The POU-HD TFs impede the replication efficiency of several human papillomavirus genomes.

Human papillomavirus (HPV) is a double-stranded DNA virus that infects cutaneous and mucosal epithelial cells. HPV replication initiates at the origin (ori), located within a noncoding region near the major early promoter. Only two viral proteins, E1 and E2, are essential for replication, with the host cell contributing other necessary factors. However, the role of host cell proteins in regulating HPV replication remains poorly understood. While several binding sites for cellular transcription factors (TFs), such as POU-HD proteins, have been mapped in the regulatory region, their functional importance is unclear. Some POU-HD TFs have been shown to influence replication in a system where E1 and E2 are provided exogenously. In this study, we investigated the impact of several POU-HD TFs on the replication of the HPV5, HPV11, and HPV18 genomes in U2OS cells and human primary keratinocytes. We demonstrated that OCT1, OCT6, BRN5A, and SKN1A are expressed in HPV host cells and that their overexpression inhibits HPV genome replication, whereas knocking down OCT1 had a positive effect. Using the replication-deficient HPV18-E1- genome, we demonstrated that OCT1-mediated inhibition of HPV replication involves modulation of HPV early promoters controlling E1 and E2 expression. Moreover, using Oct6 mutants deficient either in DNA binding or transcriptional regulation, we showed that the inhibition of HPV18 replication is solely dependent on Oct6's DNA binding activity. Our study highlights the complex regulatory roles of POU-HD factors in the HPV replication.

Characterization and comparative analysis of phosphorylation patterns in HPV18 and HPV11 E1 helicases: Implications for viral genome replication.

The genome of human papillomaviruses (HPVs) encodes the E1 replication factor, whose biological activities are regulated by cellular protein kinases. Here, the phosphorylation pattern of the E1 helicase of oncogenic mucosotropic HPV18 was investigated both in vitro and in vivo. Four serine residues located in a short peptide within a localization regulatory region were found to be phosphorylated in both experimental settings. We demonstrate that this peptide is targeted in vitro by various protein kinases, including CK2, PKA, and CKD2/cyclin A/B/E complexes. Through point mutagenesis, we show that phosphorylation of this region is essential for E1 subcellular localization, the interaction of E1 with the E2 protein, and replication of the HPV18 genome. Furthermore, we demonstrate the functional conservation of this phosphorylation across the E1 proteins of the low-risk mucosotropic HPV11 and high-risk cutaneotropic HPV5. These findings provide deeper insights into the phosphorylation-mediated regulation of biological activities of the E1 protein.

Phosphorylation of E2 Serine Residue 402 Is Required for the Transcription and Replication of the HPV5 Genome.

Cutaneous human papillomavirus type 5 (HPV5) belongs to the supposedly oncogenic ß-HPVs associated with specific types of skin and oral cavity cancers. Three viral proteins, namely, helicase E1 and transcription factors E2 and E8^E2, are master regulators of the viral life cycle. HPV5 E2 is a transcriptional activator that also participates in the E1-dependent replication and nuclear retention of the viral genome, whereas E8^E2 counterbalances the activity of E2 and inhibits HPV transcription and replication. In the present study, we demonstrate that the HPV5 E2 protein is extensively phosphorylated by cellular protein kinases, and serine residue 402 (S402) is the highest scoring phosphoacceptor site. This residue is located within a motif conserved among many ß-HPVs and in the oncogenic HPV31 α-type. Using the nonphosphorylatable and phosphomimetic mutants, we demonstrate that phosphorylation of the E2 S402 residue is required for the transcription and replication of the HPV5 genome in U2OS cells and human primary keratinocytes. Mechanistically, the E2-S402-phopshodeficient protein is unable to trigger viral gene transcription and has an impaired ability to support E1-dependent replication, but the respective E8^E2-S213 mutant displays no phenotype. However, phosphorylation of the E2 S402 residue has no impact on the E2 stability, subcellular localization, self-assembly, DNA-binding capacity, and affinity to the E1 and BRD4 proteins. Further studies are needed to identify the protein kinase(s) responsible for this phosphorylation. IMPORTANCE Human papillomavirus type 5 (HPV5) may play a role in the development of specific types of cutaneous and head and neck cancers. The persistence of the HPV genome in host cells depends on the activity of its proteins, namely, a helicase E1 and transcription/replication factor E2. The latter also facilitates the attachment of episomal viral genomes to host cell chromosomes. In the present study, we show that the HPV5 E2 protein is extensively phosphorylated by host cell protein kinases, and we identify serine residue 402 as the highest scoring phosphoacceptor site of E2. We demonstrate that the replication of the HPV5 genome may be blocked by a single point mutation that prevents phosphorylation of this serine residue and switches off the transcriptional activity of the E2 protein. The present study contributes to a better understanding of ß-HPV5 replication and its regulation by host cell protein kinases.

