Journal articles on the topic 'Gene transfer, viral genome'
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1
Hohn, Barbara, Zdena Koukolíková-Nicola, Guus Bakkeren, and Nigel Grimsley. "Agrobacterium-mediated gene transfer to monocots and dicots." Genome 31, no. 2 (January 15, 1989): 987–93. http://dx.doi.org/10.1139/g89-172.
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The interaction of the soil bacterium Agrobacterium tumefaciens with plants constitutes a unique kind of genetic flux: the bacterium transfers the T-DNA part of its Ti plasmid to plant cells, where it is integrated into the genome. Possible transfer intermediates, isolated from bacteria and from plants early after transfer, are described. Agroinfection, Agrobacterium-mediated delivery of plant viral genomes, is employed to monitor early events in T-DNA transfer in dicot plants. Graminaceous monocots, so far excluded from Agrobacterium's host range because of lack of tumor formation, have been shown to be agroinfectable. This newly discovered interaction between grasses and the pathogen is described in terms of the efficiency of gene transfer as compared with dicot hosts, the involvement of the bacterium's virulence genes, the susceptibility of various developmental stages of the host, the implications for biotechnology, and the evolutionary aspects of this host–parasite relationship.Key words: T-DNA, agroinfection, maize streak virus, plant transformation, Zea mays.
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Liu, Huiquan, Yanping Fu, Daohong Jiang, Guoqing Li, Jiatao Xie, Jiasen Cheng, Youliang Peng, Said A. Ghabrial, and Xianhong Yi. "Widespread Horizontal Gene Transfer from Double-Stranded RNA Viruses to Eukaryotic Nuclear Genomes." Journal of Virology 84, no. 22 (September 1, 2010): 11876–87. http://dx.doi.org/10.1128/jvi.00955-10.
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ABSTRACT Horizontal gene transfer commonly occurs from cells to viruses but rarely occurs from viruses to their host cells, with the exception of retroviruses and some DNA viruses. However, extensive sequence similarity searches in public genome databases for various organisms showed that the capsid protein and RNA-dependent RNA polymerase genes from totiviruses and partitiviruses have widespread homologs in the nuclear genomes of eukaryotic organisms, including plants, arthropods, fungi, nematodes, and protozoa. PCR amplification and sequencing as well as comparative evidence of junction coverage between virus and host sequences support the conclusion that these viral homologs are real and occur in eukaryotic genomes. Sequence comparison and phylogenetic analysis suggest that these genes were likely transferred horizontally from viruses to eukaryotic genomes. Furthermore, we present evidence showing that some of the transferred genes are conserved and expressed in eukaryotic organisms and suggesting that these viral genes are also functional in the recipient genomes. Our findings imply that horizontal transfer of double-stranded RNA viral genes is widespread among eukaryotes and may give rise to functionally important new genes, thus entailing that RNA viruses may play significant roles in the evolution of eukaryotes.
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Bobay, Louis-Marie, and Howard Ochman. "Biological species in the viral world." Proceedings of the National Academy of Sciences 115, no. 23 (May 21, 2018): 6040–45. http://dx.doi.org/10.1073/pnas.1717593115.
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Due to their dependence on cellular organisms for metabolism and replication, viruses are typically named and assigned to species according to their genome structure and the original host that they infect. But because viruses often infect multiple hosts and the numbers of distinct lineages within a host can be vast, their delineation into species is often dictated by arbitrary sequence thresholds, which are highly inconsistent across lineages. Here we apply an approach to determine the boundaries of viral species based on the detection of gene flow within populations, thereby defining viral species according to the biological species concept (BSC). Despite the potential for gene transfer between highly divergent genomes, viruses, like the cellular organisms they infect, assort into reproductively isolated groups and can be organized into biological species. This approach revealed that BSC-defined viral species are often congruent with the taxonomic partitioning based on shared gene contents and host tropism, and that bacteriophages can similarly be classified in biological species. These results open the possibility to use a single, universal definition of species that is applicable across cellular and acellular lifeforms.
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Shayakhmetov, Dmitry M., Zong-Yi Li, Anuj Gaggar, Helen Gharwan, Vladimir Ternovoi, Volker Sandig, and André Lieber. "Genome Size and Structure Determine Efficiency of Postinternalization Steps and Gene Transfer of Capsid-Modified Adenovirus Vectors in a Cell-Type-Specific Manner." Journal of Virology 78, no. 18 (September 15, 2004): 10009–22. http://dx.doi.org/10.1128/jvi.78.18.10009-10022.2004.
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ABSTRACT Adenovirus serotype 5 (Ad5) vectors containing Ad B-group fibers have become increasingly popular as gene transfer vectors because they efficiently transduce human cell types that are relatively refractory to Ad5 infection. So far, most B-group fiber-containing vectors have been first-generation vectors, deleted of E1 and/or E3 genes. Transduction with these vectors, however, results in viral gene expression and is associated with cytotoxicity and immune responses against transduced cells. To circumvent these problems, we developed fiber-chimeric Ad vectors devoid of all viral genes that were produced either by the homologous recombination of first-generation vectors or by using the Cre/lox-based helper virus system. In this study we compared early steps of infection between first-generation (35-kb genome) and Ad vectors devoid of all viral genes with genome sizes of 28 kb and 12.6 kb. All vectors possessed an Ad35-derived fiber knob domain, which uses CD46 as a primary attachment receptor. Using immortalized human hematopoietic cell lines and primary human CD34-positive hematopoietic cells, we found that the Ad genome size did not affect the efficiency of virus attachment to and internalization into cells. Furthermore, independently of the genome length and structure, all vectors migrated to the nucleus through late endosomal and lysosomal cellular compartments. However, the vector containing the short 12.6-kb genome was unable to efficiently escape from endosomes and deliver its DNA into the nucleus. Moreover, compared to other vectors, these Ad particles were less stable and had an abnormal capsid protein composition, including a lack of capsid-stabilizing protein IX. Our data indicate that the size and structure of the packaged viral genomes can affect the integrity of Ad particles, which in turn results in lower infectivity of Ad vectors.
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Shettima, Abubakar, Muhammad M. Ibrahim, and Musa Ibn Abbas. "Features and properties of viral and non-viral gene delivery systems towards effective gene therapy." International Journal of Medicine 5, no. 1 (February 8, 2017): 45. http://dx.doi.org/10.14419/ijm.v5i1.7189.
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Gene therapy has revolutionized the treatment of hereditary and genetic link disorders by consciously swapping, fixing, adding or deleting the genetic sequences responsible for the condition. The culprit cells are altered by inserting purposeful genes and incorporated into their genome for proper expression. Germ line therapy ensures the genotypic changes to be transferred to the next generation (offspring) while the somatic type adequately rest on corrective pedestals and as such not advantageous to the offspring. The earlier was constrained by technical difficulties as well as ethical consideration. The accomplishment of the therapeutic benefits of gene therapy requires a special ferry system “vectors”. Vectors are designed to transfer the desired gene into its target cell without exposing it to some degrading enzymes, and must allow transcription to successfully take place. A model vector must not be immunogenic, it must not trigger high immune response detrimental to the patient and a specific tropism must be a pre-requisite. The choice of a vector should be based on safety, cost and availability as well as the accessibility of possible options. Mainly for viral carriers, host immune response trigger are the main concern. Viral vectors most frequently used in gene therapy include adenoviruses, retroviruses, poxviruses, adeno-associated viruses and herpes simplex viruses.
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Yang, Grace S., Michael Schmidt, Ziying Yan, Jonathan D. Lindbloom, Thomas C. Harding, Brian A. Donahue, John F. Engelhardt, Robert Kotin, and Beverly L. Davidson. "Virus-Mediated Transduction of Murine Retina with Adeno-Associated Virus: Effects of Viral Capsid and Genome Size." Journal of Virology 76, no. 15 (August 1, 2002): 7651–60. http://dx.doi.org/10.1128/jvi.76.15.7651-7660.2002.
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ABSTRACT Gene therapy vectors based on adeno-associated viruses (AAVs) show promise for the treatment of retinal degenerative diseases. In prior work, subretinal injections of AAV2, AAV5, and AAV2 pseudotyped with AAV5 capsids (AAV2/5) showed variable retinal pigmented epithelium (RPE) and photoreceptor cell transduction, while AAV2/1 predominantly transduced the RPE. To more thoroughly compare the efficiencies of gene transfer of AAV2, AAV3, AAV5, and AAV6, we quantified, using stereological methods, the kinetics and efficiency of AAV transduction to mouse photoreceptor cells. We observed persistent photoreceptor and RPE transduction by AAV5 and AAV2 up to 31 weeks and found that AAV5 transduced a greater volume than AAV2. AAV5 containing full-length or half-length genomes and AAV2/5 transduced comparable numbers of photoreceptor cells with similar rates of onset of expression. Compared to AAV2, AAV5 transduced significantly greater numbers of photoreceptor cells at 5 and 15 weeks after surgery (greater than 1,000 times and up to 400 times more, respectively). Also, there were 30 times more genome copies in eyes injected with AAV2/5 than in eyes injected with AAV2. Comparing AAVs with half-length genomes, AAV5 transduced only four times more photoreceptor cells than AAV2 at 5 weeks and nearly equivalent numbers at 15 weeks. The enhancement of transduction was seen at the DNA level, with 50 times more viral genome copies in retinas injected with AAV having short genomes than in retinas injected with AAV containing full-length ones. Subretinal injection of AAV2/6 showed only RPE transduction at 5 and 15 weeks, while AAV2/3 did not transduce retinal cells. We conclude that varying genome length and AAV capsids may allow for improved expression and/or gene transfer to specific cell types in the retina.
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Monier, Adam, Aurélie Chambouvet, David S. Milner, Victoria Attah, Ramón Terrado, Connie Lovejoy, Hervé Moreau, Alyson E. Santoro, Évelyne Derelle, and Thomas A. Richards. "Host-derived viral transporter protein for nitrogen uptake in infected marine phytoplankton." Proceedings of the National Academy of Sciences 114, no. 36 (August 21, 2017): E7489—E7498. http://dx.doi.org/10.1073/pnas.1708097114.
