Overlapping roles of the Rous sarcoma virus Gag p10 domain in nuclear export and virion core morphology

Nucleocytoplasmic shuttling of the Rous sarcoma virus (RSV) Gag polyprotein is an integral step in virus particle assembly. A nuclear export signal (NES) was previously identified within the p10 domain of RSV Gag. Gag mutants containing deletions of the p10 NES or mutations of critical hydrophobic residues at positions 219, 222, 225, or 229 become trapped within the nucleus and exhibit defects in the efficiency of virus particle release. To investigate other potential roles for Gag nuclear trafficking in RSV replication, we created viruses bearing NES mutant Gag proteins. Viruses carrying p10 mutations produced low levels of particles, as anticipated, and those particles that were released were noninfectious. The p10 mutant viruses contained approximately normal amounts of Gag, Gag-Pol, and Env proteins and genomic viral RNA (vRNA), but several major structural defects were found. Thin-section transmission electron microscopy revealed that the mature particles appeared misshapen, while the viral cores were cylindrical, horseshoe-shaped, or fragmented, with some particles containing multiple small, electron-dense aggregates. Immature virus-like particles produced by the expression of Gag proteins bearing p10 mutations were also aberrant, with both spherical and tubular filamentous particles produced. Interestingly, the secondary structure of the encapsidated vRNA was altered; although dimeric vRNA was predominant, there was an additional high-molecular-weight fraction. Together, these results indicate that the p10 NES domain of Gag is critical for virus replication and that it plays overlapping roles required for the nuclear shuttling of Gag and for the maintenance of proper virion core morphology.     

Importin-beta family members mediate alpharetrovirus gag nuclear entry via interactions with matrix and nucleocapsid

The retroviral Gag polyprotein orchestrates the assembly and release of virus particles from infected cells. We previously reported that nuclear transport of the Rous sarcoma virus (RSV) Gag protein is intrinsic to the virus assembly pathway. To identify cis- and trans-acting factors governing nucleocytoplasmic trafficking, we developed novel vectors to express regions of Gag in Saccharomyces cerevisiae. The localization of Gag proteins was examined in the wild type and in mutant strains deficient in members of the importin-beta family. We confirmed the Crm1p dependence of the previously identified Gag p10 nuclear export signal. The known nuclear localization signal (NLS) in MA (matrix) was also functional in S. cerevisiae, and additionally we discovered a novel NLS within the NC (nucleocapsid) domain of Gag. MA utilizes Kap120p and Mtr10p import receptors while nuclear entry of NC involves the classical importin-alpha/beta (Kap60p/95p) pathway. NC also possesses nuclear targeting activity in avian cells and contains the primary signal for the import of the Gag polyprotein. Thus, the nucleocytoplasmic dynamics of RSV Gag depend upon the counterbalance of Crm1p-mediated export with two independent NLSs, each interacting with distinct nuclear import factors.     

Intermolecular interactions between retroviral Gag proteins in the nucleus

The retroviral Gag polyprotein directs virus particle assembly, resulting in the release of virions from the plasma membranes of infected cells. The earliest steps in assembly, those immediately following Gag synthesis, are very poorly understood. For Rous sarcoma virus (RSV), Gag proteins are synthesized in the cytoplasm and then undergo transient nuclear trafficking before returning to the cytoplasm for transport to the plasma membrane. Thus, RSV provides a useful model to study the initial steps in assembly because the early and later stages are spatially separated by the nuclear envelope. We previously described mutants of RSV Gag that are defective in nuclear export, thereby isolating these “trapped” Gag proteins at an early assembly step. Using the nuclear export mutants, we asked whether Gag protein-protein interactions occur within the nucleus. Complementation experiments revealed that the wild-type Gag protein could partially rescue export-defective Gag mutants into virus-like particles (VLPs). Additionally, the export mutants had a trans-dominant negative effect on wild-type Gag, interfering with its release into VLPs. Confocal imaging of wild-type and mutant Gag proteins bearing different fluorescent tags suggested that complementation between Gag proteins occurred in the nucleus. Additional evidence for nuclear Gag-Gag interactions was obtained using fluorescence resonance energy transfer, and we found that the formation of intranuclear Gag complexes was dependent on the NC domain. Bimolecular fluorescence complementation allowed the direct visualization of intranuclear Gag-Gag dimers. Together, these experimental results strongly suggest that RSV Gag proteins are capable of interacting within the nucleus.     

