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 titer using several packaging and genome plasmids. ZAP overexpression also did not inhibit lentiviral vector titer. In addition, decreasing CpG abundance in a lentiviral vector genome did not increase its titer, 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.
Highlights
Retroviral vectors are a key tool for gene delivery for a wide variety of therapeutic treatments including inherited immune or metabolic disorders and chimeric antigen receptor T cell (CAR-T cell) anticancer therapies.[1]
Endogenous ZAP in HEK293T cells does not substantially inhibit lentiviral vector production, transduction efficiency, or gene expression The role of ZAP for inhibiting retroviral vector production or gene expression has predominately been analyzed by ZAP overexpression in HEK293T cells
We sought to determine whether endogenous levels of ZAP in HEK293T cells, which are the most commonly used cell line for retroviral vector production,[49] could inhibit lentiviral vector titer
Summary
Retroviral vectors are a key tool for gene delivery for a wide variety of therapeutic treatments including inherited immune or metabolic disorders and chimeric antigen receptor T cell (CAR-T cell) anticancer therapies.[1]. Identifying cellular proteins that promote or inhibit retroviral vector production, transduction efficiency, and transgene expression is essential to optimize producer cells and identify transduction enhancers to promote efficient transgene expression in a wide range of target cell types. Retrovirus replication can be restricted by several components of the cell-autonomous innate immune system including APOBEC3 proteins, TRIM5a, tetherin/BST2, SAMHD1, IFITM proteins, MX2, and ZAP.[4] Retroviral vectors can potentially be inhibited by these proteins.[5] In producer cells, APOBEC3 proteins and tetherin could potentially restrict vector production, these proteins are not expressed in HEK293T cells.[6,7,8] In at least some types of target cells, IFITM proteins, TRIM5a, SAMHD1, and MX2 inhibit retroviral vector transduction by targeting entry, reverse transcription, or nuclear import.[9,10,11,12,13,14,15,16] Highlighting the importance of antiviral proteins in determining the susceptibility of target cells for transduction, cyclosporin H was recently identified to promote lentiviral vector transduction of human hematopoietic stem cells by inhibiting IFITM3 expression.[17]
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