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Phytoplankton community structure is shaped by both bottom–up factors, such as nutrient availability, and top–down processes, such as predation. Here we show that marine viruses can blur these distinctions, being able to amend how host cells acquire nutrients from their environment while also predating and lysing their algal hosts. Viral genomes often encode genes derived from their host. These genes may allow the virus to manipulate host metabolism to improve viral fitness. We identify in the genome of a phytoplankton virus, which infects the small green alga Ostreococcus tauri, a host-derived ammonium transporter. This gene is transcribed during infection and when expressed in yeast mutants the viral protein is located to the plasma membrane and rescues growth when cultured with ammonium as the sole nitrogen source. We also show that viral infection alters the nature of nitrogen compound uptake of host cells, by both increasing substrate affinity and allowing the host to access diverse nitrogen sources. This is important because the availability of nitrogen often limits phytoplankton growth. Collectively, these data show that a virus can acquire genes encoding nutrient transporters from a host genome and that expression of the viral gene can alter the nutrient uptake behavior of host cells. These results have implications for understanding how viruses manipulate the physiology and ecology of phytoplankton, influence marine nutrient cycles, and act as vectors for horizontal gene transfer.
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Pchelin, Ivan M., Pavel V. Tkachev, Daniil V. Azarov, Andrey N. Gorshkov, Daria O. Drachko, Vasily V. Zlatogursky, Alexander V. Dmitriev, and Artemiy E. Goncharov. "A Genome of Temperate Enterococcus Bacteriophage Placed in a Space of Pooled Viral Dark Matter Sequences." Viruses 15, no. 1 (January 12, 2023): 216. http://dx.doi.org/10.3390/v15010216.
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In the human gut, temperate bacteriophages interact with bacteria through a predation and horizontal gene transfer. Relying on taxonomic data, metagenomic studies have associated shifts in the phage abundance with a number of human diseases. The temperate bacteriophage VEsP-1 with siphovirus morphology was isolated from a sample of river water using Enterococcus faecalis as a host. Starting from the whole genome sequence of VEsP-1, we retrieved related phage genomes in blastp searches of the tail protein and large terminase sequences, and blastn searches of the whole genome sequences, with matches compiled from several different databases, and visualized a part of a viral dark matter sequence space. The genome network and phylogenomic analyses resulted in the proposal of a novel genus “Vespunovirus”, consisting of temperate, mainly metagenomic phages infecting Enterococcus spp.
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Arhab, Yani, Alexander G. Bulakhov, Tatyana V. Pestova, and Christopher U. T. Hellen. "Dissemination of Internal Ribosomal Entry Sites (IRES) Between Viruses by Horizontal Gene Transfer." Viruses 12, no. 6 (June 4, 2020): 612. http://dx.doi.org/10.3390/v12060612.
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Members of Picornaviridae and of the Hepacivirus, Pegivirus and Pestivirus genera of Flaviviridae all contain an internal ribosomal entry site (IRES) in the 5′-untranslated region (5′UTR) of their genomes. Each class of IRES has a conserved structure and promotes 5′-end-independent initiation of translation by a different mechanism. Picornavirus 5′UTRs, including the IRES, evolve independently of other parts of the genome and can move between genomes, most commonly by intratypic recombination. We review accumulating evidence that IRESs are genetic entities that can also move between members of different genera and even between families. Type IV IRESs, first identified in the Hepacivirus genus, have subsequently been identified in over 25 genera of Picornaviridae, juxtaposed against diverse coding sequences. In several genera, members have either type IV IRES or an IRES of type I, II or III. Similarly, in the genus Pegivirus, members contain either a type IV IRES or an unrelated type; both classes of IRES also occur in members of the genus Hepacivirus. IRESs utilize different mechanisms, have different factor requirements and contain determinants of viral growth, pathogenesis and cell type specificity. Their dissemination between viruses by horizontal gene transfer has unexpectedly emerged as an important facet of viral evolution.
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Bertani, Giuseppe. "Transduction-Like Gene Transfer in the MethanogenMethanococcus voltae." Journal of Bacteriology 181, no. 10 (May 15, 1999): 2992–3002. http://dx.doi.org/10.1128/jb.181.10.2992-3002.1999.
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ABSTRACT Strain PS of Methanococcus voltae (a methanogenic, anaerobic archaebacterium) was shown to generate spontaneously 4.4-kbp chromosomal DNA fragments that are fully protected from DNase and that, upon contact with a cell, transform it genetically. This activity, here called VTA (voltae transfer agent), affects all markers tested: three different auxotrophies (histidine, purine, and cobalamin) and resistance to BES (2-bromoethanesulfonate, an inhibitor of methanogenesis). VTA was most effectively prepared by culture filtration. This process disrupted a fraction of the M. voltae cells (which have only an S-layer covering their cytoplasmic membrane). VTA was rapidly inactivated upon storage. VTA particles were present in cultures at concentrations of approximately two per cell. Gene transfer activity varied from a minimum of 2 × 10−5 (BES resistance) to a maximum of 10−3 (histidine independence) per donor cell. Very little VTA was found free in culture supernatants. The phenomenon is functionally similar to generalized transduction, but there is no evidence, for the time being, of intrinsically viral (i.e., containing a complete viral genome) particles. Consideration of VTA DNA size makes the existence of such viral particles unlikely. If they exist, they must be relatively few in number;perhaps they differ from VTA particles in size and other properties and thus escaped detection. Digestion of VTA DNA with the AluI restriction enzyme suggests that it is a random sample of the bacterial DNA, except for a 0.9-kbp sequence which is amplified relative to the rest of the bacterial chromosome. A VTA-sized DNA fraction was demonstrated in a few other isolates of M. voltae.
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Li, Hai-Ou, Ya-Feng Zhu, Makoto Asakawa, Hidekazu Kuma, Takahiro Hirata, Yasuji Ueda, Yun-Sik Lee, et al. "A Cytoplasmic RNA Vector Derived from Nontransmissible Sendai Virus with Efficient Gene Transfer and Expression." Journal of Virology 74, no. 14 (July 15, 2000): 6564–69. http://dx.doi.org/10.1128/jvi.74.14.6564-6569.2000.
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ABSTRACT We have recovered a virion from defective cDNA of Sendai virus (SeV) that is capable of self-replication but incapable of transmissible-virion production. This virion delivers and expresses foreign genes in infected cells, and this is the first report of a gene expression vector derived from a defective viral genome of theParamyxoviridae. First, functional ribonucleoprotein complexes (RNPs) were recovered from SeV cloned cDNA defective in the F (envelope fusion protein) gene, in the presence of plasmids expressing nucleocapsid protein and viral RNA polymerase. Then the RNPs were transfected to the cells inducibly expressing F protein. Virion-like particles thus obtained had a titer of 0.5 × 108 to 1.0 × 108 cell infectious units/ml and contained F-defective RNA genome. This defective vector amplified specifically in an F-expressing packaging cell line in a trypsin-dependent manner but did not spread to F-nonexpressing cells. This vector infected and expressed an enhanced green fluorescent protein reporter gene in various types of animal and human cells, including nondividing cells, with high efficiency. These results suggest that this vector has great potential for use in human gene therapy and vaccine delivery systems.
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Martinez-Medina, Lourdes, Munevver Cinar, Bassel El-Rayes, Ganji Nagaraju, Jean-Michel Gries, and Leon Bernal-Mizrachi. "Horizontal Gene Transfer in Multiple Myeloma and Development of a Non-Viral Gene Delivery System." Blood 138, Supplement 1 (November 5, 2021): 3982. http://dx.doi.org/10.1182/blood-2021-154018.
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Abstract Multiple myeloma (MM) is a heterogeneous malignant plasma cell disorder with complex molecular and genetic abnormalities. While current therapies have improved outcomes in MM, many patients relapse. Part of the development of the resistance to treatment relates to the genetic heterogeneity of tumors that allows for the development of resistant clones. Our laboratory demonstrated that MM circulating tumor DNA (ctDNA) serves as a mechanism for horizontal gene transfer between cancer cells. Using novel 3-D imaging analysis methodologies, we demonstrated that ctDNA target specifically cancer cells of similar tumor phenotype. Furthermore, using novel in vitro models and whole genome sequencing, we identify certain transposable elements (TE) responsible of allowing DNA insertion into cancer cells. Additionally, our analysis discovered a particular retro-transposon sequence unique of multiple myeloma (MM zip-code, MMZC). After chemically synthesizing the MMZC and using flow cytometry methods, we demonstrated that a MMZC was capable of targeting and internalizing within MM cells after 30 minutes of incubation when compared to a control non-carrying TE sequence. Moreover, after culturing for 24 hours, we observed an increase on the number of cells that have internalized the MMZC. To evaluate genome integration, we ligated the MMZC to a linearized CMV-GFP and a linearized CMV-mCherry fragment prior to adding to MM cell culture. Within 12 hours, live imaging showed expression of GFP and mCherry in MM cells. This results are being validated in single cell sequencing. To evaluate a potential therapeutic potential of MMZC cargo delivery, we ligated MMZC to HSV-Tk-GFP linearized vector and tested the cell specificity targeting and killing effect of MMZC-HSV-Tk-GFP in MM, Pancreatic cancer and colon cancer cell lines. MMZC-HSV-Tk-GFP did not elicit a reduction in cell viability. However, MMZC-HSV-Tk-GFP reduce MM cell viability when ganciclovir was added to the cell culture. The capacity of gene delivery has been validated in-vivo using a MM xenograft mice. PCR evaluating for MMZC-HSV-Tk-GFP after 48 hours of tail injection demonstrated that tumor, but not other organs, amplified HSV-Tk. Furthermore, tumor tissue expressed GFP. To our knowledge this is the first description of the use of humanized non-viral synthetic technology capable of delivering therapeutic cargo material into MM cell. Disclosures Martinez-Medina: Kodikaz Therapeutic Solutions: Current Employment. El-Rayes: Bayer: Research Funding; Pfizer: Research Funding; Merck: Research Funding; Novartis: Research Funding; Boston biomedical: Research Funding; Bristol Myers Squibb: Research Funding; Astra Zeneca: Consultancy, Research Funding; exelixis: Consultancy; erytotech: Research Funding; dicephera pharmaceutical: Consultancy. Gries: Kodikaz Therapeutic Solutions: Current Employment; Feldan Therapeutics: Consultancy. Bernal-Mizrachi: Winship Cancer Institute of Emory University: Current Employment; Takeda Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kodikaz Therapeutic Solutions: Consultancy, Current holder of individual stocks in a privately-held company, Patents & Royalties; Bigene: Membership on an entity's Board of Directors or advisory committees.
