Peter Lischka

The Novel Anticytomegalovirus Compound AIC246 (Letermovir) Inhibits Human Cytomegalovirus Replication through a Specific Antiviral Mechanism That Involves the Viral Terminase

ABSTRACT Human cytomegalovirus (HCMV) remains the leading viral cause of birth defects and life-threatening disease in transplant recipients. All approved antiviral drugs target the viral DNA polymerase and are associated with severe toxicity issues and the emergence of drug resistance. Attempts to discover improved anti-HCMV drugs led to the identification of the small-molecular-weight compound AIC246 (Letermovir). AIC246 exhibits outstanding anti-HCMV activity in vitro and in vivo and currently is undergoing a clinical phase IIb trial. The initial mode-of-action studies suggested that the drug acts late in the HCMV replication cycle via a mechanism distinct from that of polymerase inhibitors. Here, we extend our mode-of-action analyses and report that AIC246 blocks viral replication without inhibiting the synthesis of progeny HCMV DNA or viral proteins. The genotyping of mutant viruses that escaped AIC246 inhibition uncovered distinct point mutations in the UL56 subunit of the viral terminase complex. Marker transfer analyses confirmed that these mutations were sufficient to mediate AIC246 resistance. The mapping of drug resistance to open reading frame UL56 suggests that viral DNA processing and/or packaging is targeted by AIC246. In line with this, we demonstrate that AIC246 affects the formation of proper unit-length genomes from viral DNA concatemers and interferes with virion maturation. However, since AIC246-resistant viruses do not exhibit cross-resistance to previously published terminase inhibitors, our data suggest that AIC246 interferes with HCMV DNA cleavage/packaging via a molecular mechanism that is distinct from that of other compound classes known to target the viral terminase.

Sox10 is an active nucleocytoplasmic shuttle protein, and shuttling is crucial for Sox10-mediated transactivation.

ABSTRACT Sox10 belongs to a family of transcription regulators characterized by a DNA-binding domain known as the HMG box. It plays fundamental roles in neural crest development, peripheral gliogenesis, and terminal differentiation of oligodendrocytes. In accord with its function as transcription factor, Sox10 contains two nuclear localization signals and is most frequently detected in the nucleus. In this study, we report that Sox10 is an active nucleocytoplasmic shuttle protein, competent of both entering and exiting the nucleus. We identified a functional Rev-type nuclear export signal within the DNA-binding domain of Sox10. Mutational inactivation of this nuclear export signal or treatment of cells with the CRM1-specific export inhibitor leptomycin B inhibited nuclear export and consequently nucleocytoplasmic shuttling of Sox10. Importantly, the inhibition of the nuclear export of Sox10 led to decreased transactivation of transfected reporters and endogenous target genes, arguing that continuous nucleocytoplasmic shuttling is essential for the function of Sox10. To our knowledge this is the first time that nuclear export has been reported and shown to be functionally relevant for any Sox protein.

A novel transferable nuclear export signal mediates CRM1‐independent nucleocytoplasmic shuttling of the human cytomegalovirus transactivator protein pUL69

The best studied nuclear export processes are mediated by classical leucine‐rich nuclear export signals that specify recognition by the CRM1 export receptor. However, details concerning alternative nuclear export signals and pathways are beginning to emerge. Within the family of Herpesviridae, a set of homologous regulatory proteins that are exemplified by the ICP27 of herpes simplex virus were described recently as nucleocytoplasmic shuttling proteins. Here we report that pUL69 of the β‐herpesvirus human cytomegalovirus is a nuclear protein that is able to shuttle between the nucleus and the cytoplasm independently of virus‐encoded cofactors. In contrast to proteins containing a leucine‐rich export signal, the shuttling activity of pUL69 was not affected by leptomycin B, indicating that pUL69 trafficking is not mediated by the export receptor CRM1. Importantly, we identified and characterized a novel type of transferable, leptomycin B‐insensitive export signal that is distinct from other export signals described previously and is required for pUL69‐mediated activation of gene expression. These data suggest that pUL69 is exported via a novel nuclear export pathway, based on a so far unique nuclear export signal of 28 amino acids.

