DNA topoisomerase 1 represses HIV-1 promoter activity through its interaction with a guanine quadruplex present in the LTR sequence.

BACKGROUND: Once integrated in the genome of infected cells, HIV-1 provirus is transcribed by the cellular transcription machinery. This process is regulated by both viral and cellular factors, which are necessary for an efficient viral replication as well as for the setting up of viral latency, leading to a repressed transcription of the integrated provirus. RESULTS: In this study, we examined the role of two parameters in HIV-1 LTR promoter activity. We identified DNA topoisomerase1 (TOP1) to be a potent repressor of this promoter and linked this repression to its catalytic domain. Additionally, we confirmed the folding of a Guanine quadruplex (G4) structure in the HIV-1 promoter and its repressive effect. We demonstrated a direct interaction between TOP1 and this G4 structure, providing evidence of a functional relationship between the two repressive elements. Mutations abolishing G4 folding affected TOP1/G4 interaction and hindered G4-dependent inhibition of TOP1 catalytic activity in vitro. As a result, HIV-1 promoter activity was reactivated in a native chromatin environment. Lastly, we noticed an enrichment of predicted G4 sequences in the promoter of TOP1-repressed cellular genes. CONCLUSIONS: Our results demonstrate the formation of a TOP1/G4 complex on the HIV-1 LTR promoter and its repressive effect on the promoter activity. They reveal the existence of a new mechanism of TOP1/G4-dependent transcriptional repression conserved between viral and human genes. This mechanism contrasts with the known property of TOP1 as global transcriptional activator and offers new perspectives for anti-cancer and anti-viral strategies.

Strain-Dependent Restriction of Human Cytomegalovirus by Zinc Finger Antiviral Proteins.

Cellular antiviral factors that recognize viral nucleic acid can inhibit virus replication. These include the zinc finger antiviral protein (ZAP), which recognizes high CpG dinucleotide content in viral RNA. Here, we investigated the ability of ZAP to inhibit the replication of human cytomegalovirus (HCMV). Depletion of ZAP or its cofactor KHNYN increased the titer of the high-passage HCMV strain AD169 but had little effect on the titer of the low-passage strain Merlin. We found no obvious difference in expression of several viral proteins between AD169 and Merlin in ZAP knockdown cells, but observed a larger increase in infectious virus in AD169 compared to Merlin in the absence of ZAP, suggesting that ZAP inhibited events late in AD169 replication. In addition, there was no clear difference in the CpG abundance of AD169 and Merlin RNAs, indicating that genomic content of the two virus strains was unlikely to be responsible for differences in their sensitivity to ZAP. Instead, we observed less ZAP expression in Merlin-infected cells late in replication compared to AD169-infected cells, which may be related to different abilities of the two virus strains to regulate interferon signaling. Therefore, there are strain-dependent differences in the sensitivity of HCMV to ZAP, and the ability of low-passage HCMV strain Merlin to evade inhibition by ZAP is likely related to its ability to regulate interferon signaling, not the CpG content of RNAs produced from its genome. IMPORTANCE Determining the function of cellular antiviral factors can inform our understanding of virus replication. The zinc finger antiviral protein (ZAP) can inhibit the replication of diverse viruses. Here, we examined ZAP interaction with the DNA virus human cytomegalovirus (HCMV). We found HCMV strain-dependent differences in the ability of ZAP to influence HCMV replication, which may be related to the interaction of HCMV strains with the type I interferon system. These observations affect our current understanding of how ZAP restricts HCMV and how HCMV interacts with the type I interferon system.