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Lieber, André, Dirk S. Steinwaerder, Cheryl A. Carlson, and Mark A. Kay. "Integrating Adenovirus–Adeno-Associated Virus Hybrid Vectors Devoid of All Viral Genes." Journal of Virology 73, no. 11 (November 1, 1999): 9314–24. http://dx.doi.org/10.1128/jvi.73.11.9314-9324.1999.
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ABSTRACT Recently, we demonstrated that inverted repeat sequences inserted into first-generation adenovirus (Ad) vector genomes mediate precise genomic rearrangements resulting in vector genomes devoid of all viral genes that are efficiently packaged into functional Ad capsids. As a specific application of this finding, we generated adenovirus–adeno-associated virus (AAV) hybrid vectors, first-generation Ad vectors containing AAV inverted terminal repeat sequences (ITRs) flanking a reporter gene cassette inserted into the E1 region. We hypothesized that the AAV ITRs present within the hybrid vector genome could mediate the formation of rearranged vector genomes (ΔAd.AAV) and stimulate transgene integration. We demonstrate here that ΔAd.AAV vectors are efficiently generated as by-products of first-generation adenovirus-AAV vector amplification. ΔAd.AAV genomes contain only the transgene flanked by AAV ITRs, Ad packaging signals, and Ad ITRs. ΔAd.AAV vectors can be produced at a high titer and purity. In vitro transduction properties of these deleted hybrid vectors were evaluated in direct comparison with first-generation Ad and recombinant AAV vectors (rAAVs). The ΔAd.AAV hybrid vector stably transduced cultured cells with efficiencies comparable to rAAV. Since cells transduced with ΔAd.AAV did not express cytotoxic viral proteins, hybrid viruses could be applied at very high multiplicities of infection to increase transduction rates. Southern analysis and pulsed-field gel electrophoresis suggested that ΔAd.AAV integrated randomly as head-to-tail tandems into the host cell genome. The presence of two intact AAV ITRs was crucial for the production of hybrid vectors and for transgene integration. ΔAd.AAV vectors, which are straightforward in their production, represent a promising tool for stable gene transfer in vitro and in vivo.
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Lim, Filip. "HSV-1 as a Model for Emerging Gene Delivery Vehicles." ISRN Virology 2013 (May 27, 2013): 1–12. http://dx.doi.org/10.5402/2013/397243.
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The majority of viral vectors currently used possess modest cargo capability (up to 40 kb) being based on retroviruses, lentiviruses, adenoviruses, and adenoassociated viruses. These vectors have made the most rapid transition from laboratory to clinic because their small genomes have simplified their characterization and modification. However, there is now an increasing need both in research and therapy to complement this repertoire with larger capacity vectors able to deliver multiple transgenes or to encode complex regulatory regions, constructs which can easily span more than 100 kb. Herpes Simplex Virus Type I (HSV-1) is a well-characterized human virus which is able to package about 150 kb of DNA, and several vector systems are currently in development for gene transfer applications, particularly in neurons where other systems have low efficiency. However, to reach the same level of versatility and ease of use as that of smaller genome viral vectors, simple systems for high-titer production must be developed. This paper reviews the major HSV-1 vector systems and analyses the common elements which may be most important to manipulate to achieve this goal.
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Gilbert, Rosalind, and Diane Ouwerkerk. "The Genetics of Rumen Phage Populations." Proceedings 36, no. 1 (April 7, 2020): 165. http://dx.doi.org/10.3390/proceedings2019036165.
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The microbial populations of the rumen are widely recognised as being essential for ruminant nutrition and health, utilising and breaking down fibrous plant material which would otherwise be indigestible. The dense and highly diverse viral populations which co-exist with these microbial populations are less understood, despite their potential impacts on microbial lysis and gene transfer. In recent years, studies using metagenomics, metatranscriptomics and proteomics have provided new insights into the types of viruses present in the rumen and the proteins they produce. These studies however are limited in their capacity to fully identify and classify the viral sequence information obtained, due to the absence of rumen-specific virus genomes in current sequence databases. The majority of commensal viruses found in the rumen are those infecting bacteria (phages), therefore we genome sequenced phage isolates from our phage culture collection infecting the common rumen microbial genera Bacteroides, Ruminococcus and Streptococcus. We also created a pan-genome using 39 whole genome sequences of predominantly livestock-derived Streptococcus isolates (representing S. bovis, S. equinus, S. henryi, and S. gallolyticus), to identify and characterise integrated viral genomes (prophage sequences). Collectively this approach has provided novel rumen phage sequences to increase the accuracy of rumen metagenomics analyses. It has also provided new insights into how viruses or virus-encoded proteins can potentially be used to modulate specific microbial populations within the rumen microbiome.
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Michou, Anne-Isabelle, Heike Lehrmann, Mediyha Saltik, and Matt Cotten. "Mutational Analysis of the Avian Adenovirus CELO, Which Provides a Basis for Gene Delivery Vectors." Journal of Virology 73, no. 2 (February 1, 1999): 1399–410. http://dx.doi.org/10.1128/jvi.73.2.1399-1410.1999.
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ABSTRACT The avian adenovirus CELO is being developed as a gene transfer tool. Using homologous recombination in Escherichia coli, the CELO genome was screened for regions that could be deleted and would tolerate the insertion of a marker gene (luciferase or enhanced green fluorescent protein). For each mutant genome, the production of viable virus able to deliver the transgene to target cells was monitored. A series of mutants in the genome identified a set of open reading frames that could be deleted but which must be supplied in trans for virus replication. A region of the genome which is dispensable for viral replication and allows the insertion of an expression cassette was identified and a vector based on this mutation was evaluated as a gene delivery reagent. Transduction of avian cells occurs at 10- to 100-fold greater efficiency (per virus particle) than with an adenovirus type 5 (Ad5)-based vector carrying the same expression cassette. Most important for gene transfer applications, the CELO vector transduced mammalian cells as efficiently as an Ad5 vector. The CELO vector is exceptionally stable, can be grown inexpensively in chicken embryos, and provides a useful alternative to Ad5-based vectors.
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Naldini, Luigi. "In Vivo Gene Delivery by Lentiviral Vectors." Thrombosis and Haemostasis 82, no. 08 (1999): 552–54. http://dx.doi.org/10.1055/s-0037-1615879.
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IntroductionSuccessful gene therapy requires the efficient delivery and sustained expression of a therapeutic gene into the tissues of a human body. Most of the candidate tissues for therapeutic gene transfer are made of quiescent cells, such as from the brain, liver, and muscle. Thus, the optimal vector should infect nondividing cells, become stably associated with the genome of target cells, and support a high, steady-state level of transcription.1,2 Like all vectors derived from retroviruses, lentiviral vectors integrate into the chromatin of target cells and do not transfer any viral genes. Both of these features are important for achieving sustained expression of the transgene. Moreover, lentiviral vectors infect nondividing cells, a feature sharply distinguishing them from simple or onco-retroviral vectors.3 Upon infection, retroviruses deliver a nucleoprotein complex that reverse transcribes the viral RNA and integrates the newly made DNA into the chromatin. The nucleoprotein complexes of onco-retroviruses are excluded by the nucleus, and they reach the chromatin only when the nuclear membrane is fragmented during mitosis. This explains the dependence of a productive onco-retroviral infection on cell division occurring shortly after viral entry.4,5 In contrast, the nucleoprotein complexes of lentiviruses contain nuclear localization signals that mediate their active transport through the nucleopores during interphase. This explains the capacity of lentiviruses to infect macrophages, a nondividing cell type.6-9
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Tromas, Nicolas, Mark P. Zwart, Javier Forment, and Santiago F. Elena. "Shrinkage of Genome Size in a Plant RNA Virus upon Transfer of an Essential Viral Gene into the Host Genome." Genome Biology and Evolution 6, no. 3 (February 20, 2014): 538–50. http://dx.doi.org/10.1093/gbe/evu036.
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Dunbar, Cynthia E., Katherine A. High, J. Keith Joung, Donald B. Kohn, Keiya Ozawa, and Michel Sadelain. "Gene therapy comes of age." Science 359, no. 6372 (January 11, 2018): eaan4672. http://dx.doi.org/10.1126/science.aan4672.
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After almost 30 years of promise tempered by setbacks, gene therapies are rapidly becoming a critical component of the therapeutic armamentarium for a variety of inherited and acquired human diseases. Gene therapies for inherited immune disorders, hemophilia, eye and neurodegenerative disorders, and lymphoid cancers recently progressed to approved drug status in the United States and Europe, or are anticipated to receive approval in the near future. In this Review, we discuss milestones in the development of gene therapies, focusing on direct in vivo administration of viral vectors and adoptive transfer of genetically engineered T cells or hematopoietic stem cells. We also discuss emerging genome editing technologies that should further advance the scope and efficacy of gene therapy approaches.
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Konno, Akihiro, G. Jayashree Jagadeesh, Daniele Moratto, Marita Bosticardo, Ingeborg Holt, Linda M. Muul, Shepherd H. Schurman, Tyra Wolfsberg, and Fabio Candotti. "Analysis of Retroviral Vector Insertion Sites after T-Cell Directed Gene Therapy." Blood 104, no. 11 (November 16, 2004): 289. http://dx.doi.org/10.1182/blood.v104.11.289.289.
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Abstract Gene transfer into peripheral blood lymphocytes has several potential applications including the correction of genetic diseases and therapeutic approaches for HIV-1 infection and cancer. Integrating gene transfer system based on murine oncoretroviruses are a convenient tool for such strategies. However, the recent occurrence of uncontrolled clonal T cell expansions in two patients treated with retroviral gene transfer for X-linked severe combined immune deficiency has raised the concern of the risk of insertional oncogenesis associated with the clinical use of integrating viral systems. In vitro studies have indicated that murine viral vectors tend to integrate in the vicinity of transcription start regions of the genome, thus providing a possible mechanism for oncogene activation, however, data from clinical gene transfer trials is lacking. We are following patients affected with adenosine deaminase (ADA) deficiency who have received T-lymphocyte-directed, retroviral-mediated gene transfer starting in 1990. The first treated patient received the last infusion of gene-corrected cells 12 years ago, has never shown any sign of lymphoproliferation and still carries ~20% of gene-corrected peripheral blood lymphocytes. We set out to study the integration sites in the cells of this patient with the aim of mapping the regions involved by retroviral integrations, determining their localization with respect to known genes, and assessing whether a preferred pattern could be defined. Genomic DNA was prepared from stored lymphocyte samples dating 1991, 1992, 1995, 1998, 2000, and 2003. By inverse PCR and ligation-mediated PCR, we have identified ~860 bona fide insertion sites. Search for homology within the human genome using BLAT returned ~330 unique hits that involved a variety of genes, including transcription factors and oncogenes (e.g. RUNX1, STAT5, FYN). To evaluate the distribution pattern of these integration sites, 2000 randomly generated data sets of genomic coordinates were assembled and their distribution relative to annotations of the human genome was analyzed. A preliminary comparison of the random distribution to our experimental samples showed that retroviral integrations in cells obtained from the patient were significantly skewed toward regions within 2 kb of genes (p<0.002) and CpG islands (p<0.001). These results suggest that, similar to what observed in murine fibroblast and human cancer cell lines, transcriptionally active regions of the genome may be preferred targets of retroviral vectors in human primary T lymphocytes. At the same time, our observations show that the resulting integration events are compatible with long-term, event-free in vivo survival of gene-modified cells in clinical settings.