The UL69 Transactivator Protein of Human Cytomegalovirus Interacts with DEXD/H-Box RNA Helicase UAP56 To Promote Cytoplasmic Accumulation of Unspliced RNA

ABSTRACT The UL69 gene product of human cytomegalovirus belongs to a family of regulatory proteins conserved among all herpesviruses that have in part been characterized as posttranscriptional transactivators participating in the nuclear export of RNA. Recent experiments suggested that pUL69 also acts as a posttranscriptional activator since it was demonstrated that nucleocytoplasmic shuttling via a CRM1-independent nuclear export signal is a prerequisite for its stimulatory effect on gene expression. Based on these findings we initiated studies to investigate the role of pUL69 in mRNA export and demonstrate that pUL69 efficiently promotes the cytoplasmic accumulation of unspliced RNA. Furthermore, we show that this pUL69 activity is linked to the cellular mRNA export machinery by direct protein interaction with the highly related DEXD/H-box RNA helicases UAP56 and URH49. Particularly, we identified a 12-amino-acid domain within the N terminus of pUL69 which is required for binding to UAP56 and URH49, and we could demonstrate that UAP56 interaction and nucleocytoplasmic shuttling are both prerequisites for pUL69-mediated mRNA export. Thus, we identified a novel cellular target which provides a herpesviral regulatory protein with access to a conserved cellular transport system in order to promote nuclear export of unspliced RNA.

HIV-1 Nef mimics an integrin receptor signal that recruits the polycomb group protein Eed to the plasma membrane

The Nef protein of human and simian immunodeficiency virus (HIV/SIV) is believed to interfere with T cell activation signals by forming a signaling complex at the plasma membrane. Composition and function of the complex are not fully understood. Here we report that Nef recruits the Polycomb Group (PcG) protein Eed, so far known as a nuclear factor and repressor of transcription, to the membrane of cells. The Nef-induced translocation of Eed led to a potent stimulation of Tat-dependent HIV transcription, implying that Eed removal from the nucleus is required for optimal Tat function. Similar to Nef action, activation of integrin receptors recruited Eed to the plasma membrane, also leading to enhanced Tat/Nef-mediated transcription. Our results suggest a link between membrane-associated activation processes and transcriptional derepression and demonstrate how HIV exploits this mechanism.

A Nonconventional Nuclear Localization Signal within the UL84 Protein of Human Cytomegalovirus Mediates Nuclear Import via the Importin α/β Pathway

ABSTRACT The open reading frame UL84 of human cytomegalovirus encodes a multifunctional regulatory protein which is required for viral DNA replication and binds with high affinity to the immediate-early transactivator IE2-p86. Although the exact role of pUL84 in DNA replication is unknown, the nuclear localization of this protein is a prerequisite for this function. To investigate whether the activities of pUL84 are modulated by cellular proteins we used the Saccharomyces cerevisiae two-hybrid system to screen a cDNA-library for interacting proteins. Strong interactions were found between pUL84 and four members of the importin α protein family. These interactions could be confirmed in vitro by pull down experiments and in vivo by coimmunoprecipitation analysis from transfected cells. Using in vitro transport assays we showed that the pUL84 nuclear import required importin α, importin β, and Ran, thus following the classical importin-mediated import pathway. Deletion mutagenesis of pUL84 revealed a domain of 282 amino acids which is required for binding to the importin α proteins. Its function as a nuclear localization signal (NLS) was confirmed by fusion to heterologous proteins. Although containing a cluster of basic amino acids similar to classical NLSs, this cluster did not contain the NLS activity. Thus, a complex structure appears to be essential for importin α binding and import activity.

Geno- and Phenotypic Characterization of Human Cytomegalovirus Mutants Selected In Vitro after Letermovir (AIC246) Exposure

ABSTRACT Letermovir is a novel antiviral compound currently in clinical development for the prevention of human cytomegalovirus (HCMV) infections. In contrast to all currently approved anti-HCMV drugs that target the viral DNA polymerase, letermovir acts via a distinct mode of action involving the viral terminase subunit pUL56. To extend our understanding of potential letermovir resistance mechanisms, we used marker transfer to characterize mutations identified in letermovir-resistant HCMV variants that were selected in cell culture.

