Nuclear export of cutaneous HPV8 E7 oncoprotein is mediated by a leucine-rich nuclear export signal via a CRM1 pathway.

We recently determined that the nuclear import of cutaneous beta genus HPV8 E7 oncoprotein it is mediated by its zinc-binding domain via direct hydrophobic interactions with the FG nucleoporins Nup62 and Nup153 (Onder and Moroianu, 2014). Here we investigated the nuclear export of HPV8 E7 oncoprotein using confocal microscopy after transfections of HeLa cells with EGFP-8cE7 and mutant plasmids and treatment with Ratjadone A nuclear export inhibitor. We determined that HPV8 E7 contains a leucine-rich nuclear export signal (NES), 76IRTFQELLF84, within its zinc-binding domain that mediates its nuclear export via a CRM1 pathway. We found that HPV8 E7 interacts with CRM1 and that the hydrophobic amino acid residues I76, F79 and L82 of the NES are essential for this interaction and for nuclear export of HPV8 E7 oncoprotein.

Nuclear import of cutaneous beta genus HPV8 E7 oncoprotein is mediated by hydrophobic interactions between its zinc-binding domain and FG nucleoporins.

We have previously discovered and characterized the nuclear import pathways for the E7 oncoproteins of mucosal alpha genus HPVs, type 16 and 11. Here we investigated the nuclear import of cutaneous beta genus HPV8 E7 protein using confocal microscopy after transfections of HeLa cells with EGFP-8E7 and mutant plasmids and nuclear import assays in digitonin-permeabilized HeLa cells. We determined that HPV8 E7 contains a nuclear localization signal (NLS) within its zinc-binding domain that mediates its nuclear import. Furthermore, we discovered that a mostly hydrophobic patch 65LRLFV69 within the zinc-binding domain is essential for the nuclear import and localization of HPV8 E7 via hydrophobic interactions with the FG nucleoporins Nup62 and Nup153. Substitution of the hydrophobic residues within the 65LRLFV69 patch to alanines, and not R66A mutation, disrupt the interactions between the 8E7 zinc-binding domain and Nup62 and Nup153 and consequently inhibit nuclear import of HPV8 E7.

Nuclear import of high risk HPV16 E7 oncoprotein is mediated by its zinc-binding domain via hydrophobic interactions with Nup62.

We previously discovered that nuclear import of high risk HPV16 E7 is mediated by a cNLS located within the zinc-binding domain via a pathway that is independent of karyopherins/importins (Angeline et al., 2003; Knapp et al., 2009). In this study we continued our characterization of the cNLS and nuclear import pathway of HPV16 E7. We find that an intact zinc-binding domain is essential for the cNLS function in mediating nuclear import of HPV16 E7. Mutagenesis of cysteine residues to alanine in each of the two CysXXCys motifs involved in zinc-binding changes the nuclear localization of the EGFP-16E7 and 2xEGFP-16E7 mutants. We further discover that a patch of hydrophobic residues, 65LRLCV69, within the zinc-binding domain of HPV16 E7 mediates its nuclear import via hydrophobic interactions with the FG domain of the central channel nucleoporin Nup62.

Characterization of the transport signals that mediate the nucleocytoplasmic traffic of low risk HPV11 E7.

We previously discovered that nuclear import of low risk HPV11 E7 is mediated by its zinc-binding domain via a pathway that is independent of karyopherins/importins (Piccioli et al., 2010. Virology 407, 100-109). In this study we mapped and characterized a leucine-rich nuclear export signal (NES), 76IRQLQDLLL84, within the zinc-binding domain that mediates the nuclear export of HPV11 E7 in a CRM1-dependent manner. We also identified a mostly hydrophobic patch 65VRLVV69 within the zinc-binding domain that mediates nuclear import of HPV11 E7 via hydrophobic interactions with the FG-repeats domain of Nup62. Substitutions of hydrophobic residues to alanine within the 65VRLVV69 sequence disrupt the nuclear localization of 11E7, whereas the R66A mutation has no effect. Overall the data support a model of nuclear entry of HPV11 E7 protein via hydrophobic interactions with FG nucleoporins at the nuclear pore complex.

The high risk HPV16 L2 minor capsid protein has multiple transport signals that mediate its nucleocytoplasmic traffic.

