Effect of Distance between Homologous Sequences and 3′ Homology on the Frequency of Retroviral Reverse Transcriptase Template Switching

Delviks, Krista A.; Pathak, Vinay Kumar

doi:10.1128/jvi.73.10.7923-7932.1999

Cited by 52 publications

(26 citation statements)

References 56 publications

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“…To enable the use of the thermomer system with viral gene delivery and genomic integration, we generated a nonhomologous variant of the TlpA G2A3 strand (nhTlpA G-2A3) in which all degenerate codons were mutagenized to synonymous triplets with minimal identity to the original sequence. This mutagenesis helps avoid template switchingmediated recombination during viral delivery of highhomology constructs 25 . The resulting open reading frame had 57.48% sequence identity to the parent sequence, with no more than 5 consecutive homologous nucleotides.…”

Section: Resultsmentioning

confidence: 99%

Modular Thermal Control of Protein Dimerization

Piraner

Shapiro

2019

Preprint

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Protein-protein interactions and protein localization are essential mechanisms of cellular signal transduction. The ability to externally control such interactions using chemical and optogenetic methods has facilitated biological research and provided components for the engineering of cell-based therapies and materials. However, chemical and optical methods are limited in their ability to provide spatiotemporal specificity in light-scattering tissues. To overcome these limitations, we present "thermomers," modular protein dimerization domains controlled with temperature -a form of energy that can be delivered to cells both globally and locally in a wide variety of in vitro and in vivo contexts. Thermomers are based on a sharply thermolabile coiled-coil protein, which we engineered to heterodimerize at a tunable transition temperature within the biocompatible range of 37-42 ºC. When fused to other proteins, thermomers can reversibly control their association, as demonstrated via membrane localization in mammalian cells. This technology enables remote control of intracellular protein-protein interactions with a form of energy that can be delivered with spatiotemporal precision in a wide range of biological, therapeutic and living material scenarios.

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Section: Resultsmentioning

confidence: 99%

Modular Thermal Control of Protein Dimerization

Piraner

Shapiro

2019

Preprint

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“…This view is based in part on the results of several studies that led to the conclusion that proximity between donor and acceptor templates contributes importantly to the frequency of sequence similarity-induced template switching. Examples include crossover hot spots for RdRps (19,29) and RTs (1,2,11,16,28). Thus, in our view, the antigenome must be nearby and possibly associated with the genome in a double-stranded or at least partially double-stranded structure at the time of the template switch.…”

Section: Discussionmentioning

confidence: 94%

5′-Proximal Hot Spot for an Inducible Positive-to-Negative-Strand Template Switch byCoronavirus RNA-Dependent RNA Polymerase

Brian

2007

J Virol

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Coronaviruses have a positive-strand RNA genome and replicate through the use of a 3 nested set of subgenomic mRNAs each possessing a leader (65 to 90 nucleotides [nt] in length, depending on the viral species) identical to and derived from the genomic leader. One widely supported model for leader acquisition states that a template switch takes place during the generation of negative-strand antileader-containing templates used subsequently for subgenomic mRNA synthesis. In this process, the switch is largely driven by canonical heptameric donor sequences at intergenic sites on the genome that match an acceptor sequence at the 3 end of the genomic leader. With experimentally placed 22-nt-long donor sequences within a bovine coronavirus defective interfering (DI) RNA we have shown that matching sites occurring anywhere within a 65-nt-wide 5-proximal genomic acceptor hot spot (nt 33 through 97) can be used for production of templates for subgenomic mRNA synthesis from the DI RNA. Here we report that with the same experimental approach, template switches can be induced in trans from an internal site in the DI RNA to the negative-strand antigenome of the helper virus. For these, a 3-proximal 89-nt acceptor hot spot on the viral antigenome (nt 35 through 123), largely complementary to that described above, was found. Molecules resulting from these switches were not templates for subgenomic mRNA synthesis but, rather, ambisense chimeras potentially exceeding the viral genome in length. The results suggest the existence of a coronavirus 5-proximal partially double-stranded template switch-facilitating structure of discrete width that contains both the viral genome and antigenome.Template switching by RNA-dependent RNA polymerases (RdRps) is a mechanism that contributes to genetic recombination and sequence diversity in RNA viruses (24). Template switching during both positive-and negative-strand RNA synthesis have been documented (20,30). Interestingly, some positive-strand RNA virus families in the Nidovirus order require a template switch for virus replication. Coronaviruses (for a leader of 65 to 90 nucleotides [nt], depending on the coronavirus species) (48) and arteriviruses (for a leader of 160 to 210 nt, depending on the arterivirus species) (41) appear to utilize a positive-to-positive-strand template switch (35, 36) during synthesis of antileader-containing negative-strand templates used in turn for the production of subgenomic mRNAs (sgmRNAs). The template switch in the end results in a leader on each sgmRNA that is identical to that on the viral genome. In toroviruses, only the largest of three sgmRNA species appears to gain an 18-nt leader in common with the genome, possibly by the same mechanism (42). The sgmRNAs for roniviruses do not share a leader sequence with the genome and therefore do not appear to use a discontinuous transcription step (10). In coronavirus-infected cells, sgmRNA-length negative strands (39) containing the antileader sequence (38) are found as components of sgmRNA-length double-stran...

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“…The repeats were seperated by intervening sequences ranging from 100 bp to 3.5 kb. The efficiency of deletion of repeats for vectors with an intervening sequence of 1.5 kb was over 90% (Delviks & Pathak 1999). For HIV, the recombination rate was 42.4%, 50.4%, and 47.4% for markers separated by distances of 1.0kb, 1.3 kb, and 1.9 kb (Rhodes et al 2003).…”

Section: Discussionmentioning

confidence: 98%

Peer Review #1 of "RRE-deleting self-inactivating and self-activating HIV-1 vectors for improved safety (v0.1)"

Presti¹

2013

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Retroviruses have been shown to efficiently delete sequences between repeats as a consequence of the template switching ability of the viral reverse transcriptase. To evaluate this approach for deriving safety-modified lentiviral vectors, we created HIV-1 vectors engineered to delete the Rev-response element (RRE) during reverse-transcription by sandwiching the RRE between two non-functional hygromycin phosphotransferase sequences. Deletion of the RRE during reverse-transcription lead to the reconstitution of a functional hygromycin phosphotransferase gene in the target cell. The efficiency of functional reconstitution, depending on vector configuration, was between 12% and 23%. Quantitative real-time PCR of genomic DNA of cells transduced with the RRE-deleting vectors that were selected selected using an independent drug resistance marker, which measured both functional and nonfunctional recombination events, indicated that the overall efficiency of RRE deletion of hygromycin phosphotransferase gene, was between 73.6% and 83.5%.

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Effect of Distance between Homologous Sequences and 3′ Homology on the Frequency of Retroviral Reverse Transcriptase Template Switching

Cited by 52 publications

References 56 publications

Modular Thermal Control of Protein Dimerization

Modular Thermal Control of Protein Dimerization

5′-Proximal Hot Spot for an Inducible Positive-to-Negative-Strand Template Switch byCoronavirus RNA-Dependent RNA Polymerase

Peer Review #1 of "RRE-deleting self-inactivating and self-activating HIV-1 vectors for improved safety (v0.1)"

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