SARS-CoV-2 Is Restricted by Zinc Finger Antiviral Protein despite Preadaptation to the Low-CpG Environment in Humans

Abstract

Recent evidence shows that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is sensitive to interferons (IFNs). However, the most effective types of IFNs and the underlying antiviral effectors remain to be defined. Here, we show that zinc finger antiviral protein (ZAP), which preferentially targets CpG dinucleotides in viral RNA sequences, restricts SARS-CoV-2. We further demonstrate that ZAP and its cofactors KHNYN and TRIM25 are expressed in human lung cells. Type I, II, and III IFNs all strongly inhibited SARS-CoV-2 and further induced ZAP expression. Comprehensive sequence analyses revealed that SARS-CoV-2 and its closest relatives from horseshoe bats showed the strongest CpG suppression among all known human and bat coronaviruses, respectively. Nevertheless, endogenous ZAP expression restricted SARS-CoV-2 replication in human lung cells, particularly upon treatment with IFN-α or IFN-γ. Both the long and the short isoforms of human ZAP reduced SARS-CoV-2 RNA expression levels, but the former did so with greater efficiency. Finally, we show that the ability to restrict SARS-CoV-2 is conserved in ZAP orthologues of the reservoir bat and potential intermediate pangolin hosts of human coronaviruses. Altogether, our results show that ZAP is an important effector of the innate response against SARS-CoV-2, although this pandemic pathogen emerged from zoonosis of a coronavirus that was preadapted to the low-CpG environment in humans. IMPORTANCE Although interferons inhibit SARS-CoV-2 and have been evaluated for treatment of coronavirus disease 2019 (COVID-19), the most effective types and antiviral effectors remain to be defined. Here, we show that IFN-γ is particularly potent in restricting SARS-CoV-2 and in inducing expression of the antiviral factor ZAP in human lung cells. Knockdown experiments revealed that endogenous ZAP significantly restricts SARS-CoV-2. We further show that CpG dinucleotides which are specifically targeted by ZAP are strongly suppressed in the SARS-CoV-2 genome and that the two closest horseshoe bat relatives of SARS-CoV-2 show the lowest genomic CpG content of all coronavirus sequences available from this reservoir host. Nonetheless, both the short and long isoforms of human ZAP reduced SARS-CoV-2 RNA levels, and this activity was conserved in horseshoe bat and pangolin ZAP orthologues. Our findings indicating that type II interferon is particularly efficient against SARS-CoV-2 and that ZAP restricts this pandemic viral pathogen might promote the development of effective immune therapies against COVID-19.

Coronavirus genomes carry the signatures of their habitats

“…Our observations are compatible with the notion that cytoplasmic ZAP can bind to CpG dinucleotides to facilitate the degradation of viral transcripts. This idea, in conjunction with our observations, is corroborated by a recent study that found ZAP targets CpG to restrict SARS-CoV-2 replication in human lung cells [17]. In contrast to ZAP, APOBEC3 enzymes are mostly expressed in immune cells such as CD4 + T cells residing in tissues [22].…”

“…To determine the nucleotide mutation patterns over time at each viral region, each aligned sequence was grouped into one of six time ranges, and the time range within each group was PLOS ONE determined as the number of days passed since the reference strain (Wuhan-Hu-1, 2019- [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31]. Note that the number of days between time intervals is unequal, because strains were grouped based on roughly equal sample size and not by equal number of days.…”

“…2 [51], 6 [41], and 14 [43] additionally studied MLV and 5 [44] additionally studied SIV. Consensus motif for AID enzyme editing was determined from a mutagenesis study of rpoB gene in Escherichia coli: 7 [81], and consensus motif for APOBEC1 enzyme editing was determined from mutagenesis study of chicken B-cell line DT40: 17 [88].…”

“…Recent studies have shown strong CpG deficiency in the SARS-CoV-2 genome in comparison to SARS-CoV and MERS-CoV, and they suggested that SARS-CoV-2 is adapted to evade the ZAP antiviral defense [ 17 , 18 ]. Indeed, endogenous ZAP activity has been shown to restrict SARS-CoV-2 replication in human lung cell lines as they express ZAP in abundance [ 17 ].…”

All About the RNA: Interferon-Stimulated Genes That Interfere With Viral RNA Processes

“…Solving the crystal structure of ZAP in complex with CG-rich RNA revealed that ZAP has a CGdinucleotide specific binding pocket and binds to ss nucleic acids (150,151). ZAP preference for CG-rich substrates could explain in part why many RNA viruses infecting mammals and other vertebrates, such as IAV and SARS-CoV-2, exhibit CG suppression (152)(153)(154)(155). ZAP can even sense CG dinucleotides within individual RNA transcripts of DNA viruses, as in the case of human cytomegalovirus (156).…”