Nuclear Export Function Research Articles

Cex1p is a novel cytoplasmic component of the Saccharomyces cerevisiae nuclear tRNA export machinery

The Saccharomyces cerevisiae Yor112wp, which we named Cex1p, was identified using a yeast tRNA three-hybrid interaction approach and an in vivo nuclear tRNA export assay as a cytoplasmic component of the nuclear tRNA export machinery. Cex1p binds tRNA saturably, and associates with the nuclear pore complex by interacting directly with Nup116p. Cex1p co-purifies with the nuclear tRNA export receptors Los1p and Msn5p, the eukaryotic elongation factor eEF-1A, which delivers aminoacylated tRNAs to the ribosome, and the RanGTPase Gsp1p, but not with Cca1p, a tRNA maturation enzyme that facilitates translocation of non-aminoacylated tRNAs across the nuclear pore complex. Depletion of Cex1p and eEF-1A or Los1p significantly reduced the efficiency of nuclear tRNA export. Cex1p interacts with Los1p but not with eEF-1A in vitro. These findings suggest that Cex1p is a component of the nuclear aminoacylation-dependent tRNA export pathway in S. cerevisiae. They also suggest that Cex1p collects aminoacyl-tRNAs from the nuclear export receptors at the cytoplasmic side of the nuclear pore complex, and transfers them to eEF-1A using a channelling mechanism.

Characterization of the Nuclear Localization and Nuclear Export Signals of Bovine Herpesvirus 1 VP22

The bovine herpesvirus 1 (BHV-1) tegument protein VP22 is predominantly localized in the nucleus after viral infection. To analyze subcellular localization in the absence of other viral proteins, a plasmid expressing BHV-1 VP22 fused to enhanced yellow fluorescent protein (EYFP) was constructed. The transient expression of VP22 fused to EYFP in COS-7 cells confirmed the predominant nuclear localization of VP22. Analysis of the amino acid sequence of VP22 revealed that it does not have a classical nuclear localization signal (NLS). However, by constructing a series of deletion derivatives, we mapped the nuclear targeting domain of BHV-1 VP22 to amino acids (aa) 121 to 139. Furthermore, a 4-aa motif, 130PRPR133, was able to direct EYFP and an EYFP dimer (dEYFP) or trimer (tEYFP) predominantly into the nucleus, whereas a deletion or mutation of this arginine-rich motif abrogated the nuclear localization property of VP22. Thus, 130PRPR133 is a functional nonclassical NLS. Since we observed that the C-terminal 68 aa of VP22 mediated the cytoplasmic localization of EYFP, an analysis was performed on these C-terminal amino acid sequences, and a leucine-rich motif, 204LDRMLKSAAIRIL216, was detected. Replacement of the leucines in this putative nuclear export signal (NES) with neutral amino acids resulted in an exclusive nuclear localization of VP22. Furthermore, this motif was able to localize EYFP and dEYFP in the cytoplasm, and the nuclear export function of this NES could be blocked by leptomycin B. This demonstrates that this leucine-rich motif is a functional NES. These data represent the first identification of a functional NLS and NES in a herpesvirus VP22 homologue.

Squid is required for efficient posterior localization of oskar mRNA during Drosophila oogenesis

The nuclear-cytoplasmic shuttling heterogeneous nuclear RNA-binding protein (hnRNP) Squid (Sqd) is required during Drosophila melanogaster oogenesis, where it plays a critical role in the regulation of the TGFalpha-like molecule Gurken (Grk). Three Sqd isoforms have been described, SqdA, S and B, and two of these, SqdA and SqdS, differentially function in grk mRNA nuclear export, cytoplasmic transport and translational control during oogenesis. Here, we report that Sqd is also required for the regulation of oskar (osk) mRNA, functioning in the cytoplasmic localization of the osk transcript. In oocytes from sqd females, osk mRNA is not efficiently localized to the posterior pole, but rather accumulates at the anterior cortex. Furthermore, anterior patterning defects observed in embryos from sqd females expressing only the SqdS protein isoform suggest that Sqd may also play a role in the translational regulation of the mislocalized osk mRNA. These findings provide additional support for models of mRNA regulation in which cytoplasmic events, such as localization and translational regulation, are coupled. These results also place Sqd among an emerging class of proteins, including such other members as Bruno (Bru) and Hrb27C/Hrp48, which function in multiple aspects of both grk and osk mRNA regulation during Drosophila oogenesis.

