefore encodes proteins that dismantle the cellular DNA damage machinery. Studying virus-host interactions has yielded insights into the molecular functioning of fundamental cellular mechanisms. In addition, it has suggested ways that viral cytotoxicity can be exploited to offer a selective means of restricted growth in tumor cells as a therapy against cancer. In this review, we discuss aspects of the intracellular response that are unique to adenovirus infection and how adenoviral proteins produced from the early region E4 act to neutralize antiviral defenses, with a particular focus on DNA damage signaling.
- Published
- 2005
- Full Text
- View/download PDF
248. Gene therapy: twenty-first century medicine.
- Author
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Verma IM and Weitzman MD
- Subjects
- Adenoviridae genetics, Animals, Clinical Trials as Topic, Dependovirus genetics, Gene Expression Regulation, Genetic Therapy trends, Genetic Vectors, Herpesviridae genetics, Humans, Retroviridae genetics, Transduction, Genetic, Genetic Therapy methods
- Abstract
Broadly defined, the concept of gene therapy involves the transfer of genetic material into a cell, tissue, or whole organ, with the goal of curing a disease or at least improving the clinical status of a patient. A key factor in the success of gene therapy is the development of delivery systems that are capable of efficient gene transfer in a variety of tissues, without causing any associated pathogenic effects. Vectors based upon many different viral systems, including retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses, currently offer the best choice for efficient gene delivery. Their performance and pathogenicity has been evaluated in animal models, and encouraging results form the basis for clinical trials to treat genetic disorders and acquired diseases. Despite some initial success in these trials, vector development remains a seminal concern for improved gene therapy technologies.
- Published
- 2005
- Full Text
- View/download PDF
249. Gene delivery to the mammalian heart using AAV vectors.
- Author
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Chu D, Thistlethwaite PA, Sullivan CC, Grifman MS, and Weitzman MD
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- Animals, Rats, Rats, Inbred F344, Dependovirus genetics, Gene Transfer Techniques, Genetic Vectors, Myocardium metabolism
- Abstract
There are a large number of cardiovascular diseases that could be treated by myocardial gene transfer. These include congestive heart failure, ischemic heart disease, and cardiomyopathy. In addition to its potential for treatment of disease, myocardial gene transfer is useful for the analysis of gene expression and promoter function and for generating animal models of human disease such as pulmonary hypertension. The ideal vector for myocardial gene therapy should give efficient and stable transduction of cardiomyocytes in vivo. Recombinant adenovirus vectors have been used to transduce cardiomyocytes in rodents, rabbits, pigs, and humans by both intramyocardial injection and intracoronary infusion (3-5). Although efficient transduction can be obtained with adenovirus vectors, immune responses and elimination of transduced cells results in only transient expression in immunocompetent hosts. Vectors based on recombinant adeno-associated virus (rAAV) offer a number of attractive features and are emerging as promising gene transfer vehicles for many in vivo applications.
- Published
- 2004
- Full Text
- View/download PDF
250. The Mre11 complex is required for ATM activation and the G2/M checkpoint.
- Author
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Carson CT, Schwartz RA, Stracker TH, Lilley CE, Lee DV, and Weitzman MD
- Subjects
- Ataxia Telangiectasia genetics, Ataxia Telangiectasia Mutated Proteins, Cell Line, G2 Phase, HeLa Cells, Humans, MRE11 Homologue Protein, Mitosis, Mutagenesis, Site-Directed, Polymerase Chain Reaction, Recombinant Proteins metabolism, Signal Transduction genetics, Tumor Suppressor Proteins, Virus Replication, Adenoviruses, Human physiology, Cell Cycle physiology, Cell Cycle Proteins, DNA Damage, DNA-Binding Proteins metabolism, Defective Viruses genetics, Protein Serine-Threonine Kinases genetics
- Abstract
The maintenance of genome integrity requires a rapid and specific response to many types of DNA damage. The conserved and related PI3-like protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate signal transduction pathways in response to genomic insults, such as DNA double-strand breaks (DSBs). It is unclear which proteins recognize DSBs and activate these pathways, but the Mre11/Rad50/NBS1 complex has been suggested to act as a damage sensor. Here we show that infection with an adenovirus lacking the E4 region also induces a cellular DNA damage response, with activation of ATM and ATR. Wild-type virus blocks this signaling through degradation of the Mre11 complex by the viral E1b55K/E4orf6 proteins. Using these viral proteins, we show that the Mre11 complex is required for both ATM activation and the ATM-dependent G(2)/M checkpoint in response to DSBs. These results demonstrate that the Mre11 complex can function as a damage sensor upstream of ATM/ATR signaling in mammalian cells.
- Published
- 2003
- Full Text
- View/download PDF
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