Cocompartmentalization of p53 and Mdm2 is a major determinant for Mdm2-mediated degradation of p53

The product of the Mdm2 oncogene directly interacts with p53 and promotes its ubiquitination and proteasomal degradation. Initial biological studies identified nuclear export sequences (NES), similar to that of the Rev protein from the human immunodeficiency virus, both in Mdm2 and p53. The reported phenotypes resulting from mutation of these NESs, together with results obtained using the nuclear export inhibitor leptomycin B (LMB), have led to a model according to which nuclear export of p53 (via either the NES of Mdm2 or its own NES) is required for efficient p53 degradation. In this study we demonstrate that Mdm2 can promote degradation of p53 in the nucleus or in the cytoplasm, provided both proteins are colocalized. We also investigated if nuclear export is an obligate step on the p53 degradation pathway. We find that (1) when proteasome activity is inhibited, ubiquitinated p53 accumulates in the nucleus and not in the cytoplasm; (2) Mdm2 with a mutated NES can efficiently mediate degradation of wild type p53 or p53 with a mutated NES; (3) the nuclear export inhibitor LMB can increase the steady-state level of p53 by inhibiting Mdm2-mediated ubiquitination of p53; and (4) LMB fails to inhibit Mdm2-mediated degradation of the p53NES mutant, demonstrating that Mdm2-dependent proteolysis of p53 is feasible in the nucleus in the absence of any nuclear export. Therefore, given cocompartmentalization, Mdm2 can promote ubiquitination and proteasomal degradation of p53 with no absolute requirement for nuclear to cytoplasmic transport.     

Molecular structure of the Japanese hepatitis C viral genome

The amino acid sequence of the polyprotein deduced from the nucleotide sequence of the Japanese hepatitis C virus genome (N. Kato et. al. (1990) Proc. Natl. Acad. Sci. USA 87, 9524–9528)indicated that this virus is a member of a new class of positive-stranded RNA viruses. Several domains of this polyprotein also showed weak homology with those of flaviviruses and pestiviruses including the chymotrypsin-like serine proteinase. NTPase and RNA-dependent RNA polymerase     

Leptomycin B Inhibition of Signal-Mediated Nuclear Export by Direct Binding to CRM1

Leptomycin B (LMB) is aStreptomycesmetabolite that inhibits nuclear export of the human immunodeficiency virus type 1 regulatory protein Rev at low nanomolar concentrations. Recently, LMB was shown to inhibit the function of CRM1, a receptor for the nuclear export signal (NES). Here we show evidence that LMB binds directly to CRM1 and that CRM1 is essential for NES-dependent nuclear export of proteins in both yeast and mammalian cells. Binding experiments with a biotinylated derivative of LMB and a HeLa cell extract led to identifying CRM1 as a major protein that bound to the LMB derivative. Microinjection of a purified anti-human CRM1 antibody into the mammalian nucleus specifically inhibited nuclear export of NES-containing proteins, as did LMB. Consistent with this, CRM1 was found to interact with NES, when assayed with immobilized NES and HeLa cell extracts. This association was disrupted by adding LMB or purified anti-human CRM1 antibody. The inhibition of CRM1 by LMB was also observed in fission yeast. The fission yeastcrm1mutant was defective in the nuclear export of NES-fused proteins, but not in the import of nuclear localization signal (NLS)-fused proteins. Interestingly, a protein containing both NES and NLS, which is expected to shuttle between nucleus and cytoplasm, was highly accumulated in the nucleus of thecrm1mutant cells or of cells treated with LMB. These results strongly suggest that CRM1 is the target of LMB and is an essential factor for nuclear export of proteins in eukaryotes.     