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Moreau, Hervé, Gwenael Piganeau, Yves Desdevises, Richard Cooke, Evelyne Derelle, and Nigel Grimsley. "Marine Prasinovirus Genomes Show Low Evolutionary Divergence and Acquisition of Protein Metabolism Genes by Horizontal Gene Transfer." Journal of Virology 84, no. 24 (September 22, 2010): 12555–63. http://dx.doi.org/10.1128/jvi.01123-10.
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ABSTRACT Although marine picophytoplankton are at the base of the global food chain, accounting for half of the planetary primary production, they are outnumbered 10 to 1 and are largely controlled by hugely diverse populations of viruses. Eukaryotic microalgae form a ubiquitous and particularly dynamic fraction of such plankton, with environmental clone libraries from coastal regions sometimes being dominated by one or more of the three genera Bathycoccus, Micromonas, and Ostreococcus (class Prasinophyceae). The complete sequences of two double-stranded (dsDNA) Bathycoccus, one dsDNA Micromonas, and one new dsDNA Ostreococcus virus genomes are described. Genome comparison of these giant viruses revealed a high degree of conservation, both for orthologous genes and for synteny, except for one 36-kb inversion in the Ostreococcus lucimarinus virus and two very large predicted proteins in Bathycoccus prasinos viruses. These viruses encode a gene repertoire of certain amino acid biosynthesis pathways never previously observed in viruses that are likely to have been acquired from lateral gene transfer from their host or from bacteria. Pairwise comparisons of whole genomes using all coding sequences with homologous counterparts, either between viruses or between their corresponding hosts, revealed that the evolutionary divergences between viruses are lower than those between their hosts, suggesting either multiple recent host transfers or lower viral evolution rates.
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Roy, Soumitra, David S. Clawson, Virginie S. Adam, Angelica Medina, and James M. Wilson. "Construction of gene transfer vectors based on simian adenovirus 7." Journal of General Virology 92, no. 8 (August 1, 2011): 1749–53. http://dx.doi.org/10.1099/vir.0.032300-0.
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The complete nucleotide sequence of an isolate of simian adenovirus 7 (SAdV-7) was determined. The genome organization of this isolate was found to be similar to that of other primate adenoviruses with two principal notable points: severe truncation of the E1A and E1B 19K proteins and an E3 region encoding only the 12.5K homologue. The viral gene products of SAdV-7 are most closely related to simian adenovirus 1 (SAdV-1), and like SAdV-1, are related to the human adenovirus species HAdV-F, such as the enteric adenoviruses HAdV-40 and HAdV-41 and the recently defined HAdV-G (HAdV-52). Two kinds of gene transfer vectors were made: a replication-competent SAdV-7-based vector with no genomic deletion, and a standard replication-incompetent vector deleted for E1. Importantly, the E1-deleted vector could be propagated to high titre by trans-complementation in human HEK 293 cells.
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Qiu, Wenping, and James W. Moyer. "Tomato Spotted Wilt Tospovirus Adapts to the TSWV N Gene-Derived Resistance by Genome Reassortment." Phytopathology® 89, no. 7 (July 1999): 575–82. http://dx.doi.org/10.1094/phyto.1999.89.7.575.
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Pathogen- and host-derived resistance have been shown to suppress infection by many plant viruses. Tomato spotted wilt tospovirus (TSWV) is among these systems; however, it has easily overcome nearly all host resistance genes and has recently been shown to overcome resistance mediated by the TSWV N gene. To better understand the resistance-breaking mechanisms, we have chosen TSWV N gene-derived resistance (TNDR) as a model to study how plant viruses defeat resistance genes. A defined viral population of isolates TSWV-D and TSWV-10, both suppressed by TNDR, was subjected to TNDR selection by serial passage in an N-gene transgenic plant. The genotype analysis demonstrated that the mixed viral population was driven to form a specific reassortant, L10M10SD, in the presence of TNDR selection, but remained as a heterogeneous mixture in the absence of the selection. A genotype assay of 120 local lesion isolates from the first, fourth, and seventh transfers confirmed the shift of genomic composition. Further analysis demonstrated that the individual L10, M10, and SD RNA segments were each selected independently in response to TNDR selection rather than to a mutation or recombination event. Following the seventh transfer on the N-gene transgenic plants, TSWV S RNA remained essentially identical to the S RNA from TSWV-D, indicating that no intermolecular recombination occurred between the two S RNAs from TSWV-10 and TSWV-D nor with the transferred N gene. These results support the hypothesis that TSWV utilizes genome reassortment to adapt to new host genotypes rapidly and that elements from two or more segments of the genome are involved in suppression of the resistance reaction.
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Sellner, Leopold, Marlon R. Veldwijk, Jürgen A. Kleinschmidt, Stephanie Laufs, Julian Topaly, W. Jens Zeller, Frederik Wenz, and Stefan Fruehauf. "Gene Therapy of Chronic Myelogenous Leukemia Using Pseudotyped Recombinant Adeno-Associated Viral Vectors." Blood 114, no. 22 (November 20, 2009): 4506. http://dx.doi.org/10.1182/blood.v114.22.4506.4506.
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Abstract Abstract 4506 Introduction Gene transfer into malignant leukemia cells can be of relevance to overcome conventional therapy-resistance. Either suicide or immune stimulating gene therapy vectors may be a tool for second line treatment of imatinib-resistant chronic myelogenous leukemia (CML). Unfortunately, for gene transfer into CML cells, most current vector systems either lack sufficient transduction efficiency or an acceptable safety profile. Conventional adeno-associated virus (AAV) based vectors have an advantageous safety profile, yet lack the required efficiency. Methods Pseudotyped recombinant adeno-associated viruses of the serotypes 2/1 to 2/6 (rAAV2/1 to rAAV2/6) were screened on a panel of human CML cell lines and primary CML cells to determine their gene transfer efficacy. Additionally, double-stranded self complementary rAAV (scAAV) were used to determine possible second strand synthesis limitations. Results On CML cell lines, generally rAAV2/2 and rAAV2/6 were most efficient (Fig. 1). For both, an interesting difference in transduction efficiency between the imatinib-resistant LAMA84-R and imatinib-sensitive LAMA84-S cells were observed. On primary human CML cells, rAAV2/6 proved to be significantly more efficient than the other tested vectors (4.6% ± 5.3% GFP-positive cells, p=0.011). Additionally, the transduction efficiency could be significantly increased by using scAAV vectors (scAAV2/6: 43.1% ± 25.9% GFP-positive cells, p<0.001 vs rAAV2/6; Fig. 2). Our data suggest that both the conversion of the single stranded AAV genome to double stranded DNA, as well as cell binding/entry are rate limiting steps in efficient transduction of primary human CML cells with AAV vectors. Furthermore, donor-dependent differences in gene transfer efficiency were observed. Of note, data from experiments on CML cell lines seems to provide limited information about the transduction efficiency of rAAV vectors on primary CML cells. Conclusions Overall, pseudotyped rAAV and scAAV vectors offer efficient clinically relevant gene transfer into human CML cells with a potential for future clinical application. Disclosures: No relevant conflicts of interest to declare.
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Lu, Zhuozhuang, Yongjin Wang, Xiaohui Zou, and Tao Hung. "Analysis of Fowl Adenovirus 4 Transcriptome by De Novo ORF Prediction Based on Corrected Nanopore Full-Length cDNA Sequencing Data." Viruses 15, no. 2 (February 14, 2023): 529. http://dx.doi.org/10.3390/v15020529.
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The transcriptome of fowl adenovirus has not been comprehensively revealed. Here, we attempted to analyze the fowl adenovirus 4 (FAdV-4) transcriptome by deep sequencing. RNA samples were extracted from chicken LMH cells at 12, 18 or 26 h post-FAdV-4 infection, and subjected to Illumina strand-specific RNA-seq or nanopore full-length PCR-cDNA sequencing. After removing the reads of host cells, the data of FAdV-4 nanopore full-length cDNAs (transcripts) were corrected with reads from the Illumina RNA-seq, mapped to the viral genome and then used to predict viral open reading frames (ORFs). Other than 42 known ORFs, 39 novel ORFs were annotated to the FAdV-4 genome. Different from human adenovirus 5, one FAdV-4 ORF was often encoded by several transcripts, and more FAdV-4 ORFs were located on two exons. With these data, 18 major transcription start sites and 15 major transcription termination sites were defined, implying 18 viral promoters and 15 polyadenylation signals. The temporal cascade of viral gene transcription was observed in FAdV-4-infected cells, with six promoters possessing considerable activity in the early phase. Unexpectedly, four promoters, instead of one major late promoter, were engaged in the transcription of the viral genus-common genes on the forward strand. The clarification of the FAdV-4 transcriptome laid a solid foundation for the study of viral gene function, virulence and virus evolution, and it would help construct FAdV-4 as a gene transfer vehicle. The strategy of de novo ORF prediction could be used to parse the transcriptome of other novel adenoviruses.
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Glaser, Astrid, Bradley McColl, and Jim Vadolas. "The therapeutic potential of genome editing for β-thalassemia." F1000Research 4 (December 11, 2015): 1431. http://dx.doi.org/10.12688/f1000research.7087.1.