Nuclear Import and Export Functions in the Different Isoforms of the AUF1/Heterogeneous Nuclear Ribonucleoprotein Protein Family

The heterogeneous nuclear ribonucleoprotein D family of proteins also known as AUF1 consists of four isoforms implicated in both nuclear and cytoplasmic functions. The AUF1 proteins are largely nuclear but also are found in the cytoplasm and are thought to undergo nucleocytoplasmic shuttling. The nucleocytoplasmic distribution and potential shuttling activity of the individual AUF1 isoforms have not been previously studied in detail. Therefore, we characterized the nucleocytoplasmic transport of each of the heterogeneous nuclear ribonucleoprotein D/AUF1 isoforms. All four AUF1 proteins were found to undergo rapid nucleocytoplasmic shuttling in a manner that is transcription-independent, carrier-mediated, and energy-requiring. Nucleocytoplasmic shuttling of the AUF1 proteins is shown to utilize a novel arrangement of nuclear import and export signals. Mutagenesis of the AUF1 proteins and fusion of polypeptides to a green fluorescent protein reporter demonstrated that a nuclear import signal is located in the C-terminal domain of the protein and is found only in the two smaller isoforms. Further mapping demonstrated that nuclear export is facilitated by sequences in AUF1 exon 7 found in the C-terminal domain of the two larger AUF1 isoforms. A subset of AUF1 proteins are shown to directly interact in vitro using purified recombinant proteins and in vivo in the absence of RNA. These results suggest that nuclear import of AUF1 is facilitated by sequences found only in the two smaller isoforms and that nuclear export is facilitated by sequences (exon 7 and the C-terminal domain) found only in the two larger isoforms. This novel arrangement of signals might represent a mechanism to assure co-shuttling of a subset of AUF1 proteins that interact in a heterocomplex.

Translation Regulation Meets Cell Survival and Transformation

As a regulator of protein synthesis, eukaryotic translation initiation factor 4E (eIF4E) lies downstream in pathways that control cell growth and cell survival. eIF4E stimulates the translation of specific transcripts by promoting nucleocytoplasmic transport and delivering the transcripts to the ribosomes for translation. In the nucleus, eIF4E is associated with subnuclear structures called PML bodies (so-named because they also contain the protein product of the gene responsible for promyelocytic leukemia (PML). eIF4E is regulated by interaction with binding partners that are controlled through phosphorylation in response to growth factors (phosphorylation of these negative regulators inhibits their interaction with eIF4E allowing eIF4E to function) and by PML, which inhibits eIF4E activity in response to stress. Topisirovic et al . identified homeodomain proteins, traditionally considered to be transcriptional regulators, that may also functionally interact with eIF4E to inhibit its nuclear export function. Two homeodomain proteins out of the 199 homeodomain proteins that contain the eIF4E consensus binding motif were tested and interacted with eIF4E: proline-rich homeodomain (PRH) protein and Hox-11. Detailed analysis of the PRH-eIF4E interaction showed a direct interaction between the two proteins and overexpression of PRH inhibited eIF4E-mediated cyclin D1 nuclear export and inhibited cellular transformation associated with eIF4E overexpression. Overexpression of PRH also disrupted the PML bodies and caused redistribution of eIF4E and PML to the cytoplasm. Thus, transcriptional regulators, especially those important early in development, may also regulate protein production by influencing translational efficiency through interactions with eIF4E. In addition to having a role in cellular transformation, eIF4E is downstream of cell survival pathways and overexpression of the protein can inhibit apoptosis. Li et al . studied rescue from apoptosis of cells overexpressing Myc and subjected to the apoptosis-stimulating drug lovastatin. Cytochrome c was not released from the mitochondria of cells overexpressing eIF4E, nor was caspase 3 activated. The amount of the antiapoptotic factor Bcl-X L was increased in the eIF4E cells because increased mRNA abundance and increased recruitment of the transcripts to the polyribosomes. Thus, increasing Bcl-X L may be one mechanism by which translational regulation pathways intersect cell survival pathways. I. Topisirovic, B. Culjkovic, N. Cohen, J. M. Perez, L. Skrabanek, K. L. B. Borden, The proline-rich homeodomain protein, PRH, is a tissue-specific inhibitor of eIF4E-dependent cyclin D1 mRNA transport and growth. EMBO J. 22 , 689-703 (2003). [Abstract] [Full Text] S. Li, T. Takasu, D. M. Perlman, M. S. Peterson, D. Burrichter, S. Avdulov, P. B. Bitterman, V. A. Polunovsky, Translation factor eIF4E rescues cells from Myc-dependent apoptosis by inhibiting cytochrome c release. J. Biol. Chem. 278 , 3015-3022 (2003). [Abstract] [Full Text]

