The Zinc Finger Antiviral Protein ZAP Restricts Human Cytomegalovirus and Selectively Binds and Destabilizes Viral UL4 / UL5 Transcripts

Abstract

Interferon-stimulated gene products (ISGs) play a crucial role in early infection control. The ISG zinc finger CCCH-type antiviral protein 1 (ZAP/ZC3HAV1) antagonizes several RNA viruses by binding to CG-rich RNA sequences, whereas its effect on DNA viruses is less well understood. Here, we decipher the role of ZAP in the context of human cytomegalovirus (HCMV) infection, a β-herpesvirus that is associated with high morbidity in immunosuppressed individuals and newborns. We show that expression of the two major isoforms of ZAP, ZAP-S and ZAP-L, is induced during HCMV infection and that both negatively affect HCMV replication. Transcriptome and proteome analyses demonstrated that the expression of ZAP results in reduced viral mRNA and protein levels and decelerates the progression of HCMV infection. Metabolic RNA labeling combined with high-throughput sequencing (SLAM-seq) revealed that most of the gene expression changes late in infection result from the general attenuation of HCMV. Furthermore, at early stages of infection, ZAP restricts HCMV by destabilizing a distinct subset of viral mRNAs, particularly those from the previously uncharacterized UL4-UL6 HCMV gene locus. Through enhanced cross-linking immunoprecipitation and sequencing analysis (eCLIP-seq), we identified the transcripts expressed from this HCMV locus as the direct targets of ZAP. Moreover, our data show that ZAP preferentially recognizes not only CG, but also other cytosine-rich sequences, thereby expanding its target specificity. In summary, this report is the first to reveal direct targets of ZAP during HCMV infection, which strongly indicates that transcripts from the UL4-UL6 locus may play an important role for HCMV replication. IMPORTANCE Viral infections have a large impact on society, leading to major human and economic losses and even global instability. So far, many viral infections, including human cytomegalovirus (HCMV) infection, are treated with a small repertoire of drugs, often accompanied by the occurrence of resistant mutants. There is no licensed HCMV vaccine in sight to protect those most at risk, particularly immunocompromised individuals or pregnant women who might otherwise transmit the virus to the fetus. Thus, the identification of novel intervention strategies is urgently required. In this study, we show that ZAP decelerates the viral gene expression cascade, presumably by selectively handpicking a distinct set of viral transcripts for degradation. Our study illustrates the potent role of ZAP as an HCMV restriction factor and sheds light on a possible role for UL4 and/or UL5 early during infection, paving a new avenue for the exploration of potential targets for novel therapies.

Does the Zinc Finger Antiviral Protein (ZAP) Shape the Evolution of Herpesvirus Genomes?

“…Recently, we identified ZAP and TRIM25 as host restriction factors against HCMV using an arrayed interferon-stimulated gene expression screen, the first such screen against a herpesvirus [ 34 ]. These results were later confirmed by an independent study that showed CRISPR Cas9 deletion of ZAP increased HCMV replication and that ZAP targeted transcripts from the UL4-UL6 region of the virus [ 33 ].…”

“…More recently, we and others have shown that ZAP can restrict the replication of large DNA viruses. Disruption of ZAP increases replication of the herpesvirus HCMV and the poxvirus vaccinia [ 33 , 34 , 35 ]. In the case of the vaccinia virus, the viral C16 protein was shown to antagonise ZAP antiviral activity by relocalising ZAP to punctate cytoplasmic structures.…”

“…It is also unclear why the virus has not evolved to evade targeting in this region. One possible explanation is that these transcripts act as a decoy target for ZAP, contributing to ZAP evasion, although further studies will be required to answer these questions [ 33 ].…”

S-farnesylation is essential for antiviral activity of the long ZAP isoform against RNA viruses with diverse replication strategies

“…Mutating Y108 partially decreased ZAP-L antiviral activity for SARS-CoV-2, which could indicate that ZAP interacts with other motifs in the viral RNA in addition to CpGs. While it is clear that ZAP binds CpG dinucleotides in the context of single-stranded RNA, how the surrounding sequence and structural context of the CpG affect ZAP binding and antiviral activity as well as the role for non-CpG binding sites in viral RNA is still unclear [ 6 , 14 , 18 – 20 , 30 , 33 , 35 , 39 , 40 ].…”

“…While there has been substantial progress in understanding how ZAP restricts viral replication, several key questions remain. First, the specific contribution of ZAP binding to CpGs for viral restriction is unclear, especially since ZAP can also restrict viruses with increased UpA abundance and CpG abundance does not always predict ZAP-sensitivity [ 14 , 15 , 30 , 39 , 40 ]. Second, while ZAP-L is often more antiviral than ZAP-S, depending on the experimental system and virus being studied, the specific determinants in the PARP domain that lead to increased antiviral activity and how this correlates with interactions between ZAP and cofactors remains to be resolved.…”