# Exonuclease ISG20 inhibits human cytomegalovirus replication by inducing an innate immune defense signature

## TL;DR

ISG20 inhibits HCMV and HSV-1 replication by boosting the host's innate immune response, not by directly degrading viral RNA or DNA.

## Contribution

ISG20's antiviral mechanism against DNA viruses is shown to involve inducing an innate immune defense signature rather than RNA/DNA degradation.

## Key findings

- ISG20 reduces HCMV and HSV-1 replication in fibroblasts.
- ISG20 induces an innate immune defense signature including ISGs, ZNFs, and TEs.
- ISG20's antiviral effect is mediated through enhancing IFN production and response.

## Abstract

ISG20 is an interferon-regulated protein that exhibits RNase activity thereby inhibiting the replication of a broad spectrum of RNA viruses. By single cell RNA sequencing, we identified ISG20 as an antiviral factor for human cytomegalovirus (HCMV) as it was upregulated in a population of HCMV-resistant cells. In accordance with an antiviral role on herpesviruses, overexpression of ISG20 in primary human fibroblasts led to reduced HCMV and HSV-1 replication, while knockdown of ISG20 enhanced virus growth. In Western blot kinetics, we observed that inhibition of HCMV replication by ISG20 occurs at the early stage of infection which correlated with reduced amounts of viral early and late transcripts. However, neither the half-life of viral and cellular RNAs nor of viral DNA was decreased in ISG20-expressing cells, indicating that ISG20 does not exert its antiviral effect via a degradation of RNAs or DNA. Instead, RNA-seq analysis revealed an innate immune defense signature upon ISG20 expression that comprised the upregulation of a distinct set of interferon stimulated genes (ISGs), zinc finger protein genes (ZNFs) and of transposable elements (TEs). Our data indicate that this gene signature augments both IFN production and response of the host cell. Consistently, the JAK-STAT inhibitor ruxolitinib rescued HCMV gene expression in ISG20-expressing cells. We conclude that ISG20 induces a broad immune defense signature that serves to amplify the IFN-mediated host cell defense thus explaining its extended antiviral activity.

The interferon-induced protein 20 (ISG20) is best known for its antiviral function on many RNA viruses. Since in vitro experiments detected that this protein is capable of directly degrading RNA, it is thought that this enzymatic function acts as the prevailing mechanism of inhibition. In contrast, knowledge concerning the role of ISG20 for DNA viruses is limited. Here, we identify the ISG20 gene within an antiviral signature of primary human fibroblast that are resistant to the herpesvirus human cytomegalovirus (HCMV). Further experiments demonstrated that ISG20 severely impairs the replication of both HCMV and herpes simplex virus type I, suggesting a broad antiviral activity on herpesviruses. Surprisingly, neither cellular nor viral RNAs nor viral DNA were found to be degraded upon expression of ISG20. Transcriptomic analyses revealed a distinct upregulation of type I interferon (IFN) response genes, zinc finger protein genes and endogenous retroelements indicating a profound effect of ISG20 on host cells. We show that depletion of ISG20 severely impairs the host cell’s response to IFN which is consistent with the finding that interference with IFN-signaling alleviates the antiviral effect of ISG20 overexpression. In summary, we propose a novel mechanism of ISG20 action based on the induction of an innate immune defense signature which serves to amplify the host cell’s IFN response.