The combination of pleconaril, rupintrivir, and remdesivir efficiently inhibits enterovirus infections in vitro, delaying the development of drug-resistant virus variants.

Enteroviruses are a significant global health concern, causing a spectrum of diseases from the common cold to more severe conditions like hand-foot-and-mouth disease, meningitis, myocarditis, pancreatitis, and poliomyelitis. Current treatment options for these infections are limited, underscoring the urgent need for effective therapeutic strategies. To find better treatment option we analyzed toxicity and efficacy of 12 known broad-spectrum anti-enterovirals both individually and in combinations against different enteroviruses in vitro. We identified several novel, synergistic two-drug and three-drug combinations that demonstrated significant inhibition of enterovirus infections in vitro. Specifically, the triple-drug combination of pleconaril, rupintrivir, and remdesivir exhibited remarkable efficacy against echovirus (EV) 1, EV6, EV11, and coxsackievirus (CV) B5, in human lung epithelial A549 cells. This combination surpassed the effectiveness of single-agent or dual-drug treatments, as evidenced by its ability to protect A549 cells from EV1-induced cytotoxicity across seven passages. Additionally, this triple-drug cocktail showed potent antiviral activity against EV-A71 in human intestinal organoids. Thus, our findings highlight the therapeutic potential of the pleconaril-rupintrivir-remdesivir combination as a broad-spectrum treatment option against a range of enterovirus infections. The study also paves the way towards development of strategic antiviral drug combinations with virus family coverage and high-resistance barriers.

The methyltransferase N6AMT1 participates in the cell cycle by regulating cyclin E levels.

The methyltransferase N6AMT1 has been associated with the progression of different pathological conditions, such as tumours and neurological malfunctions, but the underlying mechanism is not fully understood. Analysis of N6AMT1-depleted cells revealed that N6AMT1 is involved in the cell cycle and cell proliferation. In N6AMT1-depleted cells, the cell doubling time was increased, and cell progression out of mitosis and the G0/G1 and S phases was disrupted. It was discovered that in N6AMT1-depleted cells, the transcription of cyclin E was downregulated, which indicates that N6AMT1 is involved in the regulation of cyclin E transcription. Understanding the functions and importance of N6AMT1 in cell proliferation and cell cycle regulation is essential for developing treatments and strategies to control diseases that are associated with N6AMT1.

How the Intrinsically Disordered N-Terminus of Cancer/Testis Antigen MAGEA10 Is Responsible for Its Expression, Nuclear Localisation and Aberrant Migration.

Melanoma-associated antigen A (MAGEA) subfamily proteins are normally expressed in testis and/or placenta. However, aberrant expression is detected in the tumour cells of multiple types of human cancer. MAGEA expression is mainly observed in cancers that have acquired malignant phenotypes, invasiveness and metastasis, and the expression of MAGEA family proteins has been linked to poor prognosis in cancer patients. All MAGE proteins share the common MAGE homology domain (MHD) which encompasses up to 70% of the protein; however, the areas flanking the MHD region vary between family members and are poorly conserved. To investigate the molecular basis of MAGEA10 expression and anomalous mobility in gel, deletion and point-mutation, analyses of the MAGEA10 protein were performed. Our data show that the intrinsically disordered N-terminal domain and, specifically, the first seven amino acids containing a unique linear motif, PRAPKR, are responsible for its expression, aberrant migration in SDS-PAGE and nuclear localisation. The aberrant migration in gel and nuclear localisation are not related to each other. Hiding the N-terminus with an epitope tag strongly affected its mobility in gel and expression in cells. Our results suggest that the intrinsically disordered domains flanking the MHD determine the unique properties of individual MAGEA proteins.

Human TRMT112-Methyltransferase Network Consists of Seven Partners Interacting with a Common Co-Factor.

Methylation is an essential epigenetic modification mainly catalysed by S-Adenosyl methionine-dependent methyltransferases (MTases). Several MTases require a cofactor for their metabolic stability and enzymatic activity. TRMT112 is a small evolutionary conserved protein that acts as a co-factor and activator for different MTases involved in rRNA, tRNA and protein methylation. Using a SILAC screen, we pulled down seven methyltransferases-N6AMT1, WBSCR22, METTL5, ALKBH8, THUMPD2, THUMPD3 and TRMT11-as interaction partners of TRMT112. We showed that TRMT112 stabilises all seven MTases in cells. TRMT112 and MTases exhibit a strong mutual feedback loop when expressed together in cells. TRMT112 interacts with its partners in a similar way; however, single amino acid mutations on the surface of TRMT112 reveal several differences as well. In summary, mammalian TRMT112 can be considered as a central "hub" protein that regulates the activity of at least seven methyltransferases.