Structural Basis for the Interaction Between the Tap/NXF1 UBA Domain and FG Nucleoporins at 1 Å Resolution

The mRNA nuclear export function of Tap/NXF1 requires interactions with nuclear pore proteins (nucleoporins) that contain characteristic Phe-Gly repeats based on FG, GLFG or FxFG cores separated by hydrophilic linkers. FG-nucleoporins bind the two most C-terminal domains of Tap, which have NTF2 and UBA folds, respectively. We used a combination of NMR and X-ray crystallography to define the interaction interface between Tap UBA and FxFG nucleoporins and show that it involves primarily the two aromatic rings of the FxFG core that bind in a hydrophobic surface depression centred on Tap Cys588. NMR evidence indicates that the same depression mediates the binding of GLFG nucleoporins, which we confirmed by demonstrating competition between the two classes of repeat for binding to Tap UBA. Moreover, modification of Cys588 reduced the binding of Tap UBA to both GLFG and FxFG nucleoporins as well as to nuclear envelopes. These data underscore the central role of the conserved FG-nucleoporin repeat cores in binding to Tap UBA and indicate that functional differences between different classes of nucleoporins depend more on their spatial distribution in nuclear pores than on their binding to different sites on Tap UBA.

Characterization of the Nuclear Import and Export Functions of IκBε

Control over the nuclear localization of nuclear factor kappaB/Rel proteins is accomplished in large part through association with members of the inhibitor of kappaB (IkappaB) protein family. For example, the well studied IkappaBalpha protein actively shuttles between the nucleus and the cytoplasm and both inhibits nuclear import and mediates nuclear export of NF-kappaB/Rel proteins. In contrast, the IkappaBbeta protein can inhibit nuclear import of NF-kappaB/Rel proteins but does not remove NF-kappaB/Rel proteins from the nucleus. To further understand how the IkappaB proteins control the nuclear-cytoplasmic distribution of NF-kappaB/Rel proteins, we have characterized the nuclear import and nuclear export functions of IkappaBepsilon. Our results indicate that the IkappaBepsilon protein, like the IkappaBalpha protein, actively shuttles between the nucleus and the cytoplasm. Similar to IkappaBalpha, nuclear import of IkappaBepsilon is mediated by its ankyrin repeat domain and is not blocked by the dominant-negative RanQ69L protein. However, the nuclear import function of the IkappaBepsilon ankyrin repeat domain is markedly less efficient than that of IkappaBalpha, with the result that nuclear shuttling of IkappaBepsilon between the nucleus and the cytoplasm is significantly slower than IkappaBalpha. Nuclear export of IkappaBepsilon is mediated by a short leucine-rich nuclear export sequence (NES)-like sequence ((343)VLLPFDDLKI(352)), located between amino acids 343 and 352. This NES-like sequence is required for RanGTP-dependent binding of IkappaBepsilon to CRM1. Nuclear accumulation of IkappaB(epsilon) is increased by either leptomycin B treatment or alanine substitutions within the IkappaBepsilon-derived NES. A functional NES is required for both efficient cytoplasmic retention and post-induction control of c-Rel by IkappaBepsilon, consistent with the notion that IkappaBepsilon-mediated nuclear export contributes to control over the nucleocytoplasmic distribution of NF-kappaB/Rel proteins.

Actin-dependent membrane association of the APC tumour suppressor in polarized mammalian epithelial cells.

Adenomatous polyposis coli (APC) is mutated in most colorectal cancers. APC downregulates nuclear beta-catenin, which is thought to be critical for its tumour suppressor function. However, APC may have additional and separate functions at the cell periphery. Here, we examine polarized MDCK and WIF-B hepatoma cells and find that APC is associated with their lateral plasma membranes. This depends on the actin cytoskeleton but not on microtubules, and drug wash-out experiments suggest that APC is delivered continuously to the plasma membrane by a dynamic actin-dependent process. In polarized MDCK cells, APC also clusters at microtubule tips in their basal-most regions. Microtubule depolymerization causes APC to relocalize from these tips to the plasma membrane, indicating two distinct peripheral APC pools that are in equilibrium with each other in these cells. Truncations of APC such as those found in APC mutant cancer cells can neither associate with the plasma membrane nor with microtubule tips. The ability of APC to reach the cell periphery may thus contribute to its tumour suppressor function in the intestinal epithelium.