Analysis of the Replication Mechanisms of the Human Papillomavirus Genomes.

The life-cycle of human papillomaviruses (HPVs) includes three distinct phases of the viral genome replication. First, the viral genome is amplified in the infected cells, and this amplification is often accompanied by the oligomerization of the viral genomes. Second stage includes the replication of viral genomes in concert with the host cell genome. The viral genome is further amplified during the third stage of the viral-life cycle, which takes place only in the differentiated keratinocytes. We have previously shown that the HPV18 genomes utilize at least two distinct replication mechanisms during the initial amplification. One of these mechanisms is a well-described bidirectional replication via theta type of replication intermediates. The nature of another replication mechanism utilized by HPV18 involves most likely recombination-dependent replication. In this paper, we show that the usage of different replication mechanisms is a property shared also by other HPV types, namely HPV11 and HPV5. We further show that the emergence of the recombination dependent replication coincides with the oligomerization of the viral genomes and is dependent on the replicative DNA polymerases. We also show that the oligomeric genomes of HPV18 replicate almost exclusively using recombination dependent mechanism, whereas monomeric HPV31 genomes replicate bi-directionally during the maintenance phase of the viral life-cycle.

Cyclic AMP-Dependent Protein Kinase Exhibits Antagonistic Effects on the Replication Efficiency of Different Human Papillomavirus Types.

Several types of widespread human papillomaviruses (HPVs) may induce the transformation of infected cells, provoking the development of neoplasms. Two main genera of HPVs are classified as mucosatropic alphapapillomaviruses and cutaneotropic betapapillomaviruses (α- and ß-HPVs, respectively), and they both include high-risk cancer-associated species. The absence of antiviral drugs has driven investigations into the details of the molecular mechanisms of the HPV life cycle. HPV replication depends on the viral helicase E1 and the transcription factor E2. Their biological activities are controlled by numerous cellular proteins, including protein kinases. Here, we report that ubiquitously expressed cyclic AMP-dependent protein kinase A (PKA) differentially regulates the replication of α-HPV11, α-HPV18, and ß-HPV5. PKA stimulates the replication of both α-HPVs studied but has a more profound effect on the replication of high-risk α-HPV18. However, the replication of ß-HPV5 is inhibited by activated PKA in human primary keratinocytes and U2OS cells. We show that the activation of PKA signaling by different pharmacological agents induces the rapid proteasomal degradation of the HPV5 E2 protein, which in turn leads to the downregulation of E2-dependent transcription. In contrast, PKA-stimulated induction of HPV18 replication is the result of the downregulation of the E8^E2 transcript encoding a potent viral transcriptional inhibitor together with the rapid upregulation of E1 and E2 protein levels. IMPORTANCE Several types of human papillomaviruses (HPVs) are causative agents of various types of epithelial cancers. Here, we report that ubiquitously expressed cyclic AMP-dependent protein kinase A (PKA) differentially regulates the replication of various types of HPVs during the initial amplification and maintenance phases of the viral life cycle. The replication of the skin cancer-related pathogen HPV5 is suppressed, whereas the replication of the cervical cancer-associated pathogen HPV18 is activated, in response to elevated PKA activity. To inhibit HPV5 replication, PKA targets the viral transcriptional activator E2, inducing its rapid proteasomal degradation. PKA-dependent stimulation of HPV18 replication relies on the downregulation of another E2 gene product, E8^E2, which encodes a potent transcriptional repressor. Our findings highlight, for the first time, protein kinase-related mechanistic differences in the regulation of the replication of mucosal and cutaneous HPV types.

Uncovering the Role of the E1 Protein in Different Stages of Human Papillomavirus 18 Genome Replication.