Heterochromatin Networks: Topology, Dynamics, and Function (a Working Hypothesis).

Open systems can only exist by self-organization as pulsing structures exchanging matter and energy with the outer world. This review is an attempt to reveal the organizational principles of the heterochromatin supra-intra-chromosomal network in terms of nonlinear thermodynamics. The accessibility of the linear information of the genetic code is regulated by constitutive heterochromatin (CHR) creating the positional information in a system of coordinates. These features include scale-free splitting-fusing of CHR with the boundary constraints of the nucleolus and nuclear envelope. The analysis of both the literature and our own data suggests a radial-concentric network as the main structural organization principle of CHR regulating transcriptional pulsing. The dynamic CHR network is likely created together with nucleolus-associated chromatin domains, while the alveoli of this network, including springy splicing speckles, are the pulsing transcription hubs. CHR contributes to this regulation due to the silencing position variegation effect, stickiness, and flexible rigidity determined by the positioning of nucleosomes. The whole system acts in concert with the elastic nuclear actomyosin network which also emerges by self-organization during the transcriptional pulsing process. We hypothesize that the the transcriptional pulsing, in turn, adjusts its frequency/amplitudes specified by topologically associating domains to the replication timing code that determines epigenetic differentiation memory.

MAGEA4 Coated Extracellular Vesicles Are Stable and Can Be Assembled In Vitro.

Extracellular vesicles (EVs) are valued candidates for the development of new tools for medical applications. Vesicles carrying melanoma-associated antigen A (MAGEA) proteins, a subfamily of cancer-testis antigens, are particularly promising tools in the fight against cancer. Here, we have studied the biophysical and chemical properties of MAGEA4-EVs and show that they are stable under common storage conditions such as keeping at +4 °C and -80 °C for at least 3 weeks after purification. The MAGEA4-EVs can be freeze-thawed two times without losing MAGEA4 in detectable quantities. The attachment of MAGEA4 to the surface of EVs cannot be disrupted by high salt concentrations or chelators, but the vesicles are sensitive to high pH. The MAGEA4 protein can bind to the surface of EVs in vitro, using robust passive incubation. In addition, EVs can be loaded with recombinant proteins fused to the MAGEA4 open reading frame within the cells and also in vitro. The high stability of MAGEA4-EVs ensures their potential for the development of EV-based anti-cancer applications.

The Common Partner of Several Methyltransferases TRMT112 Regulates the Expression of N6AMT1 Isoforms in Mammalian Cells.

Methylation is a widespread modification occurring in DNA, RNA and proteins. The N6AMT1 (HEMK2) protein has DNA N6-methyladenine as well as the protein glutamine and histone lysine methyltransferase activities. The human genome encodes two different isoforms of N6AMT1, the major isoform and the alternatively spliced isoform, where the substrate binding motif is missing. Several RNA methyltransferases involved in ribosome biogenesis, tRNA methylation and translation interact with the common partner, the TRMT112 protein. In this study, we show that TRMT112 regulates the expression of N6AMT1 isoforms in mammalian cells. Both isoforms are equally expressed on mRNA level, but only isoform 1 is detected on the protein level in human cells. We show that the alternatively spliced isoform is not able to interact with TRMT112 and when translated, is rapidly degraded from the cells. This suggests that TRMT112 is involved in cellular quality control ensuring that N6AMT1 isoform with missing substrate binding domain is eliminated from the cells. The down-regulation of TRMT112 does not affect the N6AMT1 protein levels in cells, suggesting that the two proteins of TRMT112 network, WBSCR22 and N6AMT1, are differently regulated by their common cofactor.

Cancer-testis antigens MAGEA proteins are incorporated into extracellular vesicles released by cells.

Melanoma-associated antigen A (MAGEA) family proteins represent a class of tumor antigens that are expressed in a variety of malignant tumors, but their expression in normal tissues is restricted to germ cells. MAGEA family consists of eleven proteins that are highly conserved sharing the common MAGE homology domain (MHD). In the current study, we show that MAGEA4 and MAGEA10 proteins are incorporated into extracellular vesicles released by mouse fibroblast and human osteosarcoma U2OS cells and are expressed, at least partly, on the surface of released EVs. The C-terminal part of the protein containing MHD domain is required for this activity. Expression of MAGEA proteins induced the budding of cells and formation of extracellular vesicles with 150 to 1500 nm in diameter. Our data suggest that the release of MAGEA-positive EVs is at least to some extent induced by the expression of MAGEA proteins itself. This may be one of the mechanisms of MAGEA proteins to induce cancer formation and progression.