Mechanistic insights into the action of Bisphenol A on the germline using C. elegans

Feature on: Allard P, et al. Proc Natl Acad Sci USA 2010; 107:20405-10.     

Characterization of a novel transferable CRM-1-independent nuclear export signal in a herpesvirus tegument protein that shuttles between the nucleus and cytoplasm

A new group of nucleocytoplasmic shuttling proteins has recently been identified in the structural proteins encoded by several alphaherpesvirus UL47 genes. Nuclear import and export signals for the bovine herpesvirus type 1 UL47 protein (VP8 or bUL47) have been described previously. Here, we study the trafficking of bUL47 in detail and identify an import signal different from that shown before. It comprises a 20-residue N-terminal peptide that is fully transferable and targets a large, normally cytosolic protein to the nucleus. A conserved RRPRRS motif within this peptide was shown to be essential but not sufficient for nuclear targeting. Using interspecies heterokaryon assays, we further demonstrate that the export activity of the published leucine-rich nuclear export signal (NES) is also transferable to a large protein but is functionally weak compared to the activity of the HIV-1 Rev NES. We show that nuclear export dictated by this bUL47 NES is sensitive to leptomycin B (LMB) and therefore dependent on the export receptor CRM-1. However, nuclear export of full-length bUL47 is fully resistant to LMB, suggesting the presence of an additional NES. We go on to identify a second NES in bUL47 within a 28-residue peptide that is in close proximity to but entirely separable from the N-terminal import signal, and we use fluorescence loss in photobleaching to confirm its activity. This NES is resistant to leptomycin B, and therefore utilizes an export receptor other than CRM-1. As this new sequence bears little similarity to other export signals so far defined, we suggest it may be involved in bUL47 export from the nucleus via a novel cellular receptor.     

MicroRNA-dependent regulation of the microenvironment and the epithelial stromal cell interactions in the mouse mammary gland

Ohno Y, et al. Proc Natl Acad Sci USA 2010; In press.     

Role of Nedd4 and ubiquitination of Rous sarcoma virus Gag in budding of virus-like particles from cells

Rous sarcoma virus (RSV) budding requires an interaction of the L domain within the p2b region of Gag with cellular Nedd4-family E3 ubiquitin protein ligases. Members of our laboratories previously demonstrated that overexpression of a fragment of the chicken Nedd4-like protein (LDI-1 WW) inhibits Gag release in a dominant-negative manner (A. Kikonyogo, F. Bouamr, M. L. Vana, Y. Xiang, A. Aiyar, C. Carter, and J. Leis, Proc. Natl. Acad. Sci. USA 98:11199-11204, 2001). We have now identified the complete 3' end of LDI-1 and determined that it has a C-terminal ubiquitin ligase HECT domain, similar to other Nedd4 family members. While overexpression of the full-length LDI-1 clone (LDI-1 FL) had little effect on Gag budding, an LDI-1 FL mutant with a substitution in the HECT domain catalytic site blocked Gag release, similar to LDI-1 WW. The coexpression of Gag and hemagglutinin-tagged ubiquitin (HA-Ub) resulted in the detection of mono- and polyubiquitinated forms of Gag in cells and mostly monoubiquitinated Gag in virus-like particles (VLPs). When the Nedd4-binding site (L domain) was deleted, ubiquitinated Gag was not detected. Interestingly, the release of Gag with ubiquitin covalently linked to the C terminus (Gag-Ub) was still blocked by LDI-1 WW. To understand the mechanism of this inhibition, we examined cells expressing Gag and LDI-1 WW by electron microscopy. In the presence of LDI-1 WW, VLPs were found in electron-dense inclusion bodies in the cytoplasm of transfected cells. In contrast, when cells that coexpressed Gag-Ub and LDI-1 WW were examined, inclusion bodies were detected but did not contain VLPs. These results indicate that the ubiquitination of Gag is dependent upon Nedd4 binding to the L domain and suggest that Nedd4 has additional functions during RSV release besides the ubiquitination of Gag.     

