Human cytomegalovirus evades ZAP detection by suppressing CpG dinucleotides in the major immediate early 1 gene

Abstract

The genomes of RNA and small DNA viruses of vertebrates display significant suppression of CpG dinucleotide frequencies. Artificially increasing dinucleotide frequencies results in substantial attenuation of virus replication, suggesting that these compositional changes may facilitate recognition of non-self RNA sequences. Recently, the interferon inducible protein ZAP, was identified as the host factor responsible for sensing CpG in viral RNA, through direct binding and possibly downstream targeting for degradation. Using an arrayed interferon stimulated gene expression library screen, we identified ZAPS, and its associated factor TRIM25, as inhibitors of human cytomegalovirus (HCMV) replication. Exogenous expression of ZAPS and TRIM25 significantly reduced virus replication while knockdown resulted in increased virus replication. HCMV displays a strikingly heterogeneous pattern of CpG representation with specific suppression of CpG motifs within the IE1 major immediate early transcript which is absent in subsequently expressed genes. We demonstrated that suppression of CpG dinucleotides in the IE1 gene allows evasion of inhibitory effects of ZAP. We show that acute virus replication is mutually exclusive with high levels of cellular ZAP, potentially explaining the higher levels of CpG in viral genes expressed subsequent to IE1 due to the loss of pressure from ZAP in infected cells. Finally, we show that TRIM25 regulates alternative splicing between the ZAP short and long isoforms during HCMV infection and interferon induction, with knockdown of TRIM25 resulting in decreased ZAPS and corresponding increased ZAPL expression. These results demonstrate for the first time that ZAP is a potent host restriction factor against large DNA viruses and that HCMV evades ZAP detection through suppression of CpG dinucleotides within the major immediate early 1 transcript. Furthermore, TRIM25 is required for efficient upregulation of the interferon inducible short isoform of ZAP through regulation of alternative splicing.

Intrinsic Immune Mechanisms Restricting Human Cytomegalovirus Replication

“…Consistently, scanning analysis demonstrates that the IE1 gene is the only region of the HCMV genome that is suppressed for CpG content [ 76 ]. In accordance, Lin et al demonstrated that HCMV transcripts with high CpG content are specifically targeted by ZAP, while the CpG-suppressed IE1 transcript remains unaffected [ 75 ]. However, artificially increasing the IE1 CpG content by introducing mutations into the IE1 coding region renders IE1 accessible to ZAP inhibition.…”

“…A recent arrayed ISG expression screening performed by Lin and colleagues identified ZAP to restrict HCMV in a manner that is independent of IRF3. Overexpression and knockdown experiments displayed decreased or increased virus replication, respectively, further confirming ZAP as a RF against HCMV [ 75 ]. For herpesviruses, the pattern of CpG dinucleotide frequencies is distinct: while the majority of alpha-herpesviruses demonstrate little or no CpG suppression, gamma-herpesviruses exhibit substantial suppression across the genome.…”

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

“…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). Here, CG suppression within the major immediate early transcript 1 confers ZAP resistance (156).…”

“…ZAP can even sense CG dinucleotides within individual RNA transcripts of DNA viruses, as in the case of human cytomegalovirus ( 156 ). Here, CG suppression within the major immediate early transcript 1 confers ZAP resistance ( 156 ). However, CG suppression does not always confer resistance to ZAP as in the case of SARS-CoV-2 ( 154 ).…”

“…On the other hand, ZAPS was found to synergize with other ISGs, wherein 31 ISGs have a statistically significant increase in antiviral activity in the presence of ZAP ( 32 ). In addition to its role as a co-factor in ZAP translation inhibition and CG sensing, TRIM25 may also modulate expression of ZAP isoforms by regulating alternative splicing, wherein TRIM25 is required for efficient expression of ZAPS ( 156 ).…”