Influenza B and C virus NEP (NS2) proteins possess nuclear export activities.

Nucleocytoplasmic transport of viral ribonucleoproteins (vRNPs) is an essential aspect of the replication cycle for influenza A, B, and C viruses. These viruses replicate and transcribe their genomes in the nuclei of infected cells. During the late stages of infection, vRNPs must be exported from the nucleus to the cytoplasm prior to transport to viral assembly sites on the cellular plasma membrane. Previously, we demonstrated that the influenza A virus nuclear export protein (NEP, formerly referred to as the NS2 protein) mediates the export of vRNPs. In this report, we suggest that for influenza B and C viruses the nuclear export function is also performed by the orthologous NEP proteins (formerly referred to as the NS2 protein). The influenza virus B and C NEP proteins interact in the yeast two-hybrid assay with a subset of nucleoporins and with the Crm1 nuclear export factor and can functionally replace the effector domain from the human immunodeficiency virus type 1 Rev protein. We established a plasmid transfection system for the generation of virus-like particles (VLPs) in which a functional viral RNA-like chloramphenicol acetyltransferase (CAT) gene is delivered to a new cell. VLPs generated in the absence of the influenza B virus NEP protein were unable to transfer the viral RNA-like CAT gene to a new cell. From these data, we suggest that the nuclear export of the influenza B and C vRNPs are mediated through interaction between NEP proteins and the cellular nucleocytoplasmic export machinery.

Intracellular Trafficking Determinants in APOBEC-1, the Catalytic Subunit for Cytidine to Uridine Editing of Apolipoprotein B mRNA

The posttranscriptional deamination editing of apolipoprotein B (apoB) mRNA catalyzed by APOBEC-1 (apoB mRNA editing catalytic subunit 1) is a nuclear process. The signals in APOBEC-1 responsible for its dual cytoplasmic/nuclear distribution have been evaluated. Residues 97–172 in the middle of APOBEC-1 together with its N-terminal 56 residues affect nuclear localization. Mutagenesis studies however revealed no discrete nuclear localization signal in APOBEC-1. Fifteen amino acids (Leu 173–Leu 187) within the previously identified C-terminal domain of APOBEC-1 were sufficient as a determinant for cytoplasmic distribution in that context. These residues failed to demonstrate nuclear export function in a reporter assay. Further, the distribution of APOBEC-1 in the cytoplasm did not respond to leptomycin B, suggesting that APOBEC-1 did not have nuclear export activity. The data suggested that there are at least three regions in APOBEC-1 that participate in its distribution in both the nucleus and the cytoplasm of editing competent cells; however, none of these meet the functional criteria of nuclear localization or nuclear export signals. The findings are discussed in terms of their implications in the regulation of nuclear editing activity and the possibility that interactions with chaperones may play a role in the cellular distribution of APOBEC-1.

Dynamic Shuttling of Nuclear Factor κB between the Nucleus and Cytoplasm as a Consequence of Inhibitor Dissociation

Activation of the nuclear factor kappaB (NFkappaB) transcription factor is intimately associated with its translocation from the cytoplasm to the nucleus. Using the nuclear export inhibitor leptomycin B, we demonstrate shuttling of the RELA subunit of NFkappaB and the inhibitory subunit IkappaBalpha between these two compartments in unstimulated cells. Determination of the kinetics of nuclear entry shows marked differences for the two components; the entry of IkappaBalpha occurs more rapidly than RELA. The shuttling is suggested to be a consequence of the cytoplasmic dissociation of the NFkappaB.IkappaB complex rather than its direct nuclear import or degradation and resynthesis of IkappaBalpha. Using previously published kinetic data, this proposition is born out by the deduction that 17% of NFkappaB is not complexed to IkappaBalpha in a resting cell. A numerical model is presented to validate the proposed regulation of NFkappaB subcellular localization consequent in part on the nuclear export function and in part on the cytoplasmic retention function of IkappaBalpha. We suggest that the non-saturated interaction of NFkappaB with the inhibitor may enhance the specificity of action of IkappaB proteins on different NFkappaB dimers and allow additional modes of regulation of IkappaB function.

