The antiviral activity of type I interferons (IFNs) is primarily mediated by interferon-stimulated genes (ISGs). Induction of ISG transcription is achieved when type I IFNs bind to their cognate receptor and activate the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathways. Recently it has become clear that a number of viruses are capable of directly upregulating a subset of ISGs in the absence of type I IFN production. Using cells engineered to block either the response to, or production of type I IFN, the regulation of IFN-independent ISGs was examined in the context of human cytomegalovirus (HCMV) infection. Several ISGs, including IFIT1, IFIT2, IFIT3, Mx1, Mx2, CXCL10 and ISG15 were found to be upregulated transcriptionally following HCMV infection independently of type I IFN-initiated JAK-STAT signaling, but dependent on intact IRF3 signaling. ISG15 protein regulation mirrored that of its transcript with IFNβ neutralization failing to completely inhibit ISG15 expression post HCMV infection. In addition, no detectable ISG15 protein expression was observed following HCMV infection in IRF3 knockdown CRISPR/Cas-9 clones indicating that IFN-independent control of ISG expression during HCMV infection of human fibroblasts is absolutely dependent on IRF3 expression.

https://www.mdpi.com/1999-4915/11/3/246/pdf

Interferon-Independent Upregulation of Interferon-Stimulated Genes during Human Cytomegalovirus Infection is Dependent on IRF3 Expression

Viruses are the most abundant organisms on our planet, affecting all living beings: some of them are responsible for massive epidemics that concern health, national economies and the overall welfare of societies. Although advances in antiviral research have led to successful therapies against several human viruses, still some of them cannot be eradicated from the host and most of them do not have any treatment available. Consequently, innovative antiviral therapies are urgently needed. In the past few years, research on G-quadruplexes (G4s) in viruses has boomed, providing powerful evidence for the regulatory role of G4s in key viral steps. Comprehensive bioinformatics analyses have traced putative G4-forming sequences in the genome of almost all human viruses, showing that their distribution is statistically significant and their presence highly conserved. Since the genomes of viruses are remarkably variable, high conservation rates strongly suggest a crucial role of G4s in the viral replication cycle and evolution, emphasizing the possibility of targeting viral G4s as a new pharmacological approach in antiviral therapy. Recent studies have demonstrated the formation and function of G4s in pathogens responsible for serious diseases, such as HIV-1, Hepatitis B and C, Ebola viruses, to cite a few. In this chapter, we present the state of the art on the structural and functional characterization of viral G4s in RNA viruses, DNA viruses and retroviruses. We also present the G4 ligands that provide further details on the viral G4 role and which, showing promising antiviral activity, which could be exploited for the development of innovative antiviral agents.

/pdf/viral-g-quadruplexes-new-frontiers-in-virus-pathogenesis-and-4r50deexv7.pdf

Viral G-quadruplexes: New frontiers in virus pathogenesis and antiviral therapy.

How ISG15 combats viral infection.

Guanine-rich DNA and RNA sequences can fold into noncanonical nucleic acid structures called G-quadruplexes (G4s). Since the discovery that these structures may act as scaffolds for the binding of specific ligands, G4s aroused the attention of a growing number of scientists. The versatile roles of G4 structures in viral replication, transcription, and translation suggest direct applications in therapy or diagnostics. G4-interacting molecules (proteins or small molecules) may also affect the balance between latent and lytic phases, and increasing evidence reveals that G4s are implicated in generally suppressing viral processes, such as replication, transcription, translation, or reverse transcription. In this review, we focus on the discovery of G4s in viruses and the role of G4 ligands in the antiviral drug discovery process. After assessing the role of viral G4s, we argue that host G4s participate in immune modulation, viral tumorigenesis, cellular pathways involved in virus maturation, and DNA integration of viral genomes, which can be potentially employed for antiviral therapeutics. Furthermore, we scrutinize the impediments and shortcomings in the process of studying G4 ligands and drug discovery. Finally, some unanswered questions regarding viral G4s are highlighted for prospective future projects. SIGNIFICANCE STATEMENT: G-quadruplexes (G4s) are noncanonical nucleic acid structures that have gained increasing recognition during the last few decades. First identified as relevant targets in oncology, their importance in virology is now increasingly clear. A number of G-quadruplex ligands are known: viral transcription and replication are the main targets of these ligands. Both viral and cellular G4s may be targeted; this review embraces the different aspects of G-quadruplexes in both host and viral contexts.

https://pharmrev.aspetjournals.org/content/pharmrev/73/3/897.full.pdf

Unlocking G-Quadruplexes as Antiviral Targets.

Antiviral responses of interferons (IFNs) are crucial in the host immune response, playing a relevant role in controlling viralw infections. Three types of IFNs, type I (IFN-α, IFN-β), II (IFN-γ) and III (IFN-λ), are classified according to their receptor usage, mode of induction, biological activity and amino acid sequence. Here, we provide a comprehensive review of type I IFN responses and different mechanisms that viruses employ to circumvent this response. In the first part, we will give an overview of the different induction and signaling cascades induced in the cell by IFN-I after virus encounter. Next, highlights of some of the mechanisms used by viruses to counteract the IFN induction will be described. And finally, we will address different mechanism used by viruses to interference with the IFN signaling cascade and the blockade of IFN induced antiviral activities.