In this study we examined the transport signals contributing to HPV16 L2 nucleocytoplasmic traffic using confocal microscopy analysis of enhanced green fluorescent protein-L2 (EGFP-L2) fusions expressed in HeLa cells. We confirmed that both nuclear localization signals (NLSs), the nNLS (1MRHKRSAKRTKR12) and cNLS (456RKRRKR461), previously characterized in vitro (Darshan et al., 2004), function independently in vivo. We discovered that a middle region rich in arginine residues (296SRRTGIRYSRIGNKQTLRTRS316) functions as a nuclear retention sequence (NRS), as mutagenesis of critical arginine residues within this NRS reduced the fraction of L2 in the nucleus despite the presence of both NLSs. Significantly, the infectivity of HPV16 pseudoviruses containing either RR297AA or RR297EE within the L2 NRS was strongly reduced both in HaCaT cells and in a murine challenge model. Experiments using Ratjadone A nuclear export inhibitor and mutation-localization analysis lead to the discovery of a leucine-rich nuclear export signal ((462)LPYFFSDVSL) mediating 16L2 nuclear export. These data indicate that HPV16 L2 nucleocytoplasmic traffic is dependent on multiple functional transport signals.

The nuclear localization of low risk HPV11 E7 protein mediated by its zinc binding domain is independent of nuclear import receptors.

We investigated the nuclear import of low risk HPV11 E7 protein using 1) transfection assays in HeLa cells with EGFP fusion plasmids containing 11E7 and its domains and 2) nuclear import assays in digitonin-permeabilized HeLa cells with GST fusion proteins containing 11E7 and its domains. The EGFP-11E7 and EGFP-11cE7(39-98) localized mostly to the nucleus. The GST-11E7 and GST-11cE7(39-98) were imported into the nuclei in the presence of either Ran-GDP or RanG19V-GTP mutant and in the absence of nuclear import receptors. This suggests that 11E7 enters the nucleus via a Ran-dependent pathway, independent of nuclear import receptors, mediated by a nuclear localization signal located in its C-terminal domain (cNLS). This cNLS contains the zinc binding domain consisting of two copies of Cys-X-X-Cys motif. Mutagenesis of Cys residues in these motifs changed the localization of the EGFP-11cE7/-11E7 mutants to cytoplasmic, suggesting that the zinc binding domain is essential for nuclear localization of 11E7.

Identification of the nuclear localization and export signals of high risk HPV16 E7 oncoprotein.

The E7 oncoprotein of high risk human papillomavirus type 16 (HPV16) binds and inactivates the retinoblastoma (RB) family of proteins. Our previous studies suggested that HPV16 E7 enters the nucleus via a novel Ran-dependent pathway independent of the nuclear import receptors (Angeline, M., Merle, E., and Moroianu, J. (2003). The E7 oncoprotein of high-risk human papillomavirus type 16 enters the nucleus via a nonclassical Ran-dependent pathway. Virology 317(1), 13-23.). Here, analysis of the localization of specific E7 mutants revealed that the nuclear localization of E7 is independent of its interaction with pRB or of its phosphorylation by CKII. Fluorescence microscopy analysis of enhanced green fluorescent protein (EGFP) and 2xEGFP fusions with E7 and E7 domains in HeLa cells revealed that E7 contains a novel nuclear localization signal (NLS) in the N-terminal domain (aa 1-37). Interestingly, treatment of transfected HeLa cells with two specific nuclear export inhibitors, Leptomycin B and ratjadone, changed the localization of 2xEGFP-E7(38-98) from cytoplasmic to mostly nuclear. These data suggest the presence of a leucine-rich nuclear export signal (NES) and a second NLS in the C-terminal domain of E7 (aa 38-98). Mutagenesis of critical amino acids in the putative NES sequence ((76)IRTLEDLLM(84)) changed the localization of 2xEGFP-E7(38-98) from cytoplasmic to mostly nuclear suggesting that this is a functional NES. The presence of both NLSs and an NES suggests that HPV16 E7 shuttles between the cytoplasm and nucleus which is consistent with E7 having functions in both of these cell compartments.

Possible role for cellular karyopherins in regulating polyomavirus and papillomavirus capsid assembly.

Polyomavirus and papillomavirus (papovavirus) capsids are composed of 72 capsomeres of their major capsid proteins, VP1 and L1, respectively. After translation in the cytoplasm, L1 and VP1 pentamerize into capsomeres and are then imported into the nucleus using the cellular alpha and beta karyopherins. Virion assembly only occurs in the nucleus, and cellular mechanisms exist to prevent premature capsid assembly in the cytosol. We have identified the karyopherin family of nuclear import factors as possible "chaperones" in preventing the cytoplasmic assembly of papovavirus capsomeres. Recombinant murine polyomavirus (mPy) VP1 and human papillomavirus type 11 (HPV11) L1 capsomeres bound the karyopherin heterodimer alpha2beta1 in vitro in a nuclear localization signal (NLS)-dependent manner. Because the amino acid sequence comprising the NLS of VP1 and L1 overlaps the previously identified DNA binding domain, we examined the relationship between karyopherin and DNA binding of both mPy VP1 and HPV11 L1. Capsomeres of L1, but not VP1, bound by karyopherin alpha2beta1 or beta1 alone were unable to bind DNA. VP1 and L1 capsomeres could bind both karyopherin alpha2 and DNA simultaneously. Both VP1 and L1 capsomeres bound by karyopherin alpha2beta1 were unable to assemble into capsids, as shown by in vitro assembly reactions. These results support a role for karyopherins as chaperones in the in vivo regulation of viral capsid assembly.