Evolution of the 14-3-3 protein family: does the large number of isoforms in multicellular organisms reflect functional specificity?

14-3-3 proteins constitute a family of eukaryotic proteins that are key regulators of a large number of processes ranging from mitosis to apoptosis. 14-3-3s function as dimers and bind to particular motifs in their target proteins. To date, 14-3-3s have been implicated in regulation or stabilization of more than 35 different proteins. This number is probably only a fraction of the number of proteins that 14-3-3s bind to, as reports of new target proteins have become more frequent. An examination of 14-3-3 entries in the public databases reveals 153 isoforms, including alleloforms, reported in 48 different species. The number of isoforms range from 2, in the unicellular organism Saccharomyces cerevisiae, to 12 in the multicellular organism Arabidopsis thaliana. A phylogenetic analysis reveals that there are four major evolutionary lineages: Viridiplantae (plants), Fungi, Alveolata, and Metazoa (animals). A close examination of the aligned amino acid sequences identifies conserved amino acid residues and regions of importance for monomer stabilization, dimer formation, target protein binding, and the nuclear export function. Given the fact that 53% of the protein is conserved, including all amino acid residues in the target binding groove of the 14-3-3 monomer, one might expect little to no isoform specificity for target protein binding. However, using surface plasmon resonance we show that there are large differences in affinity between nine 14-3-3 isoforms of A. thaliana and a target peptide representing a novel binding motif present in the C terminus of the plant plasma membrane H(+)ATPase. Thus, our data suggest that one reason for the large number of isoforms found in multicellular organisms is isoform-specific functions.

APC's NES Helps Shuttle β-Catenin PDQ

Adenomatous polyposis coli (APC) promotes the degradation of β-catenin. When APC is mutated, as in colon cancer cells, β-catenin accumulates in the nucleus, where it functions as a transcriptional coactivator of T-cell factor. The mechanism by which APC regulates β-catenin degradation has remained elusive. Now, Rosin-Arbesfeld et al . demonstrate that APC contains a functional nuclear export signal (NES), and that APC shuttles β-catenin into the cytoplasm for subsequent proteasomal degradation. COOH-terminal fragments of APC remained exclusively cytoplasmic. Treatment of APC-expressing cells with leptomycin B, a nuclear export inhibitor, revealed that APC remains in the nucleus, suggesting that an NES exists in the COOH-terminus of APC. The authors observed that β-catenin colocalized with wild-type APC in the cytoplasm, or in the nucleus with NES-mutated APC. Gene reporter assays also indicated that wild-type APC reduced the ability of β-catenin to cotransactivate luciferase expression, whereas NES-mutated APC was unable to repress β-catenin-mediated transcription. Rosin-Arbesfeld, R., Townsley, F., and Bienz, M. (2000) The APC tumour suppressor has a nuclear export function. Nature 406 : 1009-1012. [Online Journal]

The APC tumour suppressor has a nuclear export function

The adenomatous polpyposis coli (APC) protein is mutated in most colorectal tumours. Nearly all APC mutations are truncations, and many of these terminate in the mutation cluster region located halfway through the protein. In cancer cells expressing mutant APC, beta-catenin is stabilized and translocates into the nucleus to act as a transcriptional co-activator of T-cell factor. During normal development, APC also promotes the destabilization of beta-catenin and Drosophila Armadillo. It does so by binding to the Axin complex which earmarks beta-catenin/Armadillo for degradation by the proteasome pathway. APC has a regulatory role in this process, which is poorly understood. Here we show that APC contains highly conserved nuclear export signals 3' adjacent to the mutation cluster region that enable it to exit from the nucleus. This ability is lost in APC mutant cancer cells, and we provide evidence that beta-catenin accumulates in the nucleus as a result. Thus, the ability of APC to exit from the nucleus appears to be critical for its tumour suppressor function.

An inhibitory switch derepressed by pbx, hox, and Meis/Prep1 partners regulates DNA-binding by pbx1 and E2a-pbx1 and is dispensable for myeloid immortalization by E2a-pbx1.