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The rapid advances in the field of genome editing using targeted endonucleases have called considerable attention to the potential of this technology for human gene therapy. Targeted correction of disease-causing mutations could ensure lifelong, tissue-specific expression of the relevant gene, thereby alleviating or resolving a specific disease phenotype. In this review, we aim to explore the potential of this technology for the therapy of β-thalassemia. This blood disorder is caused by mutations in the gene encoding the β-globin chain of hemoglobin, leading to severe anemia in affected patients. Curative allogeneic bone marrow transplantation is available only to a small subset of patients, leaving the majority of patients dependent on regular blood transfusions and iron chelation therapy. The transfer of gene-corrected autologous hematopoietic stem cells could provide a therapeutic alternative, as recent results from gene therapy trials using a lentiviral gene addition approach have demonstrated. Genome editing has the potential to further advance this approach as it eliminates the need for semi-randomly integrating viral vectors and their associated risk of insertional mutagenesis. In the following pages we will highlight the advantages and risks of genome editing compared to standard therapy for β-thalassemia and elaborate on lessons learned from recent gene therapy trials.
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Hauck, Bernd, Wei Zhao, Katherine High, and Weidong Xiao. "Intracellular Viral Processing, Not Single-Stranded DNA Accumulation, Is Crucial for Recombinant Adeno-Associated Virus Transduction." Journal of Virology 78, no. 24 (December 15, 2004): 13678–86. http://dx.doi.org/10.1128/jvi.78.24.13678-13686.2004.
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ABSTRACT Adeno-associated virus (AAV) is a unique gene transfer vector which takes approximately 4 to 6 weeks to reach its expression plateau. The mechanism for this slow-rise expression profile was proposed to be inefficient second-strand DNA synthesis from the input single-stranded (ss) DNA viral genome. In order to clarify the status of ss AAV genomes, we generated AAV vectors labeled with bromodeoxyuridine (BrdU), a nucleotide analog that can be incorporated into the AAV genome and packaged into infectious virions. Since BrdU-DNA can be detected only by an anti-BrdU antibody when DNA is in an ss form, not in a double-stranded (ds) form, ss AAV genomes with BrdU can be readily tracked in situ. Although ss AAV DNA was abundant by Southern blot analysis, free ss AAV genomes were not detectable after AAV transduction by this new detection method. Further Southern blot analysis of viral DNA and virions revealed that ss AAV DNA was protected within virions. Extracted cellular fractions demonstrated that viral particles in host cells remained infectious. In addition, a significant amount of AAV genomes was degraded after AAV transduction. Therefore, we conclude that the amount of free ss DNA is not abundant during AAV transduction. AAV transduction is limited by the steps that affect AAV ss DNA release (i.e., uncoating) before second-strand DNA synthesis can occur. AAV ss DNA released from viral uncoating is either converted into ds DNA efficiently or degraded by cellular DNA repair mechanisms as damaged DNA. This study elucidates a mechanism that can be exploited to develop new strategies to improve AAV vector transduction efficiency.
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Wu, Yuntao, Ge Liu, and Eric B. Carstens. "Replication, Integration, and Packaging of Plasmid DNA following Cotransfection with Baculovirus Viral DNA." Journal of Virology 73, no. 7 (July 1, 1999): 5473–80. http://dx.doi.org/10.1128/jvi.73.7.5473-5480.1999.
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ABSTRACT Infection-dependent replication assays have been used to identify numerous putative origins of baculovirus replication. However, plasmid DNA, when cotransfected into insect cells with Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) DNA, replicates independently of any viral sequence in cis (11). Cotransfection of transfer plasmids and baculovirus DNA is a common procedure used in generating recombinant viruses and in measuring the level of gene expression in transient-expression assays. We have examined the fate of a series of vector plasmids in cotransfection experiments. The data reveal that these plasmids replicate following cotransfection and the replication of plasmid DNA is not due to acquisition of viral putative origin sequences. The conformation of plasmid DNA replicating in the cotransfected cells was analyzed and found to exist as high-molecular-weight concatemers. Ten to 25% of the replicated plasmid DNA was integrated into multiple locations on the viral genome and was present in progeny virions following serial passage. Sequence analysis of plasmid-viral DNA junction sites revealed no homologous or conserved sequences in the proximity of the integration sites, suggesting that nonhomologous recombination was involved during the integration process. These data suggest that while a rolling-circle mechanism could be used for baculovirus DNA replication, recombination may also be involved in this process. Plasmid integration may generate large deletions of the viral genome, suggesting that the process of DNA replication in baculovirus may be prone to generation of defective genomes.
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Kogay, Roman, Taylor B. Neely, Daniel P. Birnbaum, Camille R. Hankel, Migun Shakya, and Olga Zhaxybayeva. "Machine-Learning Classification Suggests That Many Alphaproteobacterial Prophages May Instead Be Gene Transfer Agents." Genome Biology and Evolution 11, no. 10 (September 27, 2019): 2941–53. http://dx.doi.org/10.1093/gbe/evz206.
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Abstract Many of the sequenced bacterial and archaeal genomes encode regions of viral provenance. Yet, not all of these regions encode bona fide viruses. Gene transfer agents (GTAs) are thought to be former viruses that are now maintained in genomes of some bacteria and archaea and are hypothesized to enable exchange of DNA within bacterial populations. In Alphaproteobacteria, genes homologous to the “head–tail” gene cluster that encodes structural components of the Rhodobacter capsulatus GTA (RcGTA) are found in many taxa, even if they are only distantly related to Rhodobacter capsulatus. Yet, in most genomes available in GenBank RcGTA-like genes have annotations of typical viral proteins, and therefore are not easily distinguished from their viral homologs without additional analyses. Here, we report a “support vector machine” classifier that quickly and accurately distinguishes RcGTA-like genes from their viral homologs by capturing the differences in the amino acid composition of the encoded proteins. Our open-source classifier is implemented in Python and can be used to scan homologs of the RcGTA genes in newly sequenced genomes. The classifier can also be trained to identify other types of GTAs, or even to detect other elements of viral ancestry. Using the classifier trained on a manually curated set of homologous viruses and GTAs, we detected RcGTA-like “head–tail” gene clusters in 57.5% of the 1,423 examined alphaproteobacterial genomes. We also demonstrated that more than half of the in silico prophage predictions are instead likely to be GTAs, suggesting that in many alphaproteobacterial genomes the RcGTA-like elements remain unrecognized.
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Cheslock, Sara Rasmussen, Jeffrey A. Anderson, Carey K. Hwang, Vinay K. Pathak, and Wei-Shau Hu. "Utilization of Nonviral Sequences for Minus-Strand DNA Transfer and Gene Reconstitution during Retroviral Replication." Journal of Virology 74, no. 20 (October 15, 2000): 9571–79. http://dx.doi.org/10.1128/jvi.74.20.9571-9579.2000.
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ABSTRACT Minus-strand DNA transfer, an essential step in retroviral reverse transcription, is mediated by the two repeat (R) regions in the viral genome. It is unclear whether R simply serves as a homologous sequence to mediate the strand transfer or contains specific sequences to promote strand transfer. To test the hypothesis that the molecular mechanism by which R mediates strand transfer is based on homology rather than specific sequences, we examined whether nonviral sequences can be used to facilitate minus-strand DNA transfer. The green fluorescent protein (GFP) gene was divided into GF and FP fragments, containing the 5′ and 3′ portions of GFP, respectively, with an overlapping F fragment (85 bp). FP and GF were inserted into the 5′ and 3′ long terminal repeats, respectively, of a murine leukemia virus-based vector. Utilization of the F fragment to mediate minus-strand DNA transfer should reconstitute GFP during reverse transcription. Flow cytometry analyses demonstrated that GFP was expressed in 73 to 92% of the infected cells, depending on the structure of the viral construct. This indicated that GFP was reconstituted at a high frequency; molecular characterization further confirmed the accurate reconstitution of GFP. These data indicated that nonviral sequences could be used to efficiently mediate minus-strand DNA transfer. Therefore, placement and homology, not specific sequence context, are the important elements in R for minus-strand DNA transfer. In addition, these experiments demonstrate that minus-strand DNA transfer can be used to efficiently reconstitute genes for gene therapy applications.
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Mu, Fan, Bo Li, Shufen Cheng, Jichun Jia, Daohong Jiang, Yanping Fu, Jiasen Cheng, Yang Lin, Tao Chen, and Jiatao Xie. "Nine viruses from eight lineages exhibiting new evolutionary modes that co-infect a hypovirulent phytopathogenic fungus." PLOS Pathogens 17, no. 8 (August 24, 2021): e1009823. http://dx.doi.org/10.1371/journal.ppat.1009823.
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Mycoviruses are an important component of the virosphere, but our current knowledge of their genome organization diversity and evolution remains rudimentary. In this study, the mycovirus composition in a hypovirulent strain of Sclerotinia sclerotiorum was molecularly characterized. Nine mycoviruses were identified and assigned into eight potential families. Of them, six were close relatives of known mycoviruses, while the other three had unique genome organizations and evolutionary positions. A deltaflexivirus with a tripartite genome has evolved via arrangement and horizontal gene transfer events, which could be an evolutionary connection from unsegmented to segmented RNA viruses. Two mycoviruses had acquired a second helicase gene by two different evolutionary mechanisms. A rhabdovirus representing an independent viral evolutionary branch was the first to be confirmed to occur naturally in fungi. The major hypovirulence-associated factor, an endornavirus, was finally corroborated. Our study expands the diversity of mycoviruses and potential virocontrol agents, and also provides new insights into virus evolutionary modes including virus genome segmentation.
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Kommareddy, Sushma, Sandip B. Tiwari, and Mansoor M. Amiji. "Long-Circulating Polymeric Nanovectors for Tumor-Selective Gene Delivery." Technology in Cancer Research & Treatment 4, no. 6 (December 2005): 615–25. http://dx.doi.org/10.1177/153303460500400605.
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Significant advances in the understanding of the genetic abnormalities that lead to the development, progression, and metastasis of neoplastic diseases has raised the promise of gene therapy as an approach to medical intervention. Most of the clinical protocols that have been approved in the United States for gene therapy have used the viral vectors because of the high efficiency of gene transfer. Conventional means of gene delivery using viral vectors, however, has undesirable side effects such as insertion of mutational viral gene into the host genome and development of replication competent viruses. Among non-viral gene delivery methods, polymeric nanoparticles are increasingly becoming popular as vectors of choice. The major limitation of these nanoparticles is poor transfection efficiency at the target site after systemic administration due to uptake by the cells of reticuloendothelial system (RES). In order to reduce the uptake by the cells of the RES and improve blood circulation time, these nanoparticles are coated with hydrophilic polymers such as poly(ethylene glycol) (PEG). This article reviews the use of such hydrophilic polymers employed for improving the circulation time of the nanocarriers. The mechanism of polymer coating and factors affecting the circulation time of these nanocarriers will be discussed. In addition to the long circulating property, modifications to improve the target specificity of the particles and the limitations of steric protection will be analyzed.
