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.

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.

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).

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.