The life cycle of human papillomaviruses (HPVs) comprises three distinct phases of DNA replication: initial amplification, maintenance of the genome copy number at a constant level, and vegetative amplification. The viral helicase E1 is one of the factors required for the initiation of HPV genome replication. However, the functions of the E1 protein during other phases of the viral life cycle are largely uncharacterized. Here, we studied the role of the HPV18 E1 helicase in three phases of viral genome replication by downregulating E1 expression using RNA interference or inducing degradation of the E1 protein via inhibition of casein kinase 2α expression or catalytic activity. We generated a novel modified HPV18 genome expressing Nanoluc and tagged E1 and E2 proteins and created several stable HPV18-positive cell lines. We showed that, in contrast to initial amplification of the HPV18 genome, other phases of viral genome replication involve also an E1-independent mechanism. We characterize two distinct populations of HPV18 replicons existing during the maintenance and vegetative amplification phases. We show that a subset of these replicons, including viral genome monomers, replicate in an E1-dependent manner, while some oligomeric forms of the HPV18 genome replicate independently of E1 function.IMPORTANCE Human papillomavirus (HPV) infections pose serious medical problem. To date, there are no HPV-specific antivirals available due to poor understanding of the molecular mechanisms of virus infection cycle. The infection cycle of HPV involves initial amplification of the viral genomes and maintenance of the viral genomes with a constant copy number, followed by another round of viral genome amplification and new viral particle formation. The viral protein E1 is critical for the initial amplification of the viral genome. However, E1 involvement in other phases of the viral life cycle has remained controversial. In the present study, we show that at least two different replication modes of the HPV18 genome are undertaken simultaneously during the maintenance and vegetative amplification phases, i.e., replication of the majority of the HPV18 genome proceeds under the control of the host cell replication machinery without E1 function, whereas a minority of the genome replicates in an E1-dependent manner.

Differential phosphorylation determines the repressor and activator potencies of GLI1 proteins and their efficiency in modulating the HPV life cycle.

The Sonic Hedgehog (Shh) signalling pathway plays multiple roles during embryonic development and under pathological conditions. Although the core components of the Shh pathway are conserved, the regulation of signal transduction varies significantly among species and cell types. Protein kinases Ulk3 and Pka are involved in the Shh pathway as modulators of the activities of Gli transcription factors, which are the nuclear mediators of the signal. Here, we investigate the regulation and activities of two GLI1 isoforms, full-length GLI1 (GLI1FL) and GLI1ΔN. The latter protein lacks the first 128 amino acids including the conserved phosphorylation cluster and the binding motif for SUFU, the key regulator of GLI activity. Both GLI1 isoforms are co-expressed in all human cell lines analysed and possess similar DNA binding activity. ULK3 potentiates the transcriptional activity of both GLI1 proteins, whereas PKA inhibits the activity of GLI1ΔN, but not GLI1FL. In addition to its well-established role as a transcriptional activator, GLI1FL acts as a repressor by inhibiting transcription from the early promoters of human papillomavirus type 18 (HPV18). Additionally, compared to GLI1ΔN, GLI1FL is a more potent suppressor of replication of several HPV types. Altogether, our data show that the N-terminal part of GLI1FL is crucial for the realization of its full potential as a transcriptional regulator.

Activity of CK2α protein kinase is required for efficient replication of some HPV types.

Inhibition of human papillomavirus (HPV) replication is a promising therapeutic approach for intervening with HPV-related pathologies. Primary targets for interference are two viral proteins, E1 and E2, which are required for HPV replication. Both E1 and E2 are phosphoproteins; thus, the protein kinases that phosphorylate them might represent secondary targets to achieve inhibition of HPV replication. In the present study, we show that CX4945, an ATP-competitive small molecule inhibitor of casein kinase 2 (CK2) catalytic activity, suppresses replication of different HPV types, including novel HPV5NLuc, HPV11NLuc and HPV18NLuc marker genomes, but enhances the replication of HPV16 and HPV31. We further corroborate our findings using short interfering RNA (siRNA)-mediated knockdown of CK2 α and α' subunits in U2OS and CIN612 cells; we show that while both subunits are expressed in these cell lines, CK2α is required for HPV replication, but CK2α' is not. Furthermore, we demonstrate that CK2α acts in a kinase activity-dependent manner and regulates the stability and nuclear retention of endogenous E1 proteins of HPV11 and HPV18. This unique feature of CK2α makes it an attractive target for developing antiviral agents.

Characterization of Protein Kinase ULK3 Regulation by Phosphorylation and Inhibition by Small Molecule SU6668.