Characterization of the nuclear localization signal of high risk HPV16 E2 protein.

The E2 protein of high risk human papillomavirus type 16 (HPV16) contains an amino-terminal (N) domain, a hinge (H) region and a carboxyl-terminal (C) DNA-binding domain. Using enhanced green fluorescent protein (EGFP) fusions with full length E2 and E2 domains in transfection assays in HeLa cells, we found that the C domain is responsible for the nuclear localization of E2 in vivo, whereas the N and H domains do not contain additional nuclear localization signals (NLSs). Deletion analysis of EGFP-E2 and EGFP-cE2 determined that the C domain contains an alpha helix cNLS that overlaps with the DNA-binding region. Mutational analysis revealed that the arginine and lysine residues in this cNLS are essential for nuclear localization of HPV16 E2. Interestingly, these basic amino acid residues are well conserved among the E2 proteins of BPV-1 and some high risk HPV types but not in the low risk HPV types, suggesting that there are differences between the NLSs and corresponding nuclear import pathways between these E2 proteins.

The positively charged termini of L2 minor capsid protein required for bovine papillomavirus infection function separately in nuclear import and DNA binding.

During the papillomavirus (PV) life cycle, the L2 minor capsid protein enters the nucleus twice: in the initial phase after entry of virions into cells and in the productive phase to mediate encapsidation of the newly replicated viral genome. Therefore, we investigated the interactions of the L2 protein of bovine PV type 1 (BPV1) with the nuclear import machinery and the viral DNA. We found that BPV1 L2 bound to the karyopherin alpha2 (Kap alpha2) adapter and formed a complex with Kap alpha2beta1 heterodimers. Previous data have shown that the positively charged termini of BPV1 L2 are required for BPV1 infection after the binding of the virions to the cell surface. We determined that these BPV1 L2 termini function as nuclear localization signals (NLSs). Both the N-terminal NLS (nNLS) and the C-terminal NLS (cNLS) interacted with Kap alpha2, formed a complex with Kap alpha2beta1 heterodimers, and mediated nuclear import via a Kap alpha2beta1 pathway. Interestingly, the cNLS was also the major DNA binding site of BPV1 L2. Consistent with the promiscuous DNA encapsidation by BPV1 pseudovirions, this DNA binding occurred without nucleotide sequence specificity. Moreover, an L2 mutant encoding a scrambled version of the cNLS, which supports production of virions, rescued the DNA binding but not the Kap alpha2 interaction. These data support a model in which BPV1 L2 functions as an adapter between the viral DNA via the cNLS and the Kaps via the nNLS and facilitates nuclear import of the DNA during infection.

The l2 minor capsid protein of human papillomavirus type 16 interacts with a network of nuclear import receptors.

The L2 minor capsid proteins enter the nucleus twice during viral infection: in the initial phase after virion disassembly and in the productive phase when, together with the L1 major capsid proteins, they assemble the replicated viral DNA into virions. In this study we investigated the interactions between the L2 protein of high-risk human papillomavirus type 16 (HPV16) and nuclear import receptors. We discovered that HPV16 L2 interacts directly with both Kapbeta(2) and Kapbeta(3). Moreover, binding of Ran-GTP to either Kapbeta(2) or Kapbeta(3) inhibits its interaction with L2, suggesting that the Kapbeta/L2 complex is import competent. In addition, we found that L2 forms a complex with the Kapalpha(2)beta(1) heterodimer via interaction with the Kapalpha(2) adapter. In agreement with the binding data, nuclear import of L2 in digitonin-permeabilized cells could be mediated by either Kapalpha(2)beta(1) heterodimers, Kapbeta(2), or Kapbeta(3). Mapping studies revealed that HPV16 L2 contains two nuclear localization signals (NLSs), in the N terminus (nNLS) and C terminus (cNLS), that could mediate its nuclear import. Together the data suggest that HPV16 L2 interacts via its NLSs with a network of karyopherins and can enter the nucleus via several import pathways mediated by Kapalpha(2)beta(1) heterodimers, Kapbeta(2), and Kapbeta(3).

The E7 oncoprotein of high-risk human papillomavirus type 16 enters the nucleus via a nonclassical Ran-dependent pathway.