/pdf/viral-pathogen-induced-mechanisms-to-antagonize-mammalian-3bytip70cl.pdf

Viral pathogen-induced mechanisms to antagonize mammalian interferon (IFN) signaling pathway

G-quadruplex (G4), formed by repetitive guanosine-rich sequences, is known to play various key regulatory roles in cells. Herpesviruses containing a large double-stranded DNA genome show relatively higher density of G4-forming sequences in their genomes compared to human and mouse. However, it remains poorly understood whether all of these sequences form G4 and how they play a role in the virus life cycle. In this study, we performed genome-wide analyses of G4s present in the putative promoter or gene regulatory regions of a 235-kb human cytomegalovirus (HCMV) genome and investigated their roles in viral gene expression. We evaluated 36 putative G4-forming sequences associated with 20 genes for their ability to form G4 and for the stability of G4s in the presence or absence of G4-stabilizing ligands, by circular dichroism and melting temperature analyses. Most identified sequences formed a stable G4; 28 sequences formed parallel G4s, one formed an antiparallel G4, and four showed mixed conformations. However, when we assessed the effect of G4 on viral promoters by cloning the 20 putative viral promoter regions containing 36 G4-forming sequences into the luciferase reporter and monitoring the expression of luciferase reporter gene in the presence of G4-stabilizing chemicals, we found that only 9 genes were affected by G4 formation. These results revealed promoter context-dependent gene suppression by G4 formation. Mutational analysis of two potential regulatory G4s also demonstrated gene suppression by the sequence-specific G4 formation. Furthermore, the analysis of a mutant virus incapable of G4 formation in the UL35 promoter confirmed promoter regulation by G4 in the context of virus infection. Our analyses provide a platform for assessing G4 functions at the genomic level and demonstrate the properties of the HCMV G4s and their regulatory roles in viral gene expression.

/pdf/genome-wide-analysis-of-regulatory-g-quadruplexes-affecting-4ppis98oup.pdf

Genome-wide analysis of regulatory G-quadruplexes affecting gene expression in human cytomegalovirus.

Interferon-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that can be conjugated to proteins via an enzymatic cascade involving the E1, E2, and E3 enzymes. ISG15 expression and protein ISGylation modulate viral infection; however, the viral mechanisms regulating the function of ISG15 and ISGylation are not well understood. We recently showed that ISGylation suppresses the growth of human cytomegalovirus (HCMV) at multiple steps of the virus life cycle and that the virus-encoded pUL26 protein inhibits protein ISGylation. In this study, we demonstrate that the HCMV UL50-encoded transmembrane protein, a component of the nuclear egress complex, also inhibits ISGylation. pUL50 interacted with UBE1L, an E1-activating enzyme for ISGylation, and (to a lesser extent) with ISG15, as did pUL26. However, unlike pUL26, pUL50 caused proteasomal degradation of UBE1L. The UBE1L level induced in human fibroblast cells by interferon beta treatment or virus infection was reduced by pUL50 expression. This activity of pUL50 involved the transmembrane (TM) domain within its C-terminal region, although pUL50 could interact with UBE1L in a manner independent of the TM domain. Consistently, colocalization of pUL50 with UBE1L was observed in cells treated with a proteasome inhibitor. Furthermore, we found that RNF170, an endoplasmic reticulum (ER)-associated ubiquitin E3 ligase, interacted with pUL50 and promoted pUL50-mediated UBE1L degradation via ubiquitination. Our results demonstrate a novel role for the pUL50 transmembrane protein of HCMV in the regulation of protein ISGylation.IMPORTANCE Proteins can be conjugated covalently by ubiquitin or ubiquitin-like proteins, such as SUMO and ISG15. ISG15 is highly induced in viral infection, and ISG15 conjugation, termed ISGylation, plays important regulatory roles in viral growth. Although ISGylation has been shown to negatively affect many viruses, including human cytomegalovirus (HCMV), viral countermeasures that might modulate ISGylation are not well understood. In the present study, we show that the transmembrane protein encoded by HCMV UL50 inhibits ISGylation by causing proteasomal degradation of UBE1L, an E1-activating enzyme for ISGylation. This pUL50 activity requires membrane targeting. In support of this finding, RNF170, an ER-associated ubiquitin E3 ligase, interacts with pUL50 and promotes UL50-mediated UBE1L ubiquitination and degradation. Our results provide the first evidence, to our knowledge, that viruses can regulate ISGylation by directly targeting the ISGylation E1 enzyme.

Young-Eui Kim

Papers

Genome-wide analysis of regulatory G-quadruplexes affecting gene expression in human cytomegalovirus.

Transmembrane Protein pUL50 of Human Cytomegalovirus Inhibits ISGylation by Downregulating UBE1L.