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.
Highlights
Interferon (IFN) is a crucial first line of defence against viral infection and shapes the adaptive immune response by triggering release of cytokines and chemokines [1, 2]
This study demonstrates that in addition to targeting RNA viruses, zinc-finger antiviral protein (ZAP) can target large DNA viruses and, in turn, these viruses have evolved evasion mechanisms, ensuring efficient replication
Cells were infected at a multiplicity of infection (MOI) of three with TB40/E-GFP, a BAC derived low passage human cytomegalovirus (HCMV) strain containing an SV40 promoter driven eGFP cassette inserted between the viral TRS1 and US34 genes [45]
Summary
Interferon (IFN) is a crucial first line of defence against viral infection and shapes the adaptive immune response by triggering release of cytokines and chemokines [1, 2]. IFN expression is triggered by the recognition of pathogen-associated molecular patterns (PAMPs) [1]. These microbe-specific molecular structures are generally essential for the survival of the microbes, but fundamentally different from the host. Cells recognize PAMPs through pattern recognition receptors (PRRs) that trigger innate immune responses following recognition of the target. Upon recognition of the specific PAMP during invasion by a foreign pathogen, PRRs trigger signaling cascades that lead to relocation of IRF3/IRF7 complexes and NF-kB into the nucleus, initiating expression of type I IFN. Activation of the IFN receptor leads to up-regulation of hundreds of IFN stimulated genes (ISGs) that, together, establish an antiviral cellular environment [4]
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