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A Ran-binding protein facilitates nuclear import of human papillomavirus type 16.

ABSTRACT: Human papillomaviruses (HPVs) utilize an atypical mode of nuclear import during cell entry. Residing in the Golgi apparatus until mitosis onset, a subviral complex composed of the minor capsid protein L2 and viral DNA (L2/vDNA) is imported into the nucleus after nuclear envelope breakdown by associating with mitotic chromatin. In this complex, L2 plays a crucial role in the interactions with cellular factors that enable delivery and ultimately tethering of the viral genome to mitotic chromatin. To date, the cellular proteins facilitating these steps remain unknown. Here, we addressed which cellular proteins may be required for this process. Using label-free mass spectrometry, biochemical assays, microscopy, and functional virological assays, we discovered that L2 engages a hitherto unknown protein complex of Ran-binding protein 10 (RanBP10), karyopherin alpha2 (KPNA2), and dynein light chain DYNLT3 to facilitate transport towards mitotic chromatin. Thus, our study not only identifies novel cellular interactors and mechanism that facilitate a poorly understood step in HPV entry, but also a novel cellular transport complex.

SUBMITTER: Lai KY

PROVIDER: S-EPMC8139508 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Human papillomavirus type 16

Project description:HPV16 DNA methylation characterization (QV1)

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Role of ran binding protein 5 in nuclear import and assembly of the influenza virus RNA polymerase complex.

Project description:The influenza A virus RNA-dependent RNA polymerase is a heterotrimeric complex of polymerase basic protein 1 (PB1), PB2, and polymerase acidic protein (PA) subunits. It performs transcription and replication of the viral RNA genome in the nucleus of infected cells. We have identified a nuclear import factor, Ran binding protein 5 (RanBP5), also known as karyopherin beta3, importin beta3, or importin 5, as an interactor of the PB1 subunit. RanBP5 interacted with either PB1 alone or with a PB1-PA dimer but not with a PB1-PB2 dimer or the trimeric complex. The interaction between RanBP5 and PB1-PA was disrupted by RanGTP in vitro, allowing PB2 to bind to the PB1-PA dimer to form a functional trimeric RNA polymerase complex. We propose a model in which RanBP5 acts as an import factor for the newly synthesized polymerase by targeting the PB1-PA dimer to the nucleus. In agreement with this model, small interfering RNA (siRNA)-mediated knock-down of RanBP5 inhibited the nuclear accumulation of the PB1-PA dimer. Moreover, siRNA knock-down of RanBP5 resulted in the delayed accumulation of viral RNAs in infected cells, confirming that RanBP5 plays a biological role during the influenza virus life cycle.

| S-EPMC1676300 | biostudies-literature

Hypoxia-inducible factor-1 facilitates cervical cancer progression in human papillomavirus type 16 transgenic mice.

Project description:Advanced cervical cancer remains a vexing clinical challenge despite screening programs. Many of these cancers are hypoxic, and expression of the alpha subunit of the major regulator of the hypoxic cellular response, the transcription factor hypoxia-inducible factor-1 (HIF-1), is correlated with poor prognosis. Here, we tested a functional role for HIF-1alpha in pathogenesis of cervical cancer in estrogen-treated transgenic mice. Double-transgenic (DTG) mice developed locally invasive cervical cancers 70 times larger than K14-HPV16 mice. In vivo bromodeoxyuridine incorporation was elevated in DTG cancers without a significant increase in apoptosis. HIF-1alpha gain of function did not up-regulate canonical HIF-1 targets in premalignant DTG cervices, in contrast to elevation of these targets in K14-HIF-1alpha transgenic cervices. The DTG transcriptional signature included up-regulation of mRNAs encoding cytokines and chemokines, immune signaling molecules, extracellular proteases, and cell motility factors, as well as reduced expression of cell adhesion and epithelial differentiation genes. Importantly, a set of gene markers derived from the DTG transcriptome predicted cervical cancer progression in patients. This study suggests a novel paradigm for HIF-1 function evident in multistage carcinogenesis as opposed to established malignancies, including interaction with viral oncogenes to induce multiple genomic networks in premalignancy that fosters the development of advanced cervical cancer.

| S-EPMC1934541 | biostudies-literature

CCHCR1 interacts specifically with the E2 protein of human papillomavirus type 16 on a surface overlapping BRD4 binding.