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Wersto, Robert P., Eugene R. Rosenthal, Prem K. Seth, N. Tony Eissa, and Robert E. Donahue. "Recombinant, Replication-Defective Adenovirus Gene Transfer Vectors Induce Cell Cycle Dysregulation and Inappropriate Expression of Cyclin Proteins." Journal of Virology 72, no. 12 (December 1, 1998): 9491–502. http://dx.doi.org/10.1128/jvi.72.12.9491-9502.1998.
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ABSTRACT First-generation adenovirus (Ad) vectors that had been rendered replication defective by removal of the E1 region of the viral genome (ΔE1) or lacking the Ad E3 region in addition to E1 sequences (ΔE1ΔE3) induced G2 cell cycle arrest and inhibited traverse across G1/S in primary and immortalized human bronchial epithelial cells. Cell cycle arrest was independent of the cDNA contained in the expression cassette and was associated with the inappropriate expression and increase in cyclin A, cyclin B1, cyclin D, and cyclin-dependent kinase p34 cdc2 protein levels. In some instances, infection with ΔE1 or ΔE1ΔE3 Ad vectors produced aneuploid DNA histogram patterns and induced polyploidization as a result of successive rounds of cell division without mitosis. Cell cycle arrest was absent in cells infected with a second-generation ΔE1Ad vector in which all of the early region E4 except the sixth open reading frame was also deleted. Consequently, E4 viral gene products present in ΔE1 or ΔE1ΔE3 Ad vectors induce G2 growth arrest, which may pose new and unintended consequences for human gene transfer and gene therapy.
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Shannon-Lowe, Claire, Emily Adland, Andrew I. Bell, Henri-Jacques Delecluse, Alan B. Rickinson, and Martin Rowe. "Features Distinguishing Epstein-Barr Virus Infections of Epithelial Cells and B Cells: Viral Genome Expression, Genome Maintenance, and Genome Amplification." Journal of Virology 83, no. 15 (May 13, 2009): 7749–60. http://dx.doi.org/10.1128/jvi.00108-09.
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ABSTRACT Epstein-Barr virus (EBV) is associated with malignant diseases of lymphoid and epithelial cell origin. The tropism of EBV is due to B-cell-restricted expression of CD21, the major receptor molecule for the virus. However, efficient infection of CD21− epithelial cells can be achieved via transfer from EBV-coated B cells. We compare and contrast here the early events following in vitro infection of primary B cells and epithelial cells. Using sensitive, quantitative reverse transcription-PCR assays for several latent and lytic transcripts and two-color immunofluorescence staining to analyze expression at the single cell level, we confirmed and extended previous reports indicating that the two cell types support different patterns of transcription. Furthermore, whereas infection of B cells with one or two copies of EBV resulted in rapid amplification of the viral genome to >20 copies per cell, such amplification was not normally observed after infection of primary epithelial cells or undifferentiated epithelial lines. In epithelial cells, EBNA1 expression was detected in only ca. 40% of EBER+ cells, and the EBV genome was subsequently lost during prolonged culture. One exception was that infection of AGS, a gastric carcinoma line, resulted in maintenance of EBNA1 expression and amplification of the EBV episome. In contrast to B cells, where amplification of the EBV episome occurred even with a replication-defective BZLF1-knockout virus, amplification in AGS cells was dependent upon early lytic cycle gene expression. These data highlight the influence of the host cell on the outcome of EBV infection with regard to genome expression, amplification, and maintenance.
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Smith, Steven J., Andrea Ferris, Xuezhi Zhao, Gary Pauly, Joel P. Schneider, Terrence R. Burke, and Stephen H. Hughes. "INSTIs and NNRTIs Potently Inhibit HIV-1 Polypurine Tract Mutants in a Single Round Infection Assay." Viruses 13, no. 12 (December 14, 2021): 2501. http://dx.doi.org/10.3390/v13122501.
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Integrase strand transfer inhibitors (INSTIs) are a class of antiretroviral compounds that prevent the insertion of a DNA copy of the viral genome into the host genome by targeting the viral enzyme integrase (IN). Dolutegravir (DTG) is a leading INSTI that is given, usually in combination with nucleoside reverse transcriptase inhibitors (NRTIs), to treat HIV-1 infections. The emergence of resistance to DTG and other leading INSTIs is rare. However, there are recent reports suggesting that drug resistance mutations can occur at positions outside the integrase gene either in the HIV-1 polypurine tract (PPT) or in the envelope gene (env). Here, we used single round infectivity assays to measure the antiviral potencies of several FDA-approved INSTIs and non-nucleoside reverse transcriptase inhibitors (NNRTIs) against a panel of HIV-1 PPT mutants. We also tested several of our promising INSTIs and NNRTIs in these assays. No measurable loss in potency was observed for either INSTIs or NNRTIs against the HIV-1 PPT mutants. This suggests that HIV-1 PPT mutants are not able, by themselves, to confer resistance to INSTIs or NNRTIs.
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Davidoff, Andrew M., Catherine Y. C. Ng, Junfang Zhou, Yunyu Spence, and Amit C. Nathwani. "Sex significantly influences transduction of murine liver by recombinant adeno-associated viral vectors through an androgen-dependent pathway." Blood 102, no. 2 (July 15, 2003): 480–88. http://dx.doi.org/10.1182/blood-2002-09-2889.
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AbstractA systematic evaluation of the influence of sex on transduction by recombinant adeno-associated viral vector (rAAV) indicated that transgene expression after liver-targeted delivery of vector particles was between 5- to 13-fold higher in male mice compared with female mice, irrespective of the proviral promoter or cDNA and mouse strain. Molecular analysis revealed that the rAAV genome was stably retained in male liver at levels that were 7-fold higher than those observed in females. Further, the sex difference in transduction was observed with AAV-2– and AAV-5–based vectors, which use distinct receptor complexes for infection. In concordance with the differences in AAV transduction, gel shift analysis with nuclear extracts derived from the liver of mice and humans revealed substantially higher binding of host nuclear protein to the rep-binding site (RBS) of AAV inverted terminal repeat (ITR) in males compared with females. Transduction efficiency and binding of nuclear protein to RBS was dramatically reduced in male mice by castration. In contrast, although oophorectomy did not significantly influence rAAV transduction, administration of 5α dihydrotestosterone, prior to gene transfer, increased stable hepatocyte gene transfer in females to levels observed in male mice, implying that androgens significantly influence hepatocyte gene transfer. Interestingly, sex did not have a significant effect on AAV gene transfer into nonhepatic tissue, indicating that there are distinct tissue- and sex-specific differences in the mechanisms responsible for efficient transduction with this vector. These results have significant implications for gene therapy of autosomal and acquired disorders affecting the liver.
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Cassany, Aurélia, Jessica Ragues, Tinglu Guan, Dominique Bégu, Harald Wodrich, Michael Kann, Glen R. Nemerow, and Larry Gerace. "Nuclear Import of Adenovirus DNA Involves Direct Interaction of Hexon with an N-Terminal Domain of the Nucleoporin Nup214." Journal of Virology 89, no. 3 (November 19, 2014): 1719–30. http://dx.doi.org/10.1128/jvi.02639-14.
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ABSTRACTIn this study, we characterized the molecular basis for binding of adenovirus (AdV) to the cytoplasmic face of the nuclear pore complex (NPC), a key step during delivery of the viral genome into the nucleus. We used RNA interference (RNAi) to deplete cells of either Nup214 or Nup358, the two major Phe-Gly (FG) repeat nucleoporins localized on the cytoplasmic side of the NPC, and evaluated the impact on hexon binding and AdV infection. The accumulation of purified hexon trimers or partially disassembled AdV at the nuclear envelope (NE) was observed in digitonin-permeabilized cells in the absence of cytosolic factors. Bothin vitrohexon binding andin vivonuclear import of the AdV genome were strongly reduced in Nup214-depleted cells but still occurred in Nup358-depleted cells, suggesting that Nup214 is a major binding site of AdV during infection. The expression of an NPC-targeted N-terminal domain of Nup214 in Nup214-depleted cells restored the binding of hexon at the NE and the nuclear import of protein VII (pVII), indicating that this region is sufficient to allow AdV binding. We further narrowed the binding site to a 137-amino-acid segment in the N-terminal domain of Nup214. Together, our results have identified a specific region within the N terminus of Nup214 that acts as a direct NPC binding site for AdV.IMPORTANCEAdVs, which have the largest genome of nonenveloped DNA viruses, are being extensively explored for use in gene therapy, especially in alternative treatments for cancers that are refractory to traditional therapies. In this study, we characterized the molecular basis for binding of AdV to the cytoplasmic face of the NPC, a key step for delivery of the viral genome into the nucleus. Our data indicate that a 137-amino-acid region of the nucleoporin Nup214 is a binding site for the major AdV capsid protein, hexon, and that this interaction is required for viral DNA import. These findings provide additional insight on how AdV exploits the nuclear transport machinery for infection. The results could promote the development of new strategies for gene transfer and enhance understanding of the nuclear import of other viral DNA genomes, such as those of papillomavirus or hepatitis B virus that induce specific cancers.
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Tucker, Jessica M., Aaron M. Schaller, Ian Willis, and Britt A. Glaunsinger. "Alteration of the Premature tRNA Landscape by Gammaherpesvirus Infection." mBio 11, no. 6 (December 15, 2020): e02664-20. http://dx.doi.org/10.1128/mbio.02664-20.
