Minimal impact of ZAP on lentiviral vector production and transduction efficiency

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.

Antiviral Activity of Zinc Finger Antiviral Protein (ZAP) in Different Virus Families

“…According to Zhu et al (2011) [55], both ZAP isoforms limit vesicular stomatitis virus G protein (VSV-G)-pseudotyped HIV-1 vector NL4-3-luc infection through RNA degradation mediated by the recruitment of PARN deadenylase, exosome, and decapping complex through the p72 helicase [55]. Sertkaya et al (2021) [126] found that endogenous ZAP efficiently inhibited CpG-high HIV-1 in human cells and that the overexpression of ZAP did not inhibit lentiviral vector titer [126]. Kmiec et al (2021) [25] demonstrated that ZAP-L, due to its PARP domain and CaaX, directs this isoform to vesicular structures, regulates the binding with TRIM25 and KHNYN, and is important for the antiviral activity of CpG-enriched HIV-1 [25].…”