The Pbx/Exd family of homeodomain (HD) proteins contribute to the transcriptional and developmental roles of other Hox and Meis/Prep1/Hth HD proteins through heterodimer formation. E2a-Pbx1 is an oncogenic derrivative of Pbx1 produced by the t(1;19) translocation in pediatric pre-B cell acute lymphoblastic leukemia. E2a-Pbx1 heterodimerizes with Hox but not with Meis/Prep1 proteins, produces acute myeloid leukemia in mice, and blocks differentiation of cultured murine myeloid progenitors. Here, we characterize negative and positive regulatory sequences that flank the Pbx1 HD and determine their importance for myeloid immortalization by E2a-Pbx1. A 25 residue predicted alpha helix preceding the Pbx1 HD bound the HD and prevented both its binding to DNA and its ability to heterodimerize with Hox proteins. Addition of 39 residues N-terminal to this inhibitory helix exposed a Pbx dimerization interface that orchestrated cooperative DNA-binding of E2a-Pbx1 and all Pbx proteins as homodimers and heterdimers. Sequences inhibiting DNA-binding and mediating Pbx dimerization coincided with those reported to have nuclear export function. An additional 103 residues N-terminal to the Pbx dimerization interface restored heterodimerization with Hox and Meis1/Prep1 proteins. This negative switch domain - comprised of the inhibitory helix and N-terminal regions required for its partner-mediated derepression - was dispensable for myeloid immortalization by E2a-Pbx1. While stabilizing the heterodimer, the 310 helix C-terminal to the Pbx1 HD was also dispensable for the ability of E2a-Pbx1 to heterodimerize with Hox proteins and immortalize myeloblasts. Retention of myeloid immortalization by E2a-Pbx1 proteins lacking all Pbx1 sequences N- or C-terminal to the HD indicates that Hox proteins, or a yet undefined factor that binds the Pbx1 HD and derepresses DNA-binding by the HD, cooperate with E2a-Pbx1 in myeloid immortalization.

Two well-defined motifs in the cAMP-dependent protein kinase inhibitor (PKIalpha) correlate with inhibitory and nuclear export function.

The heat stable inhibitor of cAMP-dependent protein kinase (PKIalpha) contains both a nuclear export signal (NES) and a high affinity inhibitory region that is essential for inhibition of the catalytic subunit of the kinase. These functions are sequentially independent. Two-dimensional NMR spectroscopy was performed on uniformly [15N]-labeled PKIalpha to examine its structure free in solution. Seventy out of 75 residues were identified, and examination of the CaH chemical shifts revealed two regions of upfield chemical shifts characteristic of alpha-helices. When PKIalpha was fragmented into two functionally distinct peptides for study at higher concentrations, no significant alterations in chemical shifts or secondary structure were observed. The first ordered region, identified in PKIalpha (1-25), contains an alpha-helix from residues 1-13. This helix extends by one turn the helix observed in the crystal structure of a PKIalpha (5-24) peptide bound to the catalytic subunit. The second region of well-defined secondary structure, residues 35-47, overlaps with the nuclear export signal in the PKIalpha (26-75) fragment. This secondary structure consists of a helix with a hydrophobic face comprised of Leu37, Leu41, and Leu44, followed by a flexible turn containing Ile46. These four residues are critical for nuclear export function. The remainder of the protein in solution appears relatively unstructured, and this lack of structure surrounding a few essential and well-defined signaling elements may be characteristic of a growing family of small regulatory proteins that interact with protein kinases.

The extended packaging sequence of MoMLV contains a constitutive mRNA nuclear export function

The present report shows that incorporation of defined sequences from the Moloney murine leukaemia virus (MoMLV) into Rex dependent expression vectors based on the human T-cell leukaemia virus (HTLV-1) allows Rex independent gene expression. Deletion mutagenesis of the MoMLV derived sequences allowed this function to be localised to a 312 nt length sequence overlapping the MoMLV gag p15/p12 open reading frame. This `extended packaging sequence' has been reported to markedly increase the titre of in vitro packaged retroviral vectors. Using fluorescent in situ hybridisation combined with confocal microscopy we show that the 312 nt element can replace Rex mediated nuclear export and expression of transcripts containing HTLV-1 cis acting repressive elements. Our observations are consistent with the extended packaging sequence of MoMLV exerting a constitutive mRNA nuclear export function.

Differential requirements for alternative splicing and nuclear export functions of equine infectious anemia virus Rev protein.