Antibody response against cancer-testis antigens MAGEA4 and MAGEA10 in patients with melanoma.

Melanoma-associated antigen A (MAGEA) represent a class of tumor antigens that are expressed in a variety of malignant tumors, however, their expression in healthy normal tissues is restricted to germ cells of testis, fetal ovary and placenta. The restricted expression and immunogenicity of these antigens make them ideal targets for immunotherapy in human cancer. In the present study the presence of naturally occurring antibodies against two MAGEA subfamily proteins, MAGEA4 and MAGEA10, was analyzed in patients with melanoma at different stages of disease. Results indicated that the anti-MAGEA4/MAGEA10 immune response in melanoma patients was heterogeneous, with only ~8% of patients having a strong response. Comparing the number of strongly responding patients between different stages of disease revealed that the highest number of strong responses was detected among stage II melanoma patients. These findings support the model that the immune system is involved in the control of melanoma in the early stages of disease.

Biochemical and proteomic characterization of retrovirus Gag based microparticles carrying melanoma antigens.

Extracellular vesicles are membraneous particles released by a variety of cells into the extracellular microenvironment. Retroviruses utilize the cellular vesiculation pathway for virus budding/assembly and the retrovirus Gag protein induces the spontaneous formation of microvesicles or virus-like particles (VLPs) when expressed in the mammalian cells. In this study, five different melanoma antigens, MAGEA4, MAGEA10, MART1, TRP1 and MCAM, were incorporated into the VLPs and their localization within the particles was determined. Our data show that the MAGEA4 and MAGEA10 proteins as well as MCAM are expressed on the surface of VLPs. The compartmentalization of exogenously expressed cancer antigens within the VLPs did not depend on the localization of the protein within the cell. Comparison of the protein content of VLPs by LC-MS/MS-based label-free quantitative proteomics showed that VLPs carrying different cancer antigens are very similar to each other, but differ to some extent from VLPs without recombinant antigen. We suggest that retrovirus Gag based virus-like particles carrying recombinant antigens have a potential to be used in cancer immunotherapy.

The Stability of Ribosome Biogenesis Factor WBSCR22 Is Regulated by Interaction with TRMT112 via Ubiquitin-Proteasome Pathway.

The human WBSCR22 protein is a 18S rRNA methyltransferase involved in pre-rRNA processing and ribosome 40S subunit biogenesis. Recent studies have shown that the protein function in ribosome synthesis is independent of its enzymatic activity. In this work, we have studied the WBSCR22 protein interaction partners by SILAC-coupled co-immunoprecipitation assay and identified TRMT112 as the interaction partner of WBSCR22. Knock-down of TRMT112 expression decreased the WBSCR22 protein level in mammalian cells, suggesting that the stability of WBSCR22 is regulated through the interaction with TRMT112. The localization of the TRMT112 protein is determined by WBSCR22, and the WBSCR22-TRMT112 complex is localized in the cell nucleus. We provide evidence that the interaction between WBSCR22/Bud23 and TRMT112/Trm112 is conserved between mammals and yeast, suggesting that the function of TRMT112 as a co-activator of methyltransferases is evolutionarily conserved. Finally, we show that the transiently expressed WBSCR22 protein is ubiquitinated and degraded through the proteasome pathway, revealing the tight control of the WBSCR22 protein level in the cells.

Characterization of the nuclear matrix targeting sequence (NMTS) of the BPV1 E8/E2 protein--the shortest known NMTS.

Technological advantages in sequencing and proteomics have revealed the remarkable diversity of alternative protein isoforms. Typically, the localization and functions of these isoforms are unknown and cannot be predicted. Also the localization signals leading to particular subnuclear compartments have not been identified and thus, predicting alternative functions due to alternative subnuclear localization is limited only to very few subnuclear compartments. Knowledge of the localization and function of alternative protein isoforms allows for a greater understanding of cellular complexity. In this article, we characterize a short and well-defined signal targeting the bovine papillomavirus type 1 E8/E2 protein to the nuclear matrix. The targeting signal comprises the peptide coded by E8 ORF, which is spliced together with part of the E2 ORF to generate the E8/E2 mRNA. Localization to the nuclear matrix correlates well with the transcription repression activities of E8/E2; a single point mutation directs the E8/E2 protein into the nucleoplasm, and transcription repression activity is lost. Our data prove that adding as few as ˜10 amino acids by alternative transcription/alternative splicing drastically alters the function and subnuclear localization of proteins. To our knowledge, E8 is the shortest known nuclear matrix targeting signal.