Serine/threonine protein kinase ULK3 is implicated in a variety of cellular processes, including autophagy, cell division, and execution of the Sonic hedgehog pathway. However, very little about how its biological activity could be controlled is known. This study focuses on unraveling biochemical insights into the mechanism of inhibition and activation of ULK3. We identify novel phosphorylation sites in ULK3 and show that autophosphorylation has no impact on the kinase activity of the protein. We further demonstrate that phosphorylation of two residues in the kinase domain of ULK3 by an as yet unidentified kinase may completely abolishes its catalytic activity. We show that a low-molecular weight inhibitor SU6668, designed as an ATP competitive inhibitor for tyrosine kinases, binds in the ATP pocket of ULK3 yet inhibits ULK3 kinase activity in a partially ATP noncompetitive manner. Finally, we demonstrate that the ULK3 kinase domain, annotated in silico, is not sufficient for its kinase activity, and additional amino acids in the 271-300 region are required.

ABCE1 is a highly conserved RNA silencing suppressor.

ATP-binding cassette sub-family E member 1 (ABCE1) is a highly conserved protein among eukaryotes and archaea. Recent studies have identified ABCE1 as a ribosome-recycling factor important for translation termination in mammalian cells, yeast and also archaea. Here we report another conserved function of ABCE1. We have previously described AtRLI2, the homolog of ABCE1 in the plant Arabidopsis thaliana, as an endogenous suppressor of RNA silencing. In this study we show that this function is conserved: human ABCE1 is able to suppress RNA silencing in Nicotiana benthamiana plants, in mammalian HEK293 cells and in the worm Caenorhabditis elegans. Using co-immunoprecipitation and mass spectrometry, we found a number of potential ABCE1-interacting proteins that might support its function as an endogenous suppressor of RNA interference. The interactor candidates are associated with epigenetic regulation, transcription, RNA processing and mRNA surveillance. In addition, one of the identified proteins is translin, which together with its binding partner TRAX supports RNA interference.

Protein kinase inhibitor SU6668 attenuates positive regulation of Gli proteins in cancer and multipotent progenitor cells.

Observations that Glioma-associated transcription factors Gli1 and Gli2 (Gli1/2), executers of the Sonic Hedgehog (Shh) signaling pathway and targets of the Transforming Growth Factor ß (TGF-ß) signaling axis, are involved in numerous developmental and pathological processes unveil them as attractive pharmaceutical targets. Unc-51-like serine/threonine kinase Ulk3 has been suggested to play kinase activity dependent and independent roles in the control of Gli proteins in the context of the Shh signaling pathway. This study aimed at investigating whether the mechanism of generation of Gli1/2 transcriptional activators has similarities regardless of the signaling cascade evoking their activation. We also elucidate further the role of Ulk3 kinase in regulation of Gli1/2 proteins and examine SU6668 as an inhibitor of Ulk3 catalytic activity and a compound targeting Gli1/2 proteins in different cell-based experimental models. Here we demonstrate that Ulk3 is required not only for maintenance of basal levels of Gli1/2 proteins but also for TGF-ß or Shh dependent activation of endogenous Gli1/2 proteins in human adipose tissue derived multipotent stromal cells (ASCs) and mouse immortalized progenitor cells, respectively. We show that cultured ASCs possess the functional Shh signaling axis and differentiate towards osteoblasts in response to Shh. Also, we demonstrate that similarly to Ulk3 RNAi, SU6668 prevents de novo expression of Gli1/2 proteins and antagonizes the Gli-dependent activation of the gene expression programs induced by either Shh or TGF-ß. Our data suggest SU6668 as an efficient inhibitor of Ulk3 kinase allowing manipulation of the Gli-dependent transcriptional outcome.

CCL5/CCR1 axis regulates multipotency of human adipose tissue derived stromal cells.

Several potential clinical applications of stem cells rely on their capacity to migrate into sites of inflammation where they contribute to tissue regeneration processes. Inflammatory signals are partially mediated by chemokines acting via their receptors expressed on the target cells. Data concerning the repertoire and biological activities of chemokine receptors in human adipose tissue derived stromal cells (ADSCs) are limited. Here we show that CCR1 is one of the few chemokine receptors expressed in ADSCs at a high level. CCR1 expression varies in ADSCs derived from different donors. It sharply decreases in the early phase of ADSCs in vitro propagation, but further demonstrates relative stability. Expression of CCR1 positively correlates with expression of SOX2, OCT4 and NANOG, transcription factors responsible for maintenance of the stemness properties of the cells. We demonstrate that signaling via CCL5/CCR1 axis triggers migration of ADSCs, activates ERK and AKT kinases, stimulates NFκB transcriptional activity and culminates in increased proliferation of CCR1(+) cells accompanied with up-regulation of SOX2, OCT4 and NANOG expression. Our data suggest that chemokine signaling via CCR1 may be involved in regulation of stemness of ADSCs.