Junona Moroianu

Nuclear import and export pathways

Macromolecules enter or leave the nucleus by using nuclear localization signals (NLS), or nuclear export signals (NES), respectively. Different types of NLS and NES are recognized directly or indirectly via adapters, by transport receptors. All transport receptors identified thus far are members of the same family and share an ability to shuttle between the nucleus and the cytoplasm, and to interact with the small GTPase Ran and with nucleoporins at the nuclear pore complex (NPC). The GTPase Ran regulates the interaction of transport receptors with either cargoes, or adapters, or nucleoporins and is crucial in providing directionality to nuclear import and export. Surprisingly, GTP hydrolysis by Ran is not required for translocation of some receptor/cargo complexes through the NPC. One of the challenges for the future will be to establish the mechanisms of translocation through the NPC of different transport receptors together with their cargoes. J. Cell. Biochem. Suppls. 32/33:76–83, 1999.

The L2 minor capsid protein of human papillomavirus type 16 interacts with a network of nuclear import receptors

ABSTRACT 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 Kapβ2 and Kapβ3. Moreover, binding of Ran-GTP to either Kapβ2 or Kapβ3 inhibits its interaction with L2, suggesting that the Kapβ/L2 complex is import competent. In addition, we found that L2 forms a complex with the Kapα2β1 heterodimer via interaction with the Kapα2 adapter. In agreement with the binding data, nuclear import of L2 in digitonin-permeabilized cells could be mediated by either Kapα2β1 heterodimers, Kapβ2, or Kapβ3. 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 Kapα2β1 heterodimers, Kapβ2, and Kapβ3.

Distinct nuclear import and export pathways mediated by members of the karyopherin β family

Transport of proteins into and out of the nucleus occurs through nuclear pore complexes (NPCs) and is mediated by the interaction of transport factors with nucleoporins at the NPC. Nuclear import of proteins containing classical nuclear localization signals (NLSs) is mediated by a heterodimeric protein complex, composed of karyopherin α and β1, that docks via β1 the NLS‐protein to the NPC. The GTPase Ran; the RanGDP binding protein, p10; and the RanGTP binding protein, RanBP1 are involved in translocation of the docked NLS‐protein into the nucleus. Recently, new distinct nuclear import and export pathways that are mediated by members of the karyopherin β family have been discovered. Karyopherin β2 mediates import of mRNA binding proteins, whereas karyopherin β3 and β4 mediate import of a set of ribosomal proteins. Two other β karyopherin family members, CRM1 and CAS, mediate export of proteins containing leucine‐rich nuclear export signals (NES) and reexport of karyopherin α, respectively. This growing family contains new members that constitute potential transport factors for cargoes yet to be identified in the future. The common features of the members of karyopherin β family are the ability to bind RanGTP and the ability to interact directly with nucleoporins at the NPC. The challenge for the future will be to identify the distinct or, perhaps, overlapping cargo(es) for each member of the karyopherin β superfamily and to characterize the molecular mechanisms of translocation of karyopherins together with their cargoes through the NPC. J. Cell. Biochem. 70:231–239, 1998.© 1998 Wiley‐Liss, Inc.

Nuclear import of HPV11 L1 capsid protein is mediated by karyopherin α2β1 heterodimers

L1 major capsid proteins of human papillomaviruses (HPVs) enter the nuclei of host cells at two times during the viral life cycle: 1) after infection and 2) later during the productive phase, when they assemble the replicated HPV genomic DNA into infectious virions. L1 proteins are stable in two oligomeric configurations: as homopentameric capsomers, and as capsids composed of 72 capsomers. We found that intact L1 capsids of HPV type 11 cannot enter the nucleus, suggesting that capsid disassembly may be required for HPV11 L1 nuclear import. We established that HPV11 L1 is imported in a receptor‐mediated manner into the nuclei of digitonin‐permeabilized HeLa cells. HPV11 L1 docked at the nuclear pore complexes via karyopherin α2β1 heterodimers. Anti‐karyopherin‐β1 and anti‐karyopherin α2 antibodies specifically inhibited nuclear import of HPV11 L1. Moreover, nuclear import of HPV11 L1 could be reconstituted using karyopherin α2, β1, RanGDP and p10. In agreement with the docking and import data, we found that HPV11 L1 binds to karyopherin α2 and that this interaction is inhibited by a peptide representing the classical nuclear localization signal of SV40 T antigen. These results strongly suggest that HPV11 L1 enters the nucleus of the infected host cell via the karyopherin α2β1 pathway. J. Cell. Biochem. 74:628–637, 1999.

