ABSTRACT: Accumulation of the interferon-stimulated gene 15 (ISG15) protein product, which is reversibly conjugated to numerous polypeptide targets, impacts the proteome and physiology of uninfected and infected cells. While many viruses, including human cytomegalovirus (HCMV), blunt host antiviral defenses by limiting ISG expression, the overall abundance of ISG15 monomer and protein conjugates rises in HCMV-infected cells. However, the molecular signals underlying ISG15 accumulation and whether the ISG15 polypeptide itself influences HCMV infection biology remain unknown. Here, we establish that the ISG15 gene product itself directly regulates HCMV replication and that its accumulation restricts productive virus growth. Although ISG15 monomer and protein conjugate accumulation was induced in cells infected with UV-inactivated HCMV, it was subsequently reduced, but not eliminated, by an immediate-early (IE) or early (E) virus-encoded function(s). Instead, HCMV-induced ISG15 monomer and protein conjugate accumulation was dependent upon the double-stranded DNA (dsDNA) sensor cyclic GMP-AMP synthase (cGAS), the innate immune adaptor STING, and interferon signaling. Significantly, dsDNA itself was sufficient to induce cGAS-, STING-, and interferon signaling-dependent ISG15 monomer and conjugate protein accumulation in uninfected cells. Accumulation of ISGylated proteins in uninfected cells treated with dsDNA was prevented by expressing the HCMV multifunctional IE1 transactivator. This demonstrates that expression of a single host interferon-stimulated gene, ISG15, restricts HCMV replication, and that IE1 is sufficient to blunt ISGylation in response to dsDNA sensing in uninfected cells. Moreover, it establishes that ISGylation modifies the proteomes of virus-infected and uninfected normal cells in response to cell-intrinsic dsDNA sensing dependent upon cGAS-STING.IMPORTANCE By antagonizing type I interferon production and action, many viruses, including human cytomegalovirus (HCMV), evade host defenses. However, levels of the interferon-induced ISG15 protein, which is covalently conjugated to host and viral proteins, increase in HCMV-infected cells. How ISG15 accumulation is regulated and whether the ISG15 polypeptide influences HCMV replication remain unknown. This study establishes that ISG15 itself restricts HCMV replication and that HCMV-induced ISG15 accumulation is triggered by host defenses that detect cytoplasmic double-stranded DNA (dsDNA). Remarkably, dsDNA triggered ISG15 accumulation even in uninfected cells, and this was reduced by HCMV IE1 expression. This shows that ISG15 itself controls the replication of HCMV, which causes life-threatening disease among the immunocompromised and is a significant source of congenital morbidity and mortality among newborns. Moreover, it demonstrates that ISG15 modifies the uninfected cell proteome in response to dsDNA, potentially impacting responses to DNA vaccines, gene therapy, and autoimmune disease pathogenesis.
