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Minimal impact of ZAP on lentiviral vector production and transduction efficiency

Research output: Contribution to journalArticlepeer-review

Original languageEnglish
Pages (from-to)147-157
Number of pages11
JournalMolecular Therapy - Methods and Clinical Development
Volume23
DOIs
Accepted/In press24 Aug 2021
Published10 Dec 2021

Bibliographical note

Funding Information: We thank the members of the Swanson and Neil labs as well as Luis Apolonia for helpful discussions. We also thank Luis Apolonia, Caroline Goujon, Harmit Malik, and Jonathan Stoye for generously providing reagents. The following reagents were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: TZM-bl from Dr. John C. Kappes, Dr. Xiaoyun Wu, and Tranzyme and HIV-1 p24 hybridoma (183-H12-5C; cat. #1513) from Dr. Bruce Chesebro. These studies were funded by MRC Discovery Award MC/PC/15068 and a Wellcome Trust Senior Research Fellowship (WT098049AIA) to S.J.D.N. Wellcome Trust (106223/Z/14/Z) to M.H.M. Medical Research Council grant MR/M019756/1 to C.M.S. and Medical Research Council grant MR/S000844/1 to S.J.D.N. and C.M.S. MR/S000844/1 is part of the EDCTP2 Program supported by the European Union. H.S. is supported by the BBSRC Industrial CASE Partnerships (ICP) training grant (BB/P504609/1) with GSK. M.F. and A.W.S. were supported by the MRC-KCL Doctoral Training Partnership in Biomedical Sciences (MR/R50225X/1 and MR/N013700/1). D.K. is funded by Deutsche Forschungsgemeinschaft (DFG [German Research Foundation]) project number: KM 5/1-1. This work was also supported via a National Institute for Health Research Biomedical Research Centre Award to Guy's and St Thomas? NHS Foundation Trust in partnership with King's College London. Conceptualization, C.M.S.; investigation, H.S. L.H. M.F. D.K. A.W.S. and S.A.; visualization, H.S. L.H. M.F. and D.K.; writing ? original draft, C.M.S.; writing ? review & editing, H.S. L.H. M.F. D.K. A.W.S. H.W. S.J.D.N. M.H.M. C.A.V. and C.M.S.; funding acquisition, C.A.V. and C.M.S.; resources, H.P. H.W. and S.J.D.N.; supervision, M.H.M. C.A.V. and C.M.S. The authors declare no competing interests. Funding Information: We thank the members of the Swanson and Neil labs as well as Luis Apolonia for helpful discussions. We also thank Luis Apolonia, Caroline Goujon, Harmit Malik, and Jonathan Stoye for generously providing reagents. The following reagents were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: TZM-bl from Dr. John C. Kappes, Dr. Xiaoyun Wu, and Tranzyme and HIV-1 p24 hybridoma (183-H12-5C; cat. #1513) from Dr. Bruce Chesebro. These studies were funded by MRC Discovery Award MC/PC/15068 and a Wellcome Trust Senior Research Fellowship ( WT098049AIA ) to S.J.D.N., Wellcome Trust ( 106223/Z/14/Z ) to M.H.M., Medical Research Council grant MR/M019756/1 to C.M.S., and Medical Research Council grant MR/S000844/1 to S.J.D.N. and C.M.S. MR/S000844/1 is part of the EDCTP2 Program supported by the European Union . H.S. is supported by the BBSRC Industrial CASE Partnerships (ICP) training grant ( BB/P504609/1 ) with GSK . M.F. and A.W.S. were supported by the MRC-KCL Doctoral Training Partnership in Biomedical Sciences ( MR/R50225X/1 and MR/N013700/1 ). D.K. is funded by Deutsche Forschungsgemeinschaft ( DFG [German Research Foundation]) project number: KM 5/1-1 . This work was also supported via a National Institute for Health Research Biomedical Research Centre Award to Guy’s and St Thomas’ NHS Foundation Trust in partnership with King’s College London . Publisher Copyright: © 2021 The Authors

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    Uploaded date:21 Sep 2021

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    Licence:CC BY-NC-ND

King's Authors

Abstract

The antiviral protein ZAP binds CpG dinucleotides in viral RNA to inhibit replication. This has likely led to the CpG suppression observed in many RNA viruses, including retroviruses. Sequences added to retroviral vector genomes, such as internal promoters, transgenes or regulatory elements, substantially increase CpG abundance. Because these CpGs could allow retroviral vector RNA to be targeted by ZAP, we analyzed whether it restricts vector production, transduction efficiency and transgene expression. Surprisingly, even though CpG-high HIV-1 was efficiently inhibited by ZAP in HEK293T cells, depleting ZAP did not substantially increase lentiviral vector titre using several packaging and genome plasmids. ZAP overexpression also did not inhibit lentiviral vector titre. In addition, decreasing CpG abundance in a lentiviral vector genome did not increase its titre and a gammaretroviral vector derived from murine leukemia virus was not substantially restricted by ZAP. Overall, we show that the increased CpG abundance in retroviral vectors relative to the wild type retroviruses they are derived from does not intrinsically sensitize them to ZAP. Further understanding of how ZAP specifically targets transcripts to inhibit their expression may allow the development of CpG sequence contexts that efficiently recruit or evade this antiviral system.

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