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ABSTRACTTransfer RNAs (tRNAs) are transcribed by RNA polymerase III (RNAPIII) and play a central role in decoding our genome, yet their expression and noncanonical function remain understudied. Many DNA tumor viruses enhance the activity of RNAPIII, yet whether infection alters tRNA expression is largely unknown. Here, we present the first genome-wide analysis of how viral infection alters the tRNAome. Using a tRNA-specific sequencing method (DM-tRNA-seq), we find that the murine gammaherpesvirus MHV68 induces global changes in premature tRNA (pre-tRNA) expression, with 14% of tRNA genes upregulated more than 3-fold, indicating that differential tRNA gene induction is a characteristic of DNA virus infection. Elevated pre-tRNA expression corresponds to increased RNAPIII occupancy for the subset of tRNA genes tested; additionally, posttranscriptional mechanisms contribute to the accumulation of pre-tRNA species. We find increased abundance of tRNA fragments derived from pre-tRNAs upregulated by viral infection, suggesting that noncanonical tRNA cleavage is also affected. Furthermore, pre-tRNA accumulation, but not RNAPIII recruitment, requires gammaherpesvirus-induced degradation of host mRNAs by the virally encoded mRNA endonuclease muSOX. We hypothesize that depletion of pre-tRNA maturation or turnover machinery contributes to robust accumulation of full-length pre-tRNAs in infected cells. Collectively, these findings reveal pervasive changes to tRNA expression during DNA virus infection and highlight the potential of using viruses to explore tRNA biology.IMPORTANCE Viral infection can dramatically change the gene expression landscape of the host cell, yet little is known regarding changes in noncoding gene transcription by RNA polymerase III (RNAPIII). Among these are transfer RNAs (tRNAs), which are fundamental in protein translation, yet whose gene regulatory features remain largely undefined in mammalian cells. Here, we perform the first genome-wide analysis of tRNA expression changes during viral infection. We show that premature tRNAs accumulate during infection with the model gammaherpesvirus MHV68 as a consequence of increased transcription, but that transcripts do not undergo canonical maturation into mature tRNAs. These findings underscore how tRNA expression is a highly regulated process, especially during conditions of elevated RNAPIII activity.
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Tallóczy, Zsolt, Rebecca Mazar, Denise E. Georgopoulos, Fausto Ramos, and Michael J. Leibowitz. "The [KIL-d] Element Specifically Regulates Viral Gene Expression in Yeast." Genetics 155, no. 2 (June 1, 2000): 601–9. http://dx.doi.org/10.1093/genetics/155.2.601.
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Abstract The cytoplasmically inherited [KIL-d] element epigenetically regulates killer virus gene expression in Saccharomyces cerevisiae. [KIL-d] results in variegated defects in expression of the M double-stranded RNA viral segment in haploid cells that are “healed” in diploids. We report that the [KIL-d] element is spontaneously lost with a frequency of 10−4–10−5 and reappears with variegated phenotypic expression with a frequency of ≥10−3. This high rate of loss and higher rate of reappearance is unlike any known nucleic acid replicon but resembles the behavior of yeast prions. However, [KIL-d] is distinct from the known yeast prions in its relative guanidinium hydrochloride incurability and independence of Hsp104 protein for its maintenance. Despite its transmissibility by successive cytoplasmic transfers, multiple cytoplasmic nucleic acids have been proven not to carry the [KIL-d] trait. [KIL-d] epigenetically regulates the expression of the M double-stranded RNA satellite virus genome, but fails to alter the expression of M cDNA. This specificity remained even after a cycle of mating and meiosis. Due to its unique genetic properties and viral RNA specificity, [KIL-d] represents a new type of genetic element that interacts with a viral RNA genome.
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Sarin, L. Peter. "Learning from the Invaders: What Viruses Teach Us about RNA-Based Regulation in Microbes." Microorganisms 10, no. 11 (October 25, 2022): 2106. http://dx.doi.org/10.3390/microorganisms10112106.
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Viruses feature an evolutionary shaped minimal genome that is obligately dependent on the cellular transcription and translation machinery for propagation. To suppress host cell immune responses and ensure efficient replication, viruses employ numerous tactics to favor viral gene expression and protein synthesis. This necessitates a carefully balanced network of virus- and host-encoded components, of which the RNA-based regulatory mechanisms have emerged as particularly interesting albeit insufficiently studied, especially in unicellular organisms such as archaea, bacteria, and yeasts. Here, recent advances that further our understanding of RNA-based translation regulation, mainly through post-transcriptional chemical modification of ribonucleosides, codon usage, and (virus-encoded) transfer RNAs, will be discussed in the context of viral infection.
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Goodman, S., X. Xiao, RE Donahue, A. Moulton, J. Miller, C. Walsh, NS Young, RJ Samulski, and AW Nienhuis. "Recombinant adeno-associated virus-mediated gene transfer into hematopoietic progenitor cells [published erratum appears in Blood 1995 Feb 1;85(3):862]." Blood 84, no. 5 (September 1, 1994): 1492–500. http://dx.doi.org/10.1182/blood.v84.5.1492.1492.
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Abstract Recombinant adeno-associated viruses (rAAV) containing only the inverted terminal repeats (ITR) from the wild-type virus are capable of stable integration into the host cell genome, and expression of inserted genes in cultured cells. We have now defined the ability of rAAV to introduce genes into primary hematopoietic progenitors. A vector was constructed containing the coding sequences for beta- galactosidase (beta-gal), including a nuclear localization signal, under the control of a strong viral promotor. Infectious vector particles were prepared by cotransfection of the vector plasmid with a second plasmid that contained the coding sequences for AAV proteins into adenovirus-infected human embryonic kidney cells. These vector preparations transferred and expressed the beta-gal gene in human K562 erythroleukemia and Detroit 6 cells. Positive immunoselection yielded a population of enriched CD34+ cells that were transduced with the rAAV beta-gal vector. Nuclear localized enzyme expression was documented in 60% to 70% of infected cells. Progenitor-derived colonies that developed after 2 weeks in clonogenic cultures were shown to have viral- associated DNA at an estimated copy number of 1 to 2 per cell using a semiquantitative polymerase chain reaction (PCR) method. Integration of AAV into hematopoietic progenitors was documented using wild-type virus, as its genome may integrate at a preferred site on chromosome 19. Our data suggest that rAAV will transfer and express genes in primitive hematopoietic progenitors with high frequency, and support the development of this vector system for therapeutic gene transfer.
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Goodman, S., X. Xiao, RE Donahue, A. Moulton, J. Miller, C. Walsh, NS Young, RJ Samulski, and AW Nienhuis. "Recombinant adeno-associated virus-mediated gene transfer into hematopoietic progenitor cells [published erratum appears in Blood 1995 Feb 1;85(3):862]." Blood 84, no. 5 (September 1, 1994): 1492–500. http://dx.doi.org/10.1182/blood.v84.5.1492.bloodjournal8451492.
Full textAbstract:
Recombinant adeno-associated viruses (rAAV) containing only the inverted terminal repeats (ITR) from the wild-type virus are capable of stable integration into the host cell genome, and expression of inserted genes in cultured cells. We have now defined the ability of rAAV to introduce genes into primary hematopoietic progenitors. A vector was constructed containing the coding sequences for beta- galactosidase (beta-gal), including a nuclear localization signal, under the control of a strong viral promotor. Infectious vector particles were prepared by cotransfection of the vector plasmid with a second plasmid that contained the coding sequences for AAV proteins into adenovirus-infected human embryonic kidney cells. These vector preparations transferred and expressed the beta-gal gene in human K562 erythroleukemia and Detroit 6 cells. Positive immunoselection yielded a population of enriched CD34+ cells that were transduced with the rAAV beta-gal vector. Nuclear localized enzyme expression was documented in 60% to 70% of infected cells. Progenitor-derived colonies that developed after 2 weeks in clonogenic cultures were shown to have viral- associated DNA at an estimated copy number of 1 to 2 per cell using a semiquantitative polymerase chain reaction (PCR) method. Integration of AAV into hematopoietic progenitors was documented using wild-type virus, as its genome may integrate at a preferred site on chromosome 19. Our data suggest that rAAV will transfer and express genes in primitive hematopoietic progenitors with high frequency, and support the development of this vector system for therapeutic gene transfer.
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Martino, Ashley T., Masataka Suzuki, David M. Markusic, Irene Zolotukhin, Renee C. Ryals, Babak Moghimi, Hildegund C. J. Ertl, Daniel A. Muruve, Brendan Lee, and Roland W. Herzog. "The genome of self-complementary adeno-associated viral vectors increases Toll-like receptor 9–dependent innate immune responses in the liver." Blood 117, no. 24 (June 16, 2011): 6459–68. http://dx.doi.org/10.1182/blood-2010-10-314518.
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AbstractAlthough adeno-associated viral (AAV) vectors have been successfully used in hepatic gene transfer for treatment of hemophilia and other diseases in animals, adaptive immune responses blocked long-term transgene expression in patients on administration of single-stranded AAV serotype-2 vector. More efficient vectors have been developed using alternate capsids and self-complimentary (sc) genomes. This study investigated their effects on the innate immune profile on hepatic gene transfer to mice. A mild and transient up-regulation of myeloid differentiation primary response gene (88), TLR9, TNF-α, monocyte chemotactic protein-1, IFN-γ inducible protein-10, and IFN-α/β expression in the liver was found after single-stranded AAV vector administration, regardless of the capsid sequence. In contrast, scAAV vectors induced higher increases of these transcripts, upregulated additional proinflammatory genes, and increased circulating IL-6. Neutrophil, macrophage, and natural killer cell liver infiltrates were substantially higher on injection of scAAV. Some but not all of these responses were Kupffer cell dependent. Independent of the capsid or expression cassette, scAAV vectors induced dose-dependent innate responses by signaling through TLR9. Increased innate responses to scAAV correlated with stronger adaptive immune responses against capsid (but not against the transgene product). However, these could be blunted by transient inhibition of TLR9.
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Seckert, Christof K., Angélique Renzaho, Hanna-Mari Tervo, Claudia Krause, Petra Deegen, Birgit Kühnapfel, Matthias J. Reddehase, and Natascha K. A. Grzimek. "Liver Sinusoidal Endothelial Cells Are a Site of Murine Cytomegalovirus Latency and Reactivation." Journal of Virology 83, no. 17 (June 17, 2009): 8869–84. http://dx.doi.org/10.1128/jvi.00870-09.