DAXX modulates human papillomavirus early gene expression and genome replication in U2OS cells.

BACKGROUND: The human papillomavirus (HPV) genomes can replicate, and are maintained as autonomously replicating extrachromosomal plasmids in human U2OS cells. Previous studies have shown that HPV genomes are transcriptionally active in U2OS cells and can express the viral early proteins required for initiation and establishment of HPV replication. In the present work, we have examined the involvement of cellular DAXX protein in HPV replication in U2OS cells. METHODS: We have used indirect immunofluorescence and FISH analysis in order to study HPV replication compartments in U2OS cells. In addition, we have used siRNA knock-down for examining the effect of the DAXX protein on HPV replication and transcription in U2OS cells. RESULTS: We show that a portion of HPV replication foci are partially co-localized with components of ND10, cellular DAXX and PML proteins. In addition, we demonstrate that the knock-down of the cellular DAXX protein modulates the HPV genome replication and transcription in U2OS cells--papillomavirus replication is reduced in the absence of this component of ND10. CONCLUSIONS: The DAXX protein modulates the early gene expression and the transient replication of HPV genomes in U2OS cells.

The human WBSCR22 protein is involved in the biogenesis of the 40S ribosomal subunits in mammalian cells.

The human WBSCR22 protein was previously shown to be up-regulated in invasive breast cancer and its ectopic expression enhances tumor cell survival in the vasculature. In the current study, we show that the WBSCR22 protein is important for cell growth. Knock-down of WBSCR22 with siRNA results in slower growth of WBSCR22-depleted cells. Treatment with siWBSCR22 causes defects in the processing of pre-rRNAs and reduces the level of free 40S ribosomal subunit, suggesting that WBSCR22 is involved in ribosome small subunit biosynthesis. The human WBSCR22 partially complements the growth of WBSCR22 yeast homologue, bud23 deletion mutant suggesting that the human WBSCR22 is a functional homologue of yeast Bud23. WBSCR22 is localized throughout the cell nucleus and is not stably associated with ribosomal subunits within the cell nucleus. We also show that the WBSCR22 protein level is decreased in lymphoblastoid cell lines derived from William-Beuren Syndrome (WBS) patients compared to healthy controls. Our data suggest that the WBSCR22 protein is a ribosome biogenesis factor involved in the biosynthesis of 40S ribosomal particles in mammalian cells.

Identification and analysis of papillomavirus E2 protein binding sites in the human genome.

Papillomavirus E2 protein is required for the replication and maintenance of viral genomes and transcriptional regulation of viral genes. E2 functions through sequence-specific binding to 12-bp DNA motifs-E2 binding sites (E2BS)-in the virus genome. Papillomaviruses are able to establish persistent infection in their host and have developed a long-term relationship with the host cell in order to guarantee the propagation of the virus. In this study, we have analyzed the occurrence and functionality of E2BSs in the human genome. Our computational analysis indicates that most E2BSs in the human genome are found in repetitive DNA regions and have G/C-rich spacer sequences. Using a chromatin immunoprecipitation approach, we show that human papillomavirus type 11 (HPV11) E2 interacts with a subset of cellular E2BSs located in active chromatin regions. Two E2 activities, sequence-specific DNA binding and interaction with cellular Brd4 protein, are important for E2 binding to consensus sites. E2 binding to cellular E2BSs has a moderate or no effect on cellular transcription. We suggest that the preference of HPV E2 proteins for E2BSs with A/T-rich spacers, which are present in the viral genomes and underrepresented in the human genome, ensures E2 binding to specific binding sites in the virus genome and may help to prevent extensive and possibly detrimental changes in cellular transcription in response to the viral protein.

Human papillomavirus E2 protein with single activation domain initiates HPV18 genome replication, but is not sufficient for long-term maintenance of virus genome.

The papillomavirus life cycle is regulated by a family of proteins encoded by the E2 open reading frame; E2 proteins regulate viral gene expression, DNA replication and genome maintenance. We have previously shown that the bovine papillomavirus (BPV1) full-length E2 protein forms heterodimers with repressor forms of E2, and these E2 heterodimers serve as activators of transcription and replication during the viral life cycle. In the present study, using the single-chain E2 heterodimer as a model, we show that human papillomavirus (HPV) 11 and 18 E2 heterodimers with single activation domain are able to initiate replication of URR-containing plasmid in transient assay. Single-chain E2 heterodimer in the context of HPV18 genome initiates genome replication, but is not sufficient for long-term replication of HPV18 genome. We also show that HPV18 genome has a capacity to encode truncated E2 repressor E8/E2 which acts as a negative regulator of HPV18 genome replication.