Project description:The Human Papillomavirus E2 proteins are key regulators of the viral life cycle, whose functions are commonly mediated through protein-protein interactions with the host cell proteome. We identified an interaction between E2 and a cellular protein called CCHCR1, which proved highly specific for the HPV16 genotype, the most prevalent in HPV-associated cancers. Further characterization of the interaction revealed that CCHCR1 binds the N-terminal alpha helices of HPV16 E2 N-terminal domain. On this domain, the CCHCR1 binding interface overlaps that of BRD4, a key mediator of E2 transcriptional activity. Consequently a physical competition occurs between the two proteins for the binding to HPV16 E2, and CCHCR1 interferes with BRD4-mediated enhancement of E2-dependent transcription. In addition, we showed that the interaction with CCHCR1 induced a massive redistribution of HPV16 E2, from a predominantly nuclear to a cytoplasmic localization in dot-like structures, where E2 perfectly co-localizes with CCHCR1. Such a cytoplasmic docking likely interferes with the nuclear functions of HPV16 E2. Upon co-expression of BRD4 and CCHCR1, E2 accumulates both in the nucleus and in the cytoplasm, indicating that for HPV16, both sub-cellular localization and transcriptional functions of E2 may depend on the proportion of both factors within the cell. We provided evidence of a strong induction of the keratinocyte differentiation marker K10 by HPV16 E2, and showed that this activation is compromised by the interaction with CCHCR1. The specific interaction described here could thus impact on the pathogenesis of HPV16. We propose that it could underlie some specific traits of HPV16 infection, such as an enhanced propensity to give rise to lesions evolving toward cancer.

| S-EPMC3963918 | biostudies-literature

Nuclear factor I and epithelial cell-specific transcription of human papillomavirus type 16.

Project description:The transcription of human papillomavirus type 16 (HPV-16) is mediated by the viral enhancer. Epithelial cell-specific activation is achieved by the cooperative interaction of apparently ubiquitous transcriptional factors. One of them, nuclear factor I (NFI), binds seven sites within the HPV-16 enhancer. Point mutations on enhancer fragments, which retain epithelial cell specificity, verify the functional contribution of NFI. In band shift experiments, the epithelial cell-derived NFI proteins CTF-1, CTF-2, and CTF-3 form a characteristic pattern of heterodimeric complexes which are observed in all epithelial cells tested. Divergence from this pattern in fibroblasts, liver cells, and lymphoid cells correlates with the lack of HPV-16 enhancer activation. The HPV-16 enhancer can be activated by CTF-1 in SL-2 cells, which lack NFI-like proteins. However, exogenous CTF-1 fails to overcome the inactivity of the viral enhancer in fibroblasts. Western immunoblot and supershift analysis shows that exogenously introduced CTF-1 proteins form different heterodimer complexes with the given subset of endogenous NFI proteins in epithelial or fibroblast cells. Polymerase chain reaction analysis and cDNA library screens identified the endogenous fibroblast type NFI as NFI-X, an NFI family member originally cloned from hamster liver cells. The strict correlation between the activation or lack of activation of the HPV-16 enhancer and cell-specific subsets of NFI proteins argues for the pivotal role of NFI binding sites in the epithelial cell-specific function of the viral enhancer.

| S-EPMC237828 | biostudies-other

Regulated binding of importin-α to protein kinase Cδ in response to apoptotic signals facilitates nuclear import.

Project description:PKCδ translocates into the nucleus in response to apoptotic agents and functions as a potent cell death signal. Cytoplasmic retention of PKCδ and its transport into the nucleus are essential for cell homeostasis, but how these processes are regulated is poorly understood. We show that PKCδ resides in the cytoplasm in a conformation that precludes binding of importin-α. A structural model of PKCδ in the inactive state suggests that the nuclear localization sequence (NLS) is prevented from binding to importin-α through intramolecular contacts between the C2 and catalytic domains. We have previously shown that PKCδ is phosphorylated on specific tyrosine residues in response to apoptotic agents. Here, we show that phosphorylation of PKCδ at Tyr-64 and Tyr-155 results in a conformational change that allows exposure of the NLS and binding of importin-α. In addition, Hsp90 binds to PKCδ with similar kinetics as importin-α and is required for the interaction of importin-α with the NLS. Finally, we elucidate a role for a conserved PPxxP motif, which overlaps the NLS, in nuclear exclusion of PKCδ. Mutagenesis of the conserved prolines to alanines enhanced importin-α binding to PKCδ and induced its nuclear import in resting cells. Thus, the PPxxP motif is important for maintaining a conformation that facilitates cytosplasmic retention of PKCδ. Taken together, this study establishes a novel mechanism that retains PKCδ in the cytoplasm of resting cells and regulates its nuclear import in response to apoptotic stimuli.

| S-EPMC3195609 | biostudies-literature

Direct binding of retromer to human papillomavirus type 16 minor capsid protein L2 mediates endosome exit during viral infection.