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ABSTRACT Latent cytomegalovirus (CMV) is frequently transmitted by organ transplantation, and its reactivation under conditions of immunosuppressive prophylaxis against graft rejection by host-versus-graft disease bears a risk of graft failure due to viral pathogenesis. CMV is the most common cause of infection following liver transplantation. Although hematopoietic cells of the myeloid lineage are a recognized source of latent CMV, the cellular sites of latency in the liver are not comprehensively typed. Here we have used the BALB/c mouse model of murine CMV infection to identify latently infected hepatic cell types. We performed sex-mismatched bone marrow transplantation with male donors and female recipients to generate latently infected sex chromosome chimeras, allowing us to distinguish between Y-chromosome (gene sry or tdy)-positive donor-derived hematopoietic descendants and Y-chromosome-negative cells of recipients' tissues. The viral genome was found to localize primarily to sry-negative CD11b− CD11c− CD31+ CD146+ cells lacking major histocompatibility complex class II antigen (MHC-II) but expressing murine L-SIGN. This cell surface phenotype is typical of liver sinusoidal endothelial cells (LSECs). Notably, sry-positive CD146+ cells were distinguished by the expression of MHC-II and did not harbor latent viral DNA. In this model, the frequency of latently infected cells was found to be 1 to 2 per 104 LSECs, with an average copy number of 9 (range, 4 to 17) viral genomes. Ex vivo-isolated, latently infected LSECs expressed the viral genes m123/ie1 and M122/ie3 but not M11 2-M 1 13/e1, M55/gB, or M86/MCP. Importantly, in an LSEC transfer model, infectious virus reactivated from recipients' tissue explants with an incidence of one reactivation per 1,000 viral-genome-carrying LSECs. These findings identified LSECs as the main cellular site of murine CMV latency and reactivation in the liver.
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Dedeo, Corynne L., Carolyn M. Teschke, and Andrei T. Alexandrescu. "Keeping It Together: Structures, Functions, and Applications of Viral Decoration Proteins." Viruses 12, no. 10 (October 14, 2020): 1163. http://dx.doi.org/10.3390/v12101163.
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Decoration proteins are viral accessory gene products that adorn the surfaces of some phages and viral capsids, particularly tailed dsDNA phages. These proteins often play a “cementing” role, reinforcing capsids against accumulating internal pressure due to genome packaging, or environmental insults such as extremes of temperature or pH. Many decoration proteins serve alternative functions, including target cell recognition, participation in viral assembly, capsid size determination, or modulation of host gene expression. Examples that currently have structures characterized to high-resolution fall into five main folding motifs: β-tulip, β-tadpole, OB-fold, Ig-like, and a rare knotted α-helical fold. Most of these folding motifs have structure homologs in virus and target cell proteins, suggesting horizontal gene transfer was important in their evolution. Oligomerization states of decoration proteins range from monomers to trimers, with the latter most typical. Decoration proteins bind to a variety of loci on capsids that include icosahedral 2-, 3-, and 5-fold symmetry axes, as well as pseudo-symmetry sites. These binding sites often correspond to “weak points” on the capsid lattice. Because of their unique abilities to bind virus surfaces noncovalently, decoration proteins are increasingly exploited for technology, with uses including phage display, viral functionalization, vaccination, and improved nanoparticle design for imaging and drug delivery. These applications will undoubtedly benefit from further advances in our understanding of these versatile augmenters of viral functions.
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Kirsip, Heleri, and Aare Abroi. "Protein Structure-Guided Hidden Markov Models (HMMs) as A Powerful Method in the Detection of Ancestral Endogenous Viral Elements." Viruses 11, no. 4 (April 2, 2019): 320. http://dx.doi.org/10.3390/v11040320.
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It has been believed for a long time that the transfer and fixation of genetic material from RNA viruses to eukaryote genomes is very unlikely. However, during the last decade, there have been several cases in which “virus-to-host” gene transfer from various viral families into various eukaryotic phyla have been described. These transfers have been identified by sequence similarity, which may disappear very quickly, especially in the case of RNA viruses. However, compared to sequences, protein structure is known to be more conserved. Applying protein structure-guided protein domain-specific Hidden Markov Models, we detected homologues of the Virgaviridae capsid protein in Schizophora flies. Further data analysis supported “virus-to-host” transfer into Schizophora ancestors as a single transfer event. This transfer was not identifiable by BLAST or by other methods we applied. Our data show that structure-guided Hidden Markov Models should be used to detect ancestral virus-to-host transfers.
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Hildreth, Andrew D., Luke Riggan, Marion Rolot, Yung-Yu Wong, Will Satyadi, Ryan Sun, Chris Huerta, and Timothy E. O’Sullivan. "CRISPR Cas9 ribonucleoprotein-mediated genomic editing in primary innate immune cells." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 159.3. http://dx.doi.org/10.4049/jimmunol.204.supp.159.3.
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Abstract CRISPR (clustered, regularly interspaced, short palindromic repeats) genome engineering has become a powerful tool to functionally investigate complex mechanisms of immune system regulation. While decades of work have aimed to genetically reprogram innate immunity for therapeutic purposes, current approaches are often inefficient or nonspecific, limiting their use in primary innate immune cells. Here, we describe an optimized strategy for non-viral CRISPR-Cas9 ribonucleoprotein (cRNP) genomic editing of primary innate lymphocytes (ILCs) and myeloid lineage cells that results in an almost complete loss of single or double target gene expression from a single electroporation. Furthermore, we describe in vivo adoptive transfer mouse models that can be utilized to screen for gene function during viral infection using cRNP-edited naïve NK cells and bone marrow-derived dendritic cell precursors. Using these methods, we demonstrate that XCR1+ conventional dendritic cells (cDC1) are sufficient and necessary for host protection to mouse cytomegalovirus (MCMV) in a MyD88-intrinsic manner. This scalable method will enhance target gene discovery and offer a specific and simplified approach to gene editing in the innate immune system.
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Pichon, Apolline, Annie Bézier, Serge Urbach, Jean-Marc Aury, Véronique Jouan, Marc Ravallec, Julie Guy, et al. "Recurrent DNA virus domestication leading to different parasite virulence strategies." Science Advances 1, no. 10 (November 2015): e1501150. http://dx.doi.org/10.1126/sciadv.1501150.
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Relics of ancient infections are abundant in eukaryote genomes, but little is known about how they evolve when they confer a functional benefit on their host. We show here, for the first time, that the virus-like particles shown to protect Venturia canescens eggs against host immunity are derived from a nudivirus genome incorporated by the parasitic wasp into its own genetic material. Nudivirus hijacking was also at the origin of protective particles from braconid wasps. However, we show here that the viral genes produce “liposomes” that wrap and deliver V. canescens virulence proteins, whereas the particles are used as gene transfer agents in braconid wasps. Our findings indicate that virus domestication has occurred repeatedly during parasitic wasp evolution but with different evolutionary trajectories after endogenization, resulting in different virulence molecule delivery strategies.
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VILLANI, Guglielmo R. D., Antonia FOLLENZI, Borghina VANACORE, Carmela di DOMENICO, Luigi NALDINI, and Paola di NATALE. "Correction of mucopolysaccharidosis type IIIb fibroblasts by lentiviral vector-mediated gene transfer." Biochemical Journal 364, no. 3 (June 15, 2002): 747–53. http://dx.doi.org/10.1042/bj20011872.
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Mucopolysaccharidosis type IIIB (MPS IIIB; or Sanfilippo syndrome type B) is a lysosomal disease, due to glycosaminoglycan storage caused by mutations on the α-N-acetylglucosaminidase (NAGLU) gene. The disease is characterized by neurological dysfunction but relatively mild somatic manifestations. No effective treatment is available for affected patients. In the present study, we evaluated the role of a lentiviral vector as the transducing agent of NAGLU cDNA in MPS IIIB fibroblasts. The vector expressed high transduction efficiency and high levels of enzymic activity, 20-fold above normal levels, persisting for at least 2 months. PCR experiments confirmed the integration of the viral vector into the target genome. The NAGLU activity restored by virus infection was sufficient to normalize glycosaminoglycan accumulation, which is directly responsible for the disease phenotype. Metabolic labelling experiments on transduced fibroblasts exhibited, in the medium and in cellular lysates, polypeptide forms of 84 and 80kDa respectively related to the precursor and mature forms of the enzyme. The enzyme secreted by transduced MPS IIIB fibroblasts was endocytosed in deficient cells by the mannose 6-phosphate system. Thus we show that lentiviral vectors may provide a therapeutic approach for the treatment of MPS IIIB disease.
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Mikkola, Hanna, Niels-Bjarne Woods, Marketa Sjögren, Hildur Helgadottir, Isao Hamaguchi, Sten-Eirik Jacobsen, Didier Trono, and Stefan Karlsson. "Lentivirus Gene Transfer in Murine Hematopoietic Progenitor Cells Is Compromised by a Delay in Proviral Integration and Results in Transduction Mosaicism and Heterogeneous Gene Expression in Progeny Cells." Journal of Virology 74, no. 24 (December 15, 2000): 11911–18. http://dx.doi.org/10.1128/jvi.74.24.11911-11918.2000.
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ABSTRACT Human immunodeficiency virus type 1-based lentivirus vectors containing the green fluorescent protein (GFP) gene were used to transduce murine Lin− c-kit+ Sca1+primitive hematopoietic progenitor cells. Following transduction, the cells were plated into hematopoietic progenitor cell assays in methylcellulose and the colonies were scored for GFP positivity. After incubation for 20 h, lentivirus vectors transduced 27.3% ± 6.7% of the colonies derived from unstimulated target cells, but transduction was more efficient when the cells were supported with stem cell factor (SCF) alone (42.0% ± 5.5%) or SCF, interleukin-3 (IL-3), and IL-6 (53.3 ± 1.8%) during transduction. The, vesicular stomatitis virus glycoprotein-pseudotyped MGIN oncoretrovirus control vector required IL-3, IL-6, and SCF for significant transduction (39.3 ± 9.4%). Interestingly, only a portion of the progeny cells within the lentivirus-transduced methylcellulose colonies expressed GFP, in contrast to the homogeneous expression in oncoretrovirus-transduced colonies. Secondary plating of the primary GFP+ lentivirus vector-transduced colonies revealed vector PCR+ GFP+ (42%), vector PCR−GFP− (46%), and vector PCR+ GFP−(13%) secondary colonies, indicating true genetic mosaicism with respect to the viral genome in the progeny cells. The degree of vector mosaicism in individual colonies could be reduced by extending the culture time after transduction and before plating into the clonal progenitor cell assay, indicating a delay in the lentiviral integration process. Furthermore, supplementation with exogenous deoxynucleoside triphosphates during transduction decreased mosaicism within the colonies. Although cytokine stimulation during transduction correlates with higher transduction efficiency, rapid cell division after transduction may result in loss of the viral genome in the progeny cells. Therefore, optimal transduction may require activation without promoting intense cell proliferation prior to vector integration.