Bovine papillomavirus type 1 E2 protein heterodimer is functional in papillomavirus DNA replication in vivo.

Papillomaviruses are small DNA viruses that induce epithelial lesions in their host. The viral life cycle is regulated by the family of proteins encoded by the E2 open reading frame. In addition to the full-length E2 protein, the BPV-1 genome encodes two truncated E2 proteins, E2C and E8/E2, which maintain the DNA-binding-dimerization domains, but lack the activation domain. Heterodimers formed between the full-length E2 and truncated E2 proteins serve as activators of E2-dependent transcription and papillomavirus DNA replication. We show that the single activation domain of E2 is sufficient for interaction with viral helicase E1 and for initiation of DNA replication from different papillomavirus origins. Single-chain E2 heterodimer is able to activate papillomavirus DNA replication in the context of entire BPV genome in the absence of other E2 proteins. These data suggest that E2 heterodimers with single activation domain are functional in initiation of papillomavirus replication in vivo.

Coinfection of Chlamydia trachomatis, Ureaplasma urealyticum and human papillomavirus among patients attending STD clinics in Estonia.

Women visiting Estonian STD clinics were subjected to PCR assay for human papillomavirus (HPV), Chlamydia trachomatis and Ureaplasma urealyticum biovar 2. The overall prevalence of coinfection was 8%. The chlamydial infection was found to be associated with HPV, especially with high-risk HPV (OR=2.5, p<0.005) and most significantly in women over 41 y of age. C. trachomatis infection also occurred more frequently in U. urealyticum-infected than in U. urealyticum-free patients (OR=2.6, p=0.02). U. urealyticum infection did not associate with HPV status. The clinical significance of the association between C. trachomatis and U. urelyticum infection remains to be elucidated.

Characterization of the functional activities of the bovine papillomavirus type 1 E2 protein single-chain heterodimers.

Papillomaviruses are small DNA viruses which establish persistent infection in the epithelial tissue of various animal species. Three papillomavirus proteins encoded by the bovine papillomavirus type 1 E2 open reading frame have a common C-terminal DNA binding and dimerization domain and function as dimeric proteins in the regulation of viral gene expression, genome replication, and maintenance. The full-length E2 protein, expressed usually at the lowest level of the three, is an activator, while shorter forms of E2, lacking the transactivation domain, serve as repressors of replication and transcription. In virally infected cells, the full-length E2 protein forms heterodimers with repressor forms of the E2 protein and the biological activities of such heterodimers are poorly known. In order to study the functionality of E2 heterodimers, we joined the full-length E2 protein and E2 repressor by a flexible polypeptide hinge so that they formed a single-chain intramolecular dimer. The single-chain E2 heterodimers folded correctly to form genuine pseudodimers capable of binding to the specific E2 protein binding site with high affinity. Characterization of the activities of this protein in transcription showed that it functions as an effective transcriptional activator, which is comparable to what was found for the full-length E2 protein. The single-chain heterodimer is dependent to some extent on Brd4 protein and is able to support papillomavirus origin replication; however, it does not support the partitioning of the multimeric E2 binding site containing plasmids in dividing cells. Our results suggest that E2 heterodimers serve as activators of transcription and replication during the viral life cycle.

Association of bovine papillomavirus E2 protein with nuclear structures in vivo.

Papillomaviruses are small DNA viruses which have the capacity to establish a persistent infection in mammalian epithelial cells. The papillomavirus E2 protein is a central coordinator of viral gene expression, genome replication, and maintenance. We have investigated the distribution of bovine papillomavirus E2 protein in nuclei of proliferating cells and found that E2 is associated with cellular chromatin. This distribution does not change during the entire cell cycle. The N-terminal transactivation domain, but not the C-terminal DNA-binding domain, of the E2 protein is responsible for this association. The majority of the full-length E2 protein can only be detected in chromatin-enriched fractions but not as a free protein in the nucleus. Limited micrococcal nuclease digestion revealed that the E2 protein partitioned to different chromatin regions. A fraction of the E2 protein was located at nuclear sites that are resistant against nuclease attack, whereas the remaining E2 resided on compact chromatin accessible to micrococcal nuclease. These data suggest that there are two pools of E2 in the cell nucleus: one that localizes on transcriptionally inactive compact chromatin and the other, which compartmentalizes to transcriptionally active nuclear structures of the cell. Our data also suggest that E2 associates with chromatin through cellular protein(s), which in turn is released from chromatin at 0.4 M salt.