High-Throughput, Library-Based Selection of a Murine Leukemia Virus Variant To Infect Nondividing Cells

Yu, Jianfeng; Schaffer, David V.

doi:10.1128/jvi.00615-06

Cited by 20 publications

(20 citation statements)

References 56 publications

(66 reference statements)

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“…We first constructed a large (4.3 × 10 5 ) retroviral library where a 186 amino acid polydactyl zinc finger domain ZFD1-a six zinc finger domain previously designed to recognize an 18-bp sequence (each finger binds a 3-bp sequence) that appears proximal to the γ-globin locus in the human genome (15)-was randomly inserted through the use of a transposon system (20) into likely every position of the MLV Gag and Pol proteins (Figs. 1 and 2 and Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Sequence repeats within the DNA encoding the ZFD were minimized during the gene synthesis to avoid recombination problems, and codon optimization was performed to maximize expression in human cells, while preserving the amino acid sequence. The gag-pol.ZF insertion library was constructed by replacing the kan R gene of pCLGIT Gag-Pol-kan R plasmid library, where the kan R gene was previously randomly inserted into MLV gag-pol using the Mutation Generation System kit (Finnzymes) (20), with a PCR product containing the ZFD sequence. The resulting pCLGIT Gag-Pol.ZF plasmid library thus expressed gag-pol variants with the ZFD incorporated in random positions.…”

Section: Methodsmentioning

confidence: 99%

“…The library size was 4.3 × 10 5 , estimated by colony counting after transformation of electro-competent DH10B Escherichia coli (Invitrogen). Based on the 5,214 nucleotide positions in MLV gag-pol, the library likely covered all possible insertion sites, as our prior work has indicated (20).…”

Section: Methodsmentioning

confidence: 99%

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Specific insertions of zinc finger domains into Gag-Pol yield engineered retroviral vectors with selective integration properties

Lim¹,

Klimczak²,

Yu³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Retroviral vectors offer benefits of efficient delivery and stable gene expression; however, their clinical use raises the concerns of insertional mutagenesis and potential oncogenesis due to genomic integration preferences in transcriptional start sites (TSS). We have shifted the integration preferences of retroviral vectors by generating a library of viral variants with a DNA-binding domain inserted at random positions throughout murine leukemia virus Gag-Pol, then selecting for variants that are viable and exhibit altered integration properties. We found seven permissive zinc finger domain (ZFD) insertion sites throughout Gag-Pol, including within p12, reverse transcriptase, and integrase. Comprehensive genome integration analysis showed that several ZFD insertions yielded retroviral vector variants with shifted integration patterns that did not favor TSS. Furthermore, integration site analysis revealed selective integration for numerous mutants. For example, two retroviral variants with a given ZFD at appropriate positions in Gag-Pol strikingly integrated primarily into four common sites out of 3.1 × 10 9 possible human genome locations (P = 4.6 × 10 -29 ). Our findings demonstrate that insertion of DNA-binding motifs into multiple locations in Gag-Pol can make considerable progress toward engineering safer retroviral vectors that integrate into a significantly narrowed pool of sites on human genome and overcome the preference for TSS.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Specific insertions of zinc finger domains into Gag-Pol yield engineered retroviral vectors with selective integration properties

Lim¹,

Klimczak²,

Yu³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…The p12-PM14 isolate initially reported (11) encodes Ile at position 63, and is the prototype parental p12 sequence used in these studies, except where noted. Previous studies have identified regions of p12 at the N terminus ( 5 PS) (20) and the central region (substituting 49 G) that are tolerant to insertions (21). These positions were used as landing sites for the insertion of alternative tethering domains.…”

Section: Resultsmentioning

confidence: 99%

Viral DNA tethering domains complement replication-defective mutations in the p12 protein of MuLV Gag

Schneider¹,

Brzezinski²,

Aiyer³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The p12 protein of murine leukemia virus (MuLV) group-specific antigen (Gag) is associated with the preintegration complex, and mutants of p12 (PM14) show defects in nuclear entry or retention. Here we show that p12 proteins engineered to encode peptide sequences derived from known viral tethering proteins can direct chromatin binding during the early phase of viral replication and rescue a lethal p12-PM14 mutant. Peptides studied included segments of Kaposi sarcoma herpesvirus latency-associated nuclear antigen (LANA) 1-23 , human papillomavirus 8 E2, and prototype foamy virus chromatin-binding sequences. Amino acid substitutions in Kaposi sarcoma herpesvirus LANA and prototype foamy virus chromatin-binding sequences that blocked nucleosome association failed to rescue MuLV p12-PM14. Rescue by a larger LANA peptide, LANA 1-32 , required second-site mutations that are predicted to reduce peptide binding affinity to chromosomes, suggesting that excessively high binding affinity interfered with Gag/p12 function. This is supported by confocal microscopy of chimeric p12-GFP fusion constructs showing the reverted proteins had weaker association to condensed mitotic chromosomes. Analysis of the integration-site selection of these chimeric viruses showed no significant change in integration profile compared with wild-type MuLV, suggesting release of the tethered p12 post mitosis, before viral integration. gammaretroviral vectors | retroviral integration | nuclear retention

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“…Furthermore, the three novel insertion sites have further applications in engineering targeted VSVG variants. Adaptation of this high-throughput method for other novel properties such as enhanced intracellular trafficking has generated retroviral vectors capable of infecting nondividing cells both in vitro and in vivo and thus extended the potential of retroviral vectors (152). Random insertional mutagenesis thus offers the potential to rapidly select for peptides that confer novel functions and/or enhance multiple steps of the gene delivery process.…”

Section: Random Insertional Mutagenesismentioning

confidence: 99%

Molecular Engineering of Viral Gene Delivery Vehicles

Schaffer

Koerber

Lim

2008

Annu. Rev. Biomed. Eng.

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139

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Viruses can be engineered to efficiently deliver exogenous genes, but their natural gene delivery properties often fail to meet human therapeutic needs. Therefore, engineering viral vectors with new properties, including enhanced targeting abilities and resistance to immune responses, is a growing area of research. This review discusses protein engineering approaches to generate viral vectors with novel gene delivery capabilities. Rational design of viral vectors has yielded successful advances in vitro, and to an extent in vivo. However, there is often insufficient knowledge of viral structurefunction relationships to reengineer existing functions or create new capabilities, such as virus-cell interactions, whose molecular basis is distributed throughout the primary sequence of the viral proteins. Therefore, high-throughput library and directed evolution methods offer alternative approaches to engineer viral vectors with desired properties. Parallel and integrated efforts in rational and library-based design promise to aid the translation of engineered viral vectors toward the clinic.

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High-Throughput, Library-Based Selection of a Murine Leukemia Virus Variant To Infect Nondividing Cells

Cited by 20 publications

References 56 publications

Specific insertions of zinc finger domains into Gag-Pol yield engineered retroviral vectors with selective integration properties

Specific insertions of zinc finger domains into Gag-Pol yield engineered retroviral vectors with selective integration properties

Viral DNA tethering domains complement replication-defective mutations in the p12 protein of MuLV Gag

Molecular Engineering of Viral Gene Delivery Vehicles

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