Nuclear Exportin 1 (XPO1) Binds to the Nuclear Localization/Export Signal of the Turnip Mosaic Virus NIb to Promote Viral Infection

The nuclear localization signal (NLS) and nuclear export signal (NES) are key signatures of proteins for controlling nuclear import and export. The NIb protein of turnip mosaic virus (TuMV) is an RNA-dependent RNA polymerase (RdRP) that is absolutely required for viral genome replication. Previous studies have shown that NIb is a nucleocytoplasmic shuttling protein and contains four putative NES and four putative NLS motifs. Here, we analyzed the function of these NESs and NLSs, and identified two functional NESs and one functional NLS. Mutation of the identified functional NESs or NLS inhibited viral RNA accumulation and systemic infection. Exportin 1 (XPO1) is a nuclear export receptor that binds directly to cargo proteins harboring a leucine-rich NES and translocates them to the cytoplasm. We found that XPO1 contains two NIb-binding domains, which recognize the NLS and NES of NIb, respectively, to mediate the nucleocytoplasmic transport of NIb and promote viral infection. Taken together, these data suggest that the nucleocytoplasmic transport of NIb is modulated by XPO1 through its interactions with the functional NLS and NES of NIb to promote viral infection.

The Membrane-Anchoring Region of the AcMNPV P74 Protein Is Expendable or Interchangeable with Homologs from Other Species

Baculoviruses are insect pathogens that are characterized by assembling the viral dsDNA into two different enveloped virions during an infective cycle: occluded virions (ODVs; immersed in a protein matrix known as occlusion body) and budded virions (BVs). ODVs are responsible for the primary infection in midgut cells of susceptible larvae thanks to the per os infectivity factor (PIF) complex, composed of at least nine essential viral proteins. Among them, P74 is a crucial factor whose activity has been identified as virus-specific. In this work, the p74 gene from AcMNPV was pseudogenized using CRISPR/Cas9 technology and then complemented with wild-type alleles from SeMNPV and HearSNPV species, as well as chimeras combining the P74 amino and carboxyl domains. The results on Spodoptera exigua and Rachiplusia nu larvae showed that an amino terminal sector of P74 (lacking two potential transmembrane regions but possessing a putative nuclear export signal) is sufficient to restore the virus infectivity whether alone or fused to the P74 transmembrane regions of the other evaluated viral species. These results provide novel information about the functional role of P74 and delimit the region on which mutagenesis could be applied to enhance viral activity and, thus, produce better biopesticides.

Ubiquitination on Lysine 247 of Newcastle Disease Virus Matrix Protein Enhances Viral Replication and Virulence by Driving Nuclear-Cytoplasmic Trafficking

The Newcastle disease virus (NDV) matrix (M) protein is the pivotal element for viral assembly, budding and proliferation. It traffics through the cellular nucleus but performs its primary function in the cytoplasm. To investigate the biological importance of M’s nuclear–cytoplasmic trafficking and the mechanism involved, the regulatory motif nuclear export signal (NES) and nuclear localization signal (NLS) were deeply analyzed. Here, two types of combined NLS and NES signals were identified within NDV-M. The Herts/33-type M was found to mediate efficient nuclear export and stable virus-like particle (VLP) release, while the LaSota-type M was mostly retained in the nuclei and showed retarded VLP production. Two critical residues, 247 and 263, within the motif were identified and associated with nuclear export efficiency. We identified, for the first time, residue 247 as an important monoubiquitination site, the modification of which regulates the nuclear–cytoplasmic trafficking of NDV-M. Subsequently, mutant LaSota strains were rescued via reverse genetics, which contained either single or double amino acid substitutions that were similar to the M of Herts/33. The rescued rLaSota strains rLaSota-R247K, -S263R, and -DM (double mutation) showed about twofold higher HA titers and 10-fold higher EID 50 titers than wild-type (wt) rLaSota. Further, the MDT and ICPI values of those recombinant viruses were slightly higher than that of wt rLaSota probably due to their higher proliferation rates. Our findings contribute to a better understanding of the molecular mechanism of the replication and pathogenicity of NDV, and even those of all other paramyxoviruses. It is beneficial for the development of vaccines and therapies for paramyxoviruses. Importance Newcastle disease virus (NDV) is a pathogen that is lethal to birds and causes heavy losses in the poultry industry worldwide. The World Organization for Animal Health (OIE) ranked ND as the third most significant poultry disease and the eighth most important wildlife disease in the World Livestock Disease Atlas in 2011. The matrix (M) protein of NDV is very important for viral assembly and maturation. It is interesting that M proteins enter the cellular nucleus before performing their primary function in the cytoplasm. We found that NDV-M has a combined nuclear import and export signal. The ubiquitin modification of a lysine residue within this signal is critical for quick, efficient nuclear export and subsequent viral production. Our findings shed new light on viral replication and opens up new possibilities for therapeutics against NDV and other paramyxoviruses; furthermore, we demonstrate a novel approach to improving paramyxovirus vaccines.

A quantitative study of the Golgi retention of glycosyltransferases

How Golgi glycosyltransferases and glycosidases (hereafter glycosyltransferases) localize to the Golgi is still unclear. Here, we first investigated the post-Golgi trafficking of glycosyltransferases. We found that glycosyltransferases can escape the Golgi to the plasma membrane, where they are subsequently endocytosed to the endolysosome. Post-Golgi glycosyltransferases are probably degraded by the ectodomain shedding. We discovered that most glycosyltransferases are not retrieved from post-Golgi sites, indicating that retention but not retrieval should be the primary mechanism for their Golgi localization. We proposed to use the Golgi residence time to quantitatively and systematically study Golgi retention of glycosyltransferases. Various swapping chimeras between ST6GAL1 and either transferrin receptor or tumor necrosis factor α quantitatively revealed the contributions of three regions of ST6GAL1, namely the N-terminal cytosolic tail, transmembrane domain, and ectodomain, to Golgi retention. We found that each of the three regions is sufficient to produce retention in an additive manner. The N-terminal cytosolic tail length negatively affects the Golgi retention of ST6GAL1, similar to the effect of the transmembrane domain. Therefore, the long N-terminal cytosolic tail and transmembrane domain can be a Golgi export signal for transmembrane secretory cargos.