Role for Geminin in sustaining the activity of hematopoietic stem cells

Ohno Y, et al. Proc Natl Acad Sci USA 2010; In press.     

Cytoplasmic sequestration of wild-type p53 protein impairs the G1 checkpoint after DNA damage.

Wild-type p53 protein is abnormally sequestered in the cytoplasm of a subset of primary human tumors including neuroblastomas (NB) (U. M. Moll, M. LaQuaglia, J. Benard, and G. Riou, Proc. Natl. Acad. Sci. USA 92:4407-4411, 1995; U. M. Moll, G. Riou, and A. J. Levine, Proc. Natl. Acad. Sci.USA 89:7262-7266, 1992). This may represent a nonmutational mechanism for abrogating p53 tumor suppressor function. To test this hypothesis, we established the first available in vitro model that accurately reflects the wild-type p53 sequestration found in NB tumors. We characterized a series of human NB cell lines that overexpress wild-type p53 and show that p53 is preferentially localized to discrete cytoplasmic structures, with no detectable nuclear p53. These cell lines, when challenged with a variety of DNA strand-breaking agents, all exhibit impaired p53-mediated G1 arrest. Induction analysis of p53 and p53-responsive genes show that this impairment is due to suppression of nuclear p53 accumulation. Thus, this naturally occurring translocation defect compromises the suppressor function of p53 and likely plays a role in the tumorigenesis of these tumors previously thought to be unaffected by p53 alterations.     

MicroRNA regulation masters basal transcription: Clash of the Titans

Comment on: Portal MM. Proc Natl Acad Sci USA 2011; 108:8686-91.     

Analysis of the complex relationship between nuclear export and aryl hydrocarbon receptor-mediated gene regulation

The aryl hydrocarbon receptor (AHR) contains signals for both nuclear import and nuclear export (NES). The purpose of the studies in this report was to determine the relationship between the nuclear export of the AHR and AHR-mediated gene regulation. Blockage of nuclear export in HepG2 cells with leptomycin B (LMB) resulted in increased levels of AHR-AHR nuclear translocator (ARNT) complex in the nucleus and correlative reductions in agonist-stimulated AHR degradation. However, LMB exposure inhibited agonist-mediated induction of numerous AHR-responsive reporter genes by 75 to 89% and also inhibited induction of endogenous CYP1A1. LMB did not transform the AHR to a ligand binding species or affect activation by TCDD (2, 3,7,8-tetrachlorodibenzo-p-dioxin). Mutagenesis of leucines 66 and 71 of the putative AHR NES resulted in a protein with reduced function in dimerization to ARNT and binding to DNA, while alanine substitution at leucine 69 (AHR(A69)) resulted in an AHR that bound with ARNT and associated with DNA. AHR(A69) protein injected directly into the nuclei of E36 cells remained nuclear following 6 h of agonist stimulation. In transient-transfection assays, AHR(A69) accumulated within the nucleus was not degraded efficiently following agonist exposure. Finally, AHR(A69) supported induction of AHR-responsive reporter genes in an agonist-dependent manner. These findings show that it is possible to generate an AHR protein defective in nuclear export that is functional in agonist-mediated gene induction. This implies that the negative effect of LMB on agonist-mediated gene induction is independent of the nuclear export of the AHR.     

Transcription factor kinetics and the emerging asymmetry in the early mammalian embryo

Comment on: Chagpar RB, et al. Proc Natl Acad Sci USA 2010; 107:5471-6.     

Establishing the human rolling circle reaction

Comment on: Kang YH, et al. Proc Natl Acad Sci USA 2012; 109:6042-7.     

In the right place at the right time: Analysis of p53 serine 312 phosphorylation in vivo

Comment on: Slee EA, et al. Proc Natl Acad Sci USA 2010; 107:19479–84.