In Vitro Evaluation of the Activities of the Novel Anticytomegalovirus Compound AIC246 (Letermovir) against Herpesviruses and Other Human Pathogenic Viruses

ABSTRACT AIC246 (letermovir) is a potent anticytomegalovirus drug in clinical development. Here, we report a consistent antiviral efficacy of AIC246 against human cytomegalovirus laboratory strains, clinical isolates, and virus variants resistant to approved drugs. Furthermore, we describe a remarkable selectivity of AIC246 for human cytomegaloviruses compared to that of other alpha-, beta-, or gammaherpesviruses or nonrelated pathogenic viruses, including adeno-, hepadna-, retro-, orthomyxo-, and flaviviruses. Our data confirm and support an excellent and selective anticytomegaloviral activity of AIC246.

RNA-binding of the human cytomegalovirus transactivator protein UL69, mediated by arginine-rich motifs, is not required for nuclear export of unspliced RNA

The human cytomegalovirus protein pUL69 belongs to a family of regulatory factors that is conserved within the Herpesviridae and includes the proteins ICP27 of herpes simplex virus type 1 and EB2 of Epstein–Barr virus. ICP27 and EB2 have been shown to facilitate the nuclear export of viral mRNAs via interacting with the cellular mRNA export factor REF. Furthermore, direct RNA-binding of these proteins was found to be essential for their stimulating effects on mRNA export. Recently, we demonstrated that pUL69 shares common features with ICP27 and EB2 such as (i) nucleocytoplasmic shuttling and (ii) stimulation of nuclear RNA export via binding to the cellular mRNA export machinery. Here, we demonstrate that pUL69 can also interact with RNA both in vivo and in vitro via a complex N-terminal RNA-binding domain consisting of three arginine-rich motifs. Interestingly, the RNA-binding domain of pUL69 overlaps with both the NLS and the binding site of the cellular mRNA export factors UAP56 and URH49. While the deletion of the UAP56/URH49-binding site abolished pUL69-mediated RNA export, an RNA-binding deficient pUL69 mutant which still interacts with UAP56/URH49 retained its RNA export activity. This surprising finding suggests that, in contrast to its homologues, RNA-binding is not a prerequisite for pUL69-mediated nuclear RNA export.

Characterization of Cytomegalovirus Breakthrough Events in a Phase 2 Prophylaxis Trial of Letermovir (AIC246, MK 8228)

BACKGROUND The efficacy of different letermovir (AIC246, MK8228) doses (60, 120, and 240 mg/day) against human cytomegalovirus (HCMV) was evaluated in a recent phase 2b dose-range-finding prophylaxis study in stem-cell transplant recipients. Here we report the genotypic and phenotypic characterization of 15 viral breakthroughs considered to be virological failures. METHODS Direct sequencing of an HCMV open reading frame UL56 region that included amino acids 230-370 and thus encompassed all known letermovir resistance mutations was followed by marker-transfer experiments to assess the impact of the identified sequence polymorphisms on viral fitness and susceptibility to letermovir. RESULTS UL56 genotyping was successful for 12 of 15 patients. Six amino acid substitutions were detected in 5 patients. In 1 subject from the 60-mg-dose group, the known letermovir resistance mutation V236M was identified subsequent to a wild-type viremic episode. The remaining 5 sequence variants (L134V, S227I, Q228H, R410G, and D414N) were shown to be inert with regard to letermovir susceptibility, thus representing natural polymorphisms. CONCLUSIONS Our findings represent the first case of a letermovir resistance mutation emerging in the clinic, apparently because of a suboptimal prophylactic dose (60 mg/day). This is in agreement with the trials efficacy analyses, findings of which suggest that letermovir doses of 60 mg/day and 120 mg/day are suboptimal for prophylaxis whereas a dose of 240 mg/day appears to achieve complete suppression of viremia.