The Positively Charged Termini of L2 Minor Capsid Protein Required for Bovine Papillomavirus Infection Function Separately in Nuclear Import and DNA Binding

ABSTRACT 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 α2 (Kap α2) adapter and formed a complex with Kap α2β1 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 α2, formed a complex with Kap α2β1 heterodimers, and mediated nuclear import via a Kap α2β1 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 α2 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.

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.

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 α2β1 heterodimers via interaction with Kap α2. Accordingly, nuclear import of HPV16 L1 capsomeres was mediated by Kap α2β1 heterodimers, required RanGDP and free GTP, and was independent of GTP hydrolysis. Remarkably, HPV16 L1 capsomeres also interacted with Kap β2 and binding of RanGTP to Kap β2 did not dissociate the HPV16 L1·Kap β2 complex. Significantly, HPV16 L1 capsomeres inhibited the nuclear import of Kap β2 and of a Kap β2-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 α2β1-mediated pathway to assemble the virions, it also inhibits the Kap β2-mediated nuclear import of host hnRNP A1 protein and, in this way, favors virion formation.

Nuclear Entry of High-Risk Human Papillomavirus Type 16 E6 Oncoprotein Occurs via Several Pathways

ABSTRACT 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) α2 adapter and could enter the nucleus via a classical Kap α2β1-mediated pathway. Interestingly, HPV16 E6 also interacted, via its basic nuclear localization signal (NLS) located at the C terminus, with both Kap β1 and Kap β2 import receptors. Binding of RanGTP to these Kap βs 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 β1 or Kap β2. Nuclear import via these pathways required RanGDP and was independent of GTP hydrolysis by Ran. Significantly, an E6R124G mutant had reduced nuclear import activity, and the E6 deletion mutant lacking 121KKQR124 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 and DNA binding of human papillomavirus type 45 L1 capsid protein

During the life cycle of human papillomaviruses (HPVs), the L1 capsid proteins seem to enter the nucleus twice: once after the virions infect the cells, and later during the productive phase when they assemble the replicated HPV genomic DNA into infectious virions. We established for the high‐risk HPV45 that when digitonin‐permeabilized HeLa cells were incubated with L1 homopentameric capsomers, the HPV45 L1 protein was imported into the nucleus in a receptor‐mediated manner. In contrast, intact capsids were not able to enter the nucleus. Immunoisolation assays showed that HPV45 L1 capsomers interact with cytosolic karyopherin α2β1 heterodimers. HPV45 L1 bound strongly to karyopherin α2, and weakly to karyopherin β1, as did its nuclear localization signal (NLS). Nuclear import of HPV45 L1, or of a GST‐NLSHPV45L1 fusion protein was efficiently mediated by karyopherin α2β1 heterodimers, and only weakly by karyopherin β1. Nuclear import required RanGDP, but was independent of GTP hydrolysis by Ran. Together, these data suggest that the major nuclear import pathway for HPV45 L1 major capsid protein in infected host cells is mediated by karyopherin α2β1 heterodimers and that GTP hydrolysis by Ran is not required for import. Remarkably, HPV45 L1 capsomers can interact nonspecifically with different types of HPV‐DNA, and the DNA binding region of HPV45 L1 overlaps with its NLS sequence. J. Cell. Biochem. 79:225–238, 2000.

The L2 Minor Capsid Protein of Low-Risk Human Papillomavirus Type 11 Interacts with Host Nuclear Import Receptors and Viral DNA

ABSTRACT Analysis of the interactions of low-risk human papillomavirus type 11 (HPV11) L2 with karyopherin β (Kap β) nuclear import receptors revealed that L2 interacted with Kap β1, Kap β2, and Kap β3 and formed a complex with the Kap α2β1 heterodimer. HPV11 L2 contains two nuclear localization signals (NLSs)—in the N terminus and the C terminus—that could mediate its nuclear import via a classical pathway. Each NLS was functional in vivo, and deletion of both of them abolished L2 nuclear localization. Both NLSs interacted with the viral DNA. Thus, HPV11 L2 can interact with several karyopherins and the viral DNA and may enter the nucleus via multiple pathways.