The Rev protein of equine infectious anemia virus (ERev) exports unspliced and partially spliced viral RNAs from the nucleus. Like several cellular proteins, ERev regulates its own mRNA by mediating an alternative splicing event. To determine the requirements for these functions, we have identified ERev mutants that affect RNA export or both export and alternative splicing. Mutants were further characterized for subcellular localization, nuclear-cytoplasmic shuttling, and multimerization. None of the nuclear export signal (NES) mutants are defective for alternative splicing. Furthermore, the NES of ERev is similar in composition but distinct in spacing from other leucine-rich NESs. Basic residues at the C terminus of ERev are involved in nuclear localization, and disruption of the C-terminal residues affects both functions of ERev. ERev forms multimers, and no mutation disrupts this activity. In two mutants with substitutions of charged residues in the middle of ERev, RNA export is affected. One of these mutants is also defective for ERev-mediated alternative splicing but is identical to wild-type ERev in its localization, shuttling, and multimerization. Together, these results demonstrate that the two functions of ERev both require nuclear import and at least one other common activity, but RNA export can be separated from alternative splicing based on its requirement for a functional NES.

Characterization of the nuclear export signal of human T-cell lymphotropic virus type 1 Rex reveals that nuclear export is mediated by position-variable hydrophobic interactions.

We previously determined that amino acids 64 to 120 of human T-cell lymphotropic virus type 1 (HTLV-1) Rex can restore the function of an effector domain mutant of human immunodeficiency virus type 1 (HIV-1) Rev (T. J. Hope, B. L. Bond, D. McDonald, N. P. Klein, and T. G. Parslow, J. Virol. 65:6001-6007, 1991). In this report, we (i) identify and characterize a position-independent 17-amino-acid region of HTLV-1 Rex that fully complements HIV-1 Rev effector domain mutants and (ii) show that this 17-amino-acid region and specific hydrophobic substitutions can serve as nuclear export signals. Mutagenesis studies revealed that four leucines within the minimal region were essential for function. Alignment of the minimal Rex region with the HIV-1 Rev effector domain suggested that the position of some of the conserved leucines is flexible. We found two of the leucines could each occupy one of two positions within the context of the full-length HTLV-1 Rex protein and maintain function. The idea of flexibility within the Rex effector domain was confirmed and extended by identifying functional substitutions by screening a library of effector domain mutants in which the two regions of flexibility were randomized. Secondly, the functional roles of the minimal Rex effector domain and hydrophobic substitutions were independently confirmed by demonstrating that these effector domains could serve as nuclear export signals when conjugated with bovine serum albumin. Nuclear export of the wild-type Rex conjugates was temperature dependent and sensitive to wheat germ agglutinin and was blocked by a 20-fold excess of unlabeled conjugates. Together, these studies reveal that position-variable hydrophobic interactions within the HTLV-1 Rex effector domain mediate nuclear export function.

Two functional domains of the influenza virus NS1 protein are required for regulation of nuclear export of mRNA

The influenza virus NS1 protein is the only known example of a protein that inhibits the nuclear export of mRNA. To identify the functional domains of this protein, we introduced 18 2- or 3-amino-acid substitutions at approximately equally spaced locations along the entire length of the protein. Two functional domains were identified. The domain near the amino end (amino acids 19 through 38) was shown to be the RNA-binding domain, by using a gel shift assay with purified NS1 protein and spliced viral NS2 mRNA as the RNA target. The second domain, which is in the carboxy half of the molecule, was presumed to be the effector domain that interacts with host nuclear proteins to carry out the nuclear RNA export function, by analogy with the effector domain of the Rev proteins of human immunodeficiency virus (HIV) and other lentiviruses which facilitate rather than inhibit nuclear RNA export. The NS1 protein has a 10-amino-acid sequence that is similar to the consensus sequence in the effector domains of lentivirus Rev proteins, specifically including two crucial leucines at positions 7 and 9 of this sequence. However, the effector domains of the NS1 and Rev (HIV type 1 [HIV-1]) proteins differed in several significant ways including the following: (i) unlike the HIV-1 Rev protein, NS1 effector domain mutants were negative recessive rather than negative dominant, (ii) the NS1 effector domain is about three times larger than the effector domain of the HIV-1 Rev protein, and (iii) unlike the HIV-1 protein, NS1 effector domain mutants exhibited a surprising property, a changed intracellular/intranuclear distribution, compared with the wild-type protein. These differences strongly suggest that the effector domains of the NS1 and Rev proteins interact with different nuclear protein targets, which likely explains the opposite effects of these two proteins on nuclear mRNA export.