Caspase-mediated nuclear pore complex trimming in cell differentiation and endoplasmic reticulum stress

Introductory ParagraphDuring programmed cell death, caspases degrade 7 out of ∼30 nucleoporins (Nups) to irreversibly demolish the nuclear pore complex (NPC)1. However, for poorly understood reasons, caspases are also activated in differentiating cells in a non-apoptotic manner2,3. Here, we describe reversible, caspase-mediated NPC “trimming” during early myogenesis. We find that sublethal levels of caspases selectively proteolyze 4 peripheral Nups, Nup358, Nup214, Nup153, and Tpr, resulting in the transient block of nuclear export pathways. Several nuclear export signal (NES)-containing focal adhesion proteins concomitantly accumulate in the nucleus where they function as transcription cofactors4. We show that one such protein, FAK (focal adhesion kinase), drives a global reconfiguration of MBD2 (methyl CpG binding domain protein 2)-mediated genome regulation. We also observe caspase-mediated NPC trimming during neurogenesis and endoplasmic reticulum (ER) stress. Our results illustrate that the NPC can be proteolytically regulated in response to non-apoptotic cues, and call for a reassessment of the death-centric view of caspases.

NOVEL NPM1 EXON 5 MUTATIONS AND GENE FUSIONS LEADING TO ABERRANT CYTOPLASMIC NUCLEOPHOSMIN IN AML

Nucleophosmin (NPM1) mutations in acute myeloid leukemia (AML) affect exon 12, but sporadically also exon 9 and 11, all causing changes at protein C-terminal end (loss of tryptophans and creation of a nuclear export signal-NES motif) that lead to aberrant cytoplasmic NPM1 (NPM1c+), detectable by immunohistochemistry. Combining immunohistochemistry and molecular analyses in 929 AML patients, we found non-exon 12 NPM1 mutations in 5/387 (1.3%) NPM1c+ cases. Besides mutations in exon 9 (n=1) and exon 11 (n=1), novel mutations in exon 5 were discovered (n=3). One more exon 5 mutation was identified in additional 141 AML patients selected for wild-type NPM1 exon 12. Furthermore, 3 NPM1 rearrangements (i.e. NPM1/RPP30, NPM1/SETBP1, NPM1/CCDC28A) were detected and characterized among 13,979 AML samples screened by cytogenetic/FISH and RNA sequencing. Functional studies demonstrated that in AML cases the new NPM1 proteins harboured an efficient extra NES, either newly created or already present in the fusion partner, ensuring its cytoplasmic accumulation. Our findings support NPM1 cytoplasmic relocation as critical for leukemogenesis and reinforce the role of immunohistochemistry in predicting any AML-associated NPM1 genetic lesions. Also, this study highlights the need for developing new specific assays for molecular diagnosis and monitoring of NPM1-mutated AML.

Metabolite trafficking enables programmed terpene secretion by yeast

Metabolites are often unable to permeate cell membranes and are thus accumulated inside cells 1. We investigated whether engineered microbes could exclusively secrete intracellular metabolites because sustainable metabolite secretion holds a great potential for mass-production of high-value chemicals in an efficient and continuous manner 2,3. In this study, we demonstrated a synthetic pathway for a metabolite trafficking system that enables lipophilic terpene secretion by yeast cells. When metabolite-binding proteins are tagged with signal peptides, metabolite trafficking becomes programmable; loaded metabolites can be precisely delivered to a desired location within or outside the cell. As a proof of concept, we systematically coupled a terpene-specific protein with an export signal peptide and subsequently demonstrated exceptionally efficient, yet selective terpene secretion by yeast (~225 mg/L for squalene and ~1.6 mg/L for β-carotene). Other carrier proteins could also be readily fused with desired signal peptides, thereby tailoring different metabolite trafficking pathways in different microbes. To the best of our knowledge, this is the first and most efficient cognate pathway for metabolite secretion by microorganisms.

Distinct mutations in importin-β family nucleocytoplasmic transport receptors transportin-SR and importin-13 affect specific cargo binding

Importin-(Imp)β family nucleocytoplasmic transport receptors (NTRs) are supposed to bind to their cargoes through interaction between a confined interface on an NTR and a nuclear localization or export signal (NLS/NES) on a cargo. Although consensus NLS/NES sequence motifs have been defined for cargoes of some NTRs, many experimentally identified cargoes of those NTRs lack those motifs, and consensus NLSs/NESs have been reported for only a few NTRs. Crystal structures of NTR–cargo complexes have exemplified 3D structure-dependent binding of cargoes lacking a consensus NLS/NES to different sites on an NTR. Since only a limited number of NTR–cargo interactions have been studied, whether most cargoes lacking a consensus NLS/NES bind to the same confined interface or to various sites on an NTR is still unclear. Addressing this issue, we generated four mutants of transportin-(Trn)SR, of which many cargoes lack a consensus NLS, and eight mutants of Imp13, where no consensus NLS has been defined, and we analyzed their binding to as many as 40 cargo candidates that we previously identified by a nuclear import reaction-based method. The cargoes bind differently to the NTR mutants, suggesting that positions on an NTR contribute differently to the binding of respective cargoes.