Project description:Trafficking of human papillomaviruses to the Golgi apparatus during virus entry requires retromer, an endosomal coat protein complex that mediates the vesicular transport of cellular transmembrane proteins from the endosome to the Golgi apparatus or the plasma membrane. Here we show that the HPV16 L2 minor capsid protein is a retromer cargo, even though L2 is not a transmembrane protein. We show that direct binding of retromer to a conserved sequence in the carboxy-terminus of L2 is required for exit of L2 from the early endosome and delivery to the trans-Golgi network during virus entry. This binding site is different from known retromer binding motifs and can be replaced by a sorting signal from a cellular retromer cargo. Thus, HPV16 is an unconventional particulate retromer cargo, and retromer binding initiates retrograde transport of viral components from the endosome to the trans-Golgi network during virus entry. We propose that the carboxy-terminal segment of L2 protein protrudes through the endosomal membrane and is accessed by retromer in the cytoplasm.

| S-EPMC4334968 | biostudies-literature

Importin-β facilitates nuclear import of human GW proteins and balances cytoplasmic gene silencing protein levels.

Project description:MicroRNAs (miRNAs) guide Argonaute (Ago) proteins to distinct target mRNAs leading to translational repression and mRNA decay. Ago proteins interact with a member of the GW protein family, referred to as TNRC6A-C in mammals, which coordinate downstream gene-silencing processes. The cytoplasmic functions of TNRC6 and Ago proteins are reasonably well established. Both protein families are found in the nucleus as well. Their detailed nuclear functions, however, remain elusive. Furthermore, it is not clear which import routes Ago and TNRC6 proteins take into the nucleus. Using different nuclear transport assays, we find that Ago as well as TNRC6 proteins shuttle between the cytoplasm and the nucleus. While import receptors might function redundantly to transport Ago2, we demonstrate that TNRC6 proteins are imported by the Importin-β pathway. Finally, we show that nuclear localization of both Ago2 and TNRC6 proteins can depend on each other suggesting actively balanced cytoplasmic Ago - TNRC6 levels.

| S-EPMC4551936 | biostudies-literature

The ubiquitin-specific peptidase USP15 regulates human papillomavirus type 16 E6 protein stability.

Project description:Proteomic identification of human papillomavirus type 16 (HPV16) E6-interacting proteins revealed several proteins involved in ubiquitin-mediated proteolysis. In addition to the well-characterized E6AP ubiquitin-protein ligase, a second HECT domain protein (HERC2) and a deubiquitylating enzyme (USP15) were identified by tandem affinity purification of HPV16 E6-associated proteins. This study focuses on the functional consequences of the interaction of E6 with USP15. Overexpression of USP15 resulted in increased levels of the E6 protein, and the small interfering RNA-mediated knockdown of USP15 decreased E6 protein levels. These results implicate USP15 directly in the regulation of E6 protein stability and suggest that ubiquitylated E6 could be a substrate for USP15 ubiquitin peptidase activity. It remains possible that E6 could affect the activity of USP15 on specific cellular substrates, a hypothesis that can be tested as more is learned about the substrates and pathways controlled by USP15.

| S-EPMC2738190 | biostudies-literature

Signature sequence validation of human papillomavirus type 16 (HPV-16) in clinical specimens.

Project description:AIMS: Persistent infection indicated by detection of human papillomavirus 16 (HPV-16) on repeat testing over a period of time poses the greatest cervical cancer risk. However, variants of HPV-16, HPV-31 and HPV-33 may share several short sequence homologies in the hypervariable L1 gene commonly targeted for HPV genotyping. The purpose of this study was to introduce a robust laboratory procedure to validate HPV-16 detected in clinical specimens, using the GenBank sequence database as the standard reference for genotyping. METHODS: A nested PCR with two pairs of consensus primers was used to amplify the HPV DNA released in crude proteinase K digest of the cervicovaginal cells in liquid-based Papanicolaou cytology specimens. The positive nested PCR products were used for direct automated DNA sequencing. RESULTS: A 48-base sequence downstream of the GP5+ priming site, or a 34-base sequence upstream thereof, was needed for unequivocal validation of an HPV-16 isolate. Selection of a 45-base, or shorter, sequence immediately downstream of the GP5+ site for Basic Local Alignment Search Tool sequence analysis invariably led to ambiguous genotyping results. CONCLUSIONS: DNA sequence analysis may be used for differential genotyping of HPV-16, HPV-31 and HPV-33 in clinical specimens. However, selection of the signature sequence for Basic Local Alignment Search Tool algorithms is crucial to distinguish certain HPV-16 variants from other closely related HPV genotypes.

| S-EPMC2921263 | biostudies-literature

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