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

The zinc finger antiviral protein (ZAP) is a broad inhibitor of virus replication. Its best-characterized function is to bind CpG dinucleotides present in viral RNAs and, through the recruitment of TRIM25, KHNYN and other cofactors, target them for degradation or prevent their translation. The long and short isoforms of ZAP (ZAP-L and ZAP-S) have different intracellular localization and it is unclear how this regulates their antiviral activity against viruses with different sites of replication. Using ZAP-sensitive and ZAP-insensitive human immunodeficiency virus type I (HIV-1), which transcribe the viral RNA in the nucleus and assemble virions at the plasma membrane, we show that the catalytically inactive poly-ADP-ribose polymerase (PARP) domain in ZAP-L is essential for CpG-specific viral restriction. Mutation of a crucial cysteine in the C-terminal CaaX box that mediates S-farnesylation and, to a lesser extent, the residues in place of the catalytic site triad within the PARP domain, disrupted the activity of ZAP-L. Addition of the CaaX box to ZAP-S partly restored antiviral activity, explaining why ZAP-S lacks antiviral activity for CpG-enriched HIV-1 despite conservation of the RNA-binding domain. Confocal microscopy confirmed the CaaX motif mediated localization of ZAP-L to vesicular structures and enhanced physical association with intracellular membranes. Importantly, the PARP domain and CaaX box together jointly modulate the interaction between ZAP-L and its cofactors TRIM25 and KHNYN, implying that its proper subcellular localisation is required to establish an antiviral complex. The essential contribution of the PARP domain and CaaX box to ZAP-L antiviral activity was further confirmed by inhibition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, which replicates in double-membrane vesicles derived from the endoplasmic reticulum. Thus, compartmentalization of ZAP-L on intracellular membranes provides an essential effector function in ZAP-L-mediated antiviral activity against divergent viruses with different subcellular replication sites.

Novel cationic bis(acylhydrazones) as modulators of Epstein-Barr virus immune evasion acting through disruption of interaction between nucleolin and G-quadruplexes of EBNA1 mRNA.

The oncogenic Epstein-Barr virus (EBV) evades the immune system through limiting the expression of its highly antigenic and essential genome maintenance protein, EBNA1, to the minimal level to ensure viral genome replication, thereby also minimizing the production of EBNA1-derived antigenic peptides. This regulation is based on inhibition of translation of the virally-encoded EBNA1 mRNA, and involves the interaction of host protein nucleolin (NCL) with G-quadruplex (G4) structures that form in the glycine-alanine repeat (GAr)-encoding sequence of the EBNA1 mRNA. Ligands that bind to these G4-RNA can prevent their interaction with NCL, leading to disinhibition of EBNA1 expression and antigen presentation, thereby interfering with the immune evasion of EBNA1 and therefore of EBV (M.J. Lista et al., Nature Commun., 2017, 8, 16043). In this work, we synthesized and studied a series of 20 cationic bis(acylhydrazone) derivatives designed as G4 ligands. The in vitro evaluation showed that most derivatives based on central pyridine (Py), naphthyridine (Naph) or phenanthroline (Phen) units were efficient G4 binders, in contrast to their pyrimidine (Pym) counterparts, which were poor G4 binders due to a significantly different molecular geometry. The influence of lateral heterocyclic units (N-substituted pyridinium or quinolinium residues) on G4-binding properties was also investigated. Two novel compounds, namely PyDH2 and PhenDH2, used at a 5 µM concentration, were able to significantly enhance EBNA1 expression in H1299 cells in a GAr-dependent manner, while being significantly less toxic than the prototype drug PhenDC3 (GI50 > 50 µM). Antigen presentation, RNA pull-down and proximity ligation assays confirmed that the effect of both drugs was related to the disruption of NCL-EBNA1 mRNA interaction and the subsequent promotion of GAr-restricted antigen presentation. Our work provides a novel modular scaffold for the development of G-quadruplex-targeting drugs acting through interference with G4-protein interaction.

Nuclear processing of nascent transcripts determines synthesis of full-length proteins and antigenic peptides.

