Restriction of Human Cytomegalovirus Replication by ISG15, a Host Effector Regulated by cGAS-STING Double-Stranded-DNA Sensing

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.

ISG15 in antiviral immunity and beyond

“…Ectopic expression of HCMV IE1 limited ISG15 protein conjugation, presumably through the decreased expression of components of the ISG15 conjugation machinery, such as HERC5 (ref. 108 ). An IE1 deletion virus robustly induced interferon signalling, including the expression of ISG15 and ISG15 conjugates 64 .…”

Regulation of HMGB2 integrates ribosome biogenesis and innate immune responses to DNA

“…Normal Human Dermal Fibroblasts (NHDFs) and HCMV AD169GFP have been previously described (Bianco and Mohr, 2017). VSV-M51R-GFP (kindly provided by the laboratory of Benjamin TenOever, Mt.…”

“…RNA was isolated from cells using TRIzol Reagent (15596026; ThermoFisher Scientific) as previously described (Bianco and Mohr, 2017). cDNA was synthesized from 250ng of purified RNA using qScript XLT Supermix (Quanta; 95161) according to the manufacturer's instructions.…”

Transmembrane Protein pUL50 of Human Cytomegalovirus Inhibits ISGylation by Downregulating UBE1L

“…ISG15 or ISGylation represses the growth of diverse viruses, including influenza virus (types A and B), human immunodeficiency virus, avian sarcoma/leukosis virus, hepatitis C virus, Japanese encephalitis virus, Sindbis virus, vesicular stomatitis virus, dengue virus, West Nile virus, Ebola virus, porcine reproductive and respiratory syndrome virus, Chikungunya virus, herpes simplex virus 1 (HSV-1), murine gammaherpesvirus 68, and vaccinia virus (VACV) (6, 7; reviewed in reference 8). Recently, the antiviral activities of ISG15 or ISGylation were also demonstrated for Kaposi's sarcoma-associated herpesvirus (KSHV) (9), respiratory syncytial virus (10), and human cytomegalovirus (HCMV) (11,12). In contrast to the antiviral activities of ISG15, free ISG15 has also been shown to negatively regulate the type I IFN response in humans by promoting sustained expression of USP18, which negatively regulates IFN signaling, suggesting an ISGylationindependent role for ISG15 in IFN pathway regulation (13)(14)(15)(16).…”

“…The stability and phosphorylation of STAT proteins are modulated during HCMV infection (40,41). ISG15 expression and levels of ISGylation are upregulated immediately after HCMV infection via signaling pathways, including one mediated by cGAS/STING (11). However, HCMV appears to be armed with countermeasures, such as pUL26, which inhibits ISGylation at later stages of infection (12).…”