Virus origin of the oat sterile dwarf disease

To observe the effects of polyoma virus DNA on the expression of the herpes simplex virus (HSV) thymidine kinase (TK) gene early after transfer into TK-deficient mouse cells and the subsequent development of stable TK-positive transformants, we constructed a series of recombinant plasmids containing the herpes simplex virus TK gene joined with various segments of the polyoma virus genome and microinjected them into the nuclei or cytoplasm of LTK-A cells (TK − , APRT − ). The frequency of nucleus-injected cells expressing TK after 1 day, measured by autoradiography of cells incubated with [ 3 H]thymidine, increased approximately 30-fold when the plasmids contained the polyoma virus origin of replication. The origin includes sequences with homology to the simian virus 40 origin of replication and adjoining sequences, including a recently defined transcription-enhancing sequence. After microinjection of a single origin-containing plasmid molecule per cell, TK expression was detected in approximately 50% of the injected cells. When a larger number of origin-containing plasmid molecules were injected per cell, all cells showed early TK activity. When the entire polyoma virus early region was present, neighboring uninjected cells became TK positive. When plasmids were injected into the cell cytoplasm, approximately 400 times as many molecules per cell were needed to cause early TK activity. The frequency of stable transformation observed 2 weeks after nuclear injection of 10 to 20 polyoma virus origin-containing plasmid molecules per cell was at least 2 orders of magnitude greater than with plasmids containing the TK gene alone. The greatest enhancement of stable TK transformation was obtained with plasmids containing the origin alone, when the maximum frequency of stable transformation was 5%. The addition of the coding regions for the small and medium T antigens or the entire early region significantly decreased TK transformation frequency in a copy-dependent fashion. The timing of stabilization of TK-positive transformation was analyzed by releasing hypoxanthine-aminopterin-thymidine selection pressure at various times after microinjection, culturing the cells in nonselective medium, and assaying for TK activity. Stabilization was found to occur between 3 and 6 days after nuclear injection. Cells injected with a plasmid containing the origin and the early region were examined for expression of the large T antigen with polyoma virus antitumor serum and immunofluorescent staining. The expression of the large T antigen was clearly associated with a cytopathic effect. TK-positive clones observed 2 weeks after injection of the plasmid were uniformly T antigen negative. Cytotoxicity may be the result of plasmid replication and toxic levels of T antigen or TK. In addition, expression of the large T antigen may block stabilization by preventing the integration of origin-containing plasmid molecules.

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Discovery of a novel geminivirus associated with camellia chlorotic dwarf disease

Archives of Virology ◽

10.1007/s00705-018-3780-3 ◽

2018 ◽

Vol 163 (6) ◽

pp. 1709-1712 ◽

Cited By ~ 13

Author(s):

Song Zhang ◽

Pan Shen ◽

Min Li ◽

Xin Tian ◽

Changyong Zhou ◽

...

Keyword(s):

Dwarf Disease

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Rationally designed chemokine-based toxin targeting the viral G protein-coupled receptor US28 potently inhibits cytomegalovirus infection in vivo

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1509392112 ◽

2015 ◽

Vol 112 (27) ◽

pp. 8427-8432 ◽

Cited By ~ 27

Author(s):

Katja Spiess ◽

Mads G. Jeppesen ◽

Mikkel Malmgaard-Clausen ◽

Karen Krzywkowski ◽

Kalpana Dulal ◽

...

Keyword(s):

G Protein ◽

Pseudomonas Exotoxin ◽

Viral Pathogen ◽

Virus Origin ◽

Ligand Interactions ◽

Toxin Protein ◽

Novel Strategy ◽

G Protein Coupled

The use of receptor–ligand interactions to direct toxins to kill diseased cells selectively has shown considerable promise for treatment of a number of cancers and, more recently, autoimmune disease. Here we move the fusion toxin protein (FTP) technology beyond cancer/autoimmune therapeutics to target the human viral pathogen, human cytomegalovirus (HCMV), on the basis of its expression of the 7TM G protein-coupled chemokine receptor US28. The virus origin of US28 provides an exceptional chemokine-binding profile with high selectivity and improved binding for the CX3C chemokine, CX3CL1. Moreover, US28 is constitutively internalizing by nature, providing highly effective FTP delivery. We designed a synthetic CX3CL1 variant engineered to have ultra-high affinity for US28 and greater specificity for US28 than the natural sole receptor for CX3CL1, CX3CR1, and we fused the synthetic variant with the cytotoxic domain of Pseudomonas Exotoxin A. This novel strategy of a rationally designed FTP provided unparalleled anti-HCMV efficacy and potency in vitro and in vivo.

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Mechanism of Rep-Mediated Adeno-Associated Virus Origin Nicking

Journal of Virology ◽

10.1128/jvi.74.17.7762-7771.2000 ◽

2000 ◽

Vol 74 (17) ◽

pp. 7762-7771 ◽

Cited By ~ 48

Author(s):

J. Rodney Brister ◽

Nicholas Muzyczka

Keyword(s):

Specific Activity ◽

Dna Helicase ◽

Loop Structure ◽

Stem Loop ◽

Adeno Associated Virus ◽

Stem Loop Structure ◽

Virus Origin ◽

Rep Proteins ◽

Sequence Elements ◽

Origin Function

ABSTRACT The single-stranded adeno-associated virus type 2 (AAV) genome is flanked by terminal repeats (TRs) that fold back on themselves to form hairpinned structures. During AAV DNA replication, the TRs are nicked by the virus-encoded Rep proteins at the terminal resolution site (trs). This origin function apparently requires three sequence elements, the Rep binding element (RBE), a small palindrome that comprises a single tip of an internal hairpin within the TR (RBE′), and the trs. Previously, we determined the sequences at the trs required for Rep-mediated cleavage and demonstrated that the trs endonuclease reaction occurs in two discrete steps. In the first step, the Rep DNA helicase activity unwinds the TR, thereby extruding a stem-loop structure at thetrs. In the second step, Rep transesterification activity cleaves the trs. Here we investigate the contribution of the RBE and RBE′ during this process. Our data indicate that Rep is tethered to the RBE in a specific orientation duringtrs nicking. This orientation appears to align Rep on the AAV TR, allowing specific nucleotide contacts with the RBE′ and directing nicking to the trs. Accordingly, alterations in the polarity or position of the RBE relative to the trsgreatly inhibit Rep nicking. Substitutions within the RBE′ also reduce Rep specific activity, but to a lesser extent. Interestingly, Rep interactions with the RBE and RBE′ during nicking seem to be functionally distinct. Rep contacts with the RBE appear necessary for both the DNA helicase and trs cleavage steps of the endonuclease reaction. On the other hand, RBE′ contacts seem to be required primarily for TR unwinding and formation of thetrs stem-loop structure, not cleavage. Together, these results suggest a model of Rep interaction with the AAV TR during origin nicking through a tripartite cleavage signal comprised of the RBE, the RBE′, and the trs.

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