E7, the major transforming protein of high-risk human papillomavirus (HPV), type 16, binds and inactivates the retinoblastoma protein (pRb), and the Rb-related proteins p107 and p130. HPV16 E7 is a nuclear protein lacking a classical basic nuclear localization signal. In this study we investigated the nuclear import of HPV16 E7 oncoprotein in digitonin-permeabilized HeLa cells. HPV16 E7 nuclear import was independent of pRb, as an E7(DeltaDLYC) variant defective in pRb binding was imported into the nuclei of digitonin-permeabilized cells as efficiently as wild-type E7 in the presence of exogenous cytosol. Interestingly, we discovered that HPV16 E7 is imported into the nuclei via a novel pathway different from those mediated by Kap alpha2beta1 heterodimers, Kap beta1, or Kap beta2. Nuclear accumulation of E7 required Ran and was not inhibited by the RanG19V-GTP variant, an inhibitor of Kap beta mediated import pathways. Together the data suggest that HPV16 E7 translocates through the nuclear pores via a nonclassical Ran-dependent pathway, independent of the main cytosolic Kap beta import receptors.

The L1 major capsid protein of human papillomavirus type 11 interacts with Kap beta2 and Kap beta3 nuclear import receptors.

We have previously shown that the L1 major capsid protein of low-risk HPV11 binds to the Kap alpha2 adapter and enters the nucleus via a Kap alpha2beta1-mediated pathway. In this study, we discovered that HPV11 L1 capsomeres bind to Kap beta2 import receptor, known to mediate nuclear import of hnRNP A1 via interaction with its nuclear localization signal termed M9. Significantly, binding of HPV11 L1 capsomeres to Kap beta2 inhibited the nuclear import of Kap beta2, and its specific M9-containing cargo. Interestingly, HPV11 L1 capsomeres also interacted with Kap beta3 import receptor and inhibited Kap beta3 nuclear import. Moreover, the L1 capsomeres of high-risk HPV-16 shared these activities. These data suggest that HPV L1 major capsid proteins interact with Kap beta2 and Kap beta3, and they may inhibit the Kap beta2- and Kap beta3-mediated nuclear import pathways during the productive phase of the viral life cycle when the virions are assembled and released.

Nuclear entry of high-risk human papillomavirus type 16 E6 oncoprotein occurs via several pathways.

The E6 oncoprotein of high-risk human papillomavirus type 16 (HPV16) interacts with several nuclear transcription factors and coactivators in addition to cytoplasmic proteins, suggesting that nuclear import of HPV16 E6 plays a role in the cellular transformation process. In this study we have investigated the nuclear import pathways of HPV16 E6 in digitonin-permeabilized HeLa cells. We found that HPV16 E6 interacted with the karyopherin (Kap) alpha2 adapter and could enter the nucleus via a classical Kap alpha2beta1-mediated pathway. Interestingly, HPV16 E6 also interacted, via its basic nuclear localization signal (NLS) located at the C terminus, with both Kap beta1 and Kap beta2 import receptors. Binding of RanGTP to these Kap betas inhibited their interaction with HPV16 E6 NLS. In agreement with these binding data, nuclear import of the HPV16 E6 oncoprotein in digitonin-permeabilized HeLa cells could be mediated by either Kap beta1 or Kap beta2. Nuclear import via these pathways required RanGDP and was independent of GTP hydrolysis by Ran. Significantly, an E6(R124G) mutant had reduced nuclear import activity, and the E6 deletion mutant lacking (121)KKQR(124) was not imported into the nucleus. The data reveal that the HPV16 E6 oncoprotein interacts via its C-terminal NLS with several karyopherins and exploits these interactions to enter the nucleus of host cells via multiple pathways.

Nuclear import strategies of high risk HPV16 L1 major capsid protein.

During the late phase of human papillomavirus (HPV) infection, the L1 major capsid proteins enter the nuclei of host epithelial cells and, together with the L2 minor capsid proteins, assemble the replicated viral DNA into virions. We investigated the nuclear import of the L1 major capsid protein of high risk HPV16. When digitonin-permeabilized HeLa cells were incubated with HPV16 L1 capsomeres, the L1 protein was imported into the nucleus in a receptor-mediated manner. HPV16 L1 capsomeres formed complexes with Kap alpha2beta1 heterodimers via interaction with Kap alpha2. Accordingly, nuclear import of HPV16 L1 capsomeres was mediated by Kap alpha2beta1 heterodimers, required RanGDP and free GTP, and was independent of GTP hydrolysis. Remarkably, HPV16 L1 capsomeres also interacted with Kap beta2 and binding of RanGTP to Kap beta2 did not dissociate the HPV16 L1.Kap beta2 complex. Significantly, HPV16 L1 capsomeres inhibited the nuclear import of Kap beta2 and of a Kap beta2-specific M9-containing cargo. These data suggest that, during the productive stage of infection, while the HPV16 L1 major capsid protein enters the nucleus via the Kap alpha2beta1-mediated pathway to assemble the virions, it also inhibits the Kap beta2-mediated nuclear import of host hnRNP A1 protein and, in this way, favors virion formation.