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

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. Author summary The evolutionary success of viruses is dependent on their ability to circumvent defence mechanisms of the hosts they infect. These defence mechanisms rely on the ability of the host to discriminate between self and non-self, allowing identification and inhibition of invading pathogens, with the ultimate goal of containing or eradicating the infection. The specific nucleotide composition of viral genomes has been recognised as a potential target of host defences. Endogenous viruses have evolved to mirror underrepresentation of CpG motifs in mammalian genomes. Recently, the host factor ZAP has been shown to recognise and inhibit RNA viruses with artificially high CpG content, demonstrating a novel host defence mechanism. Here, we show that ZAP can potently inhibit human cytomegalovirus. In turn, the virus has evolved to reduce the levels of CpG in viral genes expressed immediately after infection of host cells, allowing evasion of ZAP recognition. This study demonstrates that in addition to targeting RNA viruses, ZAP can target large DNA viruses and, in turn, these viruses have evolved evasion mechanisms, ensuring efficient replication.