Structure of the zinc-finger antiviral protein in complex with RNA reveals a mechanism for selective targeting of CG-rich viral sequences

Abstract

Infection of animal cells by numerous viruses is detected and countered by a variety of means, including recognition of nonself nucleic acids. The zinc finger antiviral protein (ZAP) depletes cytoplasmic RNA that is recognized as foreign in mammalian cells by virtue of its elevated CG dinucleotide content compared with endogenous mRNAs. Here, we determined a crystal structure of a protein-RNA complex containing the N-terminal, 4-zinc finger human (h) ZAP RNA-binding domain (RBD) and a CG dinucleotide-containing RNA target. The structure reveals in molecular detail how hZAP is able to bind selectively to CG-rich RNA. Specifically, the 4 zinc fingers create a basic patch on the hZAP RBD surface. The highly basic second zinc finger contains a pocket that selectively accommodates CG dinucleotide bases. Structure guided mutagenesis, cross-linking immunoprecipitation sequencing assays, and RNA affinity assays show that the structurally defined CG-binding pocket is not required for RNA binding per se in human cells. However, the pocket is a crucial determinant of high-affinity, specific binding to CG dinucleotide-containing RNA. Moreover, variations in RNA-binding specificity among a panel of CG-binding pocket mutants quantitatively predict their selective antiviral activity against a CG-enriched HIV-1 strain. Overall, the hZAP RBD RNA structure provides an atomic-level explanation for how ZAP selectively targets foreign, CG-rich RNA.

Association of zinc-finger antiviral protein (ZAP) binding to viral genomic RNA with attenuation of replication of echovirus 7

“…RNA transcripts of an E7 viral cDNA with an UpA-high insert in the coding region showed a 1000-fold greater binding to ZAP compared to WT sequences, similar to that observed for a similarly constructed CpG-high viral RNA (1). While co-crystallisation of ZAP with CpG-high ligands (22,23) has recently provided information on the interaction of the zinc finger domains with CpG dinucleotides (and the stoichiometry of bases surrounding the motif), it is conceivable that UpA, as a similarly shaped pyrimidine (Y) -purine (R) dinucleotide might fit into the binding site. This degree of target flexibility would imply that other YpR dinucleotides (UpG and CpA) might be also recognised.…”

“…These findings suggest that there may be specifically favoured bases around the CpG binding ZAP that promotes binding. It is indeed intriguing, although not commented on in the studies, that both currently published structures of RNA bound to ZAP have U residues 5' to CpG (23) or at both 5' and 3'U sites (22), perhaps selected from other candidates because of their propensity to co-crystallise with ZAP.…”

“…From analysis of the topology and size constraints of the CpG binding site and immediate upstream and downstream bases (22,23) It was concluded that there should be no base constraints on the immediate 5' (-1) base to CpG and not to be part of the CpG binding site (23). Similarly, the topology of both 5' (-1) are 3' (+1) bases were considered to not impose any base constraints.…”

Human cytomegalovirus evades ZAP detection by suppressing CpG dinucleotides in the major immediate early genes

“…However, a study using a focused siRNA screen identified ZAPS and TRIM25 as host factors responsible for inhibition of HIV-1 constructs with artificially raised CpG dinucleotide frequencies and it was shown that ZAPS specifically binds to RNA sequences at CpG dinucleotide motifs 13 . The molecular mechanism for this binding has now been established through X-ray crystallography of the ZAP RNA binding domain complexed with a target CpG RNA, which demonstrates the second of four zinc fingers creates a highly basic patch that is required for specific binding of CpG dinucleotides 57 .…”

“…Mammalian ZAP is expressed in two major isoforms, ZAPS (short) and ZAPL (long) which are generated by differential splicing, with ZAPL encoding an additional catalytically inactive poly (ADP-ribose) polymerase (PARP) domain 12 . ZAP had previously been identified as a host antiviral factor and is capable of binding to viral RNA through a pocket created by the second of four zinc fingers within the RNA binding domain 13–15 . TRIM25 is an E3 ubiquitin ligase and member of the tripartite motif (TRIM) family, many of which have been associated with antiviral functions 16,17 .…”