Peptides presented on major histocompatibility (MHC) class I molecules form an essential part of the immune system's capacity to detect virus-infected or transformed cells. Earlier works have shown that pioneer translation peptides (PTPs) for the MHC class I pathway are as efficiently produced from introns as from exons, or from mRNAs targeted for the nonsense-mediated decay pathway. The production of PTPs is a target for viral immune evasion but the underlying molecular mechanisms that govern this non-canonical translation are unknown. Here, we have used different approaches to show how events taking place on the nascent transcript control the synthesis of PTPs and full-length proteins. By controlling the subcellular interaction between the G-quadruplex structure (G4) of a gly-ala encoding mRNA and nucleolin (NCL) and by interfering with mRNA maturation using multiple approaches, we demonstrate that antigenic peptides derive from a nuclear non-canonical translation event that is independently regulated from the synthesis of full-length proteins. Moreover, we show that G4 are exploited to control mRNA localization and translation by distinguishable mechanisms that are targets for viral immune evasion.

A yeast model for the mechanism of the Epstein-Barr virus immune evasion identifies a new therapeutic target to interfere with the virus stealthiness.

The oncogenic Epstein-Barr virus (EBV) evades the immune system but has an Achilles heel: its genome maintenance protein EBNA1. Indeed, EBNA1 is essential for viral genome replication and maintenance but also highly antigenic. Hence, EBV evolved a system in which the glycine-alanine repeat (GAr) of EBNA1 limits the translation of its own mRNA at a minimal level to ensure its essential function thereby, at the same time, minimizing immune recognition. Defining intervention points where to interfere with EBNA1 immune evasion is an important step to trigger an immune response against EBV-carrying cancers. Thanks to a yeast-based assay that recapitulates all the aspects of EBNA1 self-limitation of expression, a recent study by Lista et al. [Nature Communications (2017) 7, 435-444] has uncovered the role of the host cell nucleolin (NCL) in this process via a direct interaction of this protein with G-quadruplexes (G4) formed in GAr-encoding sequence of EBNA1 mRNA. In addition, the G4 ligand PhenDC3 prevents NCL binding on EBNA1 mRNA and reverses GAr-mediated repression of translation and antigen presentation. This shows that the NCL-EBNA1 mRNA interaction is a relevant therapeutic target to unveil EBV-carrying cancers to the immune system and that the yeast model can be successfully used for uncovering drugs and host factors that interfere with EBV stealthiness.

Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to G-quadruplexes of EBNA1 mRNA.

The oncogenic Epstein-Barr virus (EBV) evades the immune system but has an Achilles heel: its genome maintenance protein EBNA1, which is essential for viral genome maintenance but highly antigenic. EBV has seemingly evolved a system in which the mRNA sequence encoding the glycine-alanine repeats (GAr) of the EBNA1 protein limits its expression to the minimal level necessary for function while minimizing immune recognition. Here, we identify nucleolin (NCL) as a host factor required for this process via a direct interaction with G-quadruplexes formed in GAr-encoding mRNA sequence. Overexpression of NCL enhances GAr-based inhibition of EBNA1 protein expression, whereas its downregulation relieves the suppression of both expression and antigen presentation. Moreover, the G-quadruplex ligand PhenDC3 prevents NCL binding to EBNA1 mRNA and reverses GAr-mediated repression of EBNA1 expression and antigen presentation. Hence the NCL-EBNA1 mRNA interaction is a relevant therapeutic target to trigger an immune response against EBV-carrying cancers.

The long-lasting love affair between the budding yeast Saccharomyces cerevisiae and the Epstein-Barr virus.

The Epstein-Barr gammaherpesvirus (EBV) is the first oncogenic virus discovered in human. Indeed, EBV has been known for more than 50 years to be tightly associated with certain human cancers. As such, EBV has been the subject of extensive studies aiming at deciphering various aspects of its biological cycle, ranging from the regulation of its genome replication and maintenance to the induction of its lytic cycle, including the mechanisms that allow its immune evasion or that are related to its tumorogenicity. For more than 30 years the budding yeast Saccharomyces cerevisiae has fruitfully contributed to a number of these studies. The aim of this article is to review the various aspects of EBV biology for which yeast has been instrumental, and to propose new possible applications for these yeast-based assays, as well as the creation of further yeast models dedicated to EBV. This review article illustrates the tremendous potential of S. cerevisiae in integrated chemobiological approaches for the biomedical research.