Nuclear Import Mechanism Research Articles

Structural insights into Influenza A virus RNA polymerase PB1 binding to nuclear import host factor RanBP5

The genome of influenza A viruses consists of eight RNA segments that form a heterotrimer, and the viral genome undergoes transcription and replication in the nucleus. Thus, during infection, newly synthesized RNA polymerase subunits must be imported into the nucleus. Although several models have been proposed for this process, the consensus is that the RNA polymerase subunits PB1 and PA form a dimer in the cytoplasm and are transported into the nucleus by Ran binding protein 5 (RanBP5). The PB2 subunit undergoes separate transport to complete the nuclear import. However, the molecular mechanism of nuclear import by host factors and their interactions with proteins are largely unknown. Here we present the structural analysis of the RanBP5 and PB1 NLS domain complex by cryo-EM at 3.2 Å resolution. The pattern shows that the NLS domain of PB1 does not exist in a secondary structure and interacts with RanBP5 in a wrapped state. In addition, biochemical analyses of the mutant have identified critical amino acid sites involved in complex binding. The results suggest a stepwise assembly of influenza virus structural components regulated by nuclear import mechanisms and host factor binding, with important implications for drug discovery research.

Nucleoporin 153 deficiency in adult neural stem cells defines a pathological protein-network signature and defective neurogenesis in a mouse model of AD

BackgroundReduction of adult hippocampal neurogenesis is an early critical event in Alzheimer’s disease (AD), contributing to progressive memory loss and cognitive decline. Reduced levels of the nucleoporin 153 (Nup153), a key epigenetic regulator of NSC stemness, characterize the neural stem cells isolated from a mouse model of AD (3×Tg) (AD-NSCs) and determine their altered plasticity and gene expression.MethodsNup153-regulated mechanisms contributing to NSC function were investigated: (1) in cultured NSCs isolated from AD and wild type (WT) mice by proteomics; (2) in vivo by lentiviral-mediated delivery of Nup153 or GFP in the hippocampus of AD and control mice analyzing neurogenesis and cognitive function; (3) in human iPSC-derived brain organoids obtained from AD patients and control subjects as a model of neurodevelopment.ResultsProteomic approach identified Nup153 interactors in WT- and AD-NSCs potentially implicated in neurogenesis regulation. Gene ontology (GO) analysis showed that Nup153-bound proteins in WT-NSCs were involved in RNA metabolism, nuclear import and epigenetic mechanisms. Nup153-bound proteins in AD-NSCs were involved in pathways of neurodegeneration, mitochondrial dysfunction, proteasomal processing and RNA degradation. Furthermore, recovery of Nup153 levels in AD-NSCs reduced the levels of oxidative stress markers and recovered proteasomal activity. Lentiviral-mediated delivery of Nup153 in the hippocampal niche of AD mice increased the proliferation of early progenitors, marked by BrdU/DCX and BrdU/PSANCAM positivity and, later, the integration of differentiating neurons in the cell granule layer (BrdU/NeuN+ cells) compared with GFP-injected AD mice. Consistently, Nup153-injected AD mice showed an improvement of cognitive performance in comparison to AD-GFP mice at 1 month after virus delivery assessed by Morris Water Maze. To validate the role of Nup153 in neurogenesis we took advantage of brain organoids derived from AD-iPSCs characterized by fewer neuroepithelial progenitor loops and reduced differentiation areas. The upregulation of Nup153 in AD organoids recovered the formation of neural-like tubes and differentiation.ConclusionsOur data suggest that the positive effect of Nup153 on neurogenesis is based on a complex regulatory network orchestrated by Nup153 and that this protein is a valuable disease target.

The balance between nuclear import and export of NLRC5 regulates MHC class I transactivation

MHC (major histocompatibility complex) class I molecules play an essential role in regulating the adaptive immune system by presenting antigens to CD8 T cells. CITA (MHC class I transactivator), also known as NLRC5 (NLR family, CARD domain-containing 5), regulates the expression of MHC class I and essential components involved in the MHC class I antigen presentation pathway. While the critical role of the nuclear distribution of NLRC5 in its transactivation activity has been known, the regulatory mechanism to determine the nuclear localization of NLRC5 remains poorly understood. In this study, a comprehensive analysis of all domains in NLRC5 revealed that the regulatory mechanisms for nuclear import and export of NLRC5 coexist and counterbalance each other. Moreover, GCN5 (general control non-repressed 5 protein), a member of HATs (histone acetyltransferases), was found to be a key player to retain NLRC5 in the nucleus, thereby contributing to the expression of MHC class I. Therefore, the balance between import and export of NLRC5 has emerged as an additional regulatory mechanism for MHC class I transactivation, which would be a potential therapeutic target for the treatment of cancer and virus-infected diseases.

Nuclear localization signal-tagged systems: relevant nuclear import principles in the context of current therapeutic design.

Nuclear targeting of therapeutics provides a strategy for enhancing efficacy of molecules active in the nucleus and minimizing off-target effects. 'Active' nuclear-directed transport and efficient translocations across nuclear pore complexes provide the most effective means of maximizing nuclear localization. Nuclear-targeting systems based on nuclear localization signal (NLS) motifs have progressed significantly since the beginning of the current millennium. Here, we offer a roadmap for understanding the basic mechanisms of nuclear import in the context of actionable therapeutic design for developing NLS-therapeutics with improved treatment efficacy.

Exploiting the endogenous yeast nuclear proteome to identify short linear motifs in vivo

Peptide-domain interactions mediated by short linear motifs (SLiMs) play crucial roles in cellular biology. The simplicity of SLiMs poses challenges in their computational identification. Existing high-throughput methods for discovering SLiMs lack cellular context as they are typically performed invitro. We developed a functional selection method using yeast to identify peptides that interact with the endogenous yeast nuclear proteome. Remarkably, peptides selected for in yeast also mediated nuclear import in human cells. Notably, the identified peptides did not resemble classical nuclear localization sequences. This platform has the potential to identify and investigate motifs that interact with the nuclear proteome of yeast and human and to aid in the identification and understanding of alternative protein nuclear import mechanisms.

An ivermectin – atorvastatin combination impairs nuclear transport inhibiting dengue infection in vitro and in vivo

Dengue virus (DENV) uses cellular nuclear transport machinery to import some proteins into the nucleus. Recently, the non-structural protein 3 (NS3) of DENV was localized in the nucleus of infected cells; however, its nuclear import mechanism is still unknown. In this study, we demonstrate that Ivermectin (IVM) inhibits the nuclear localization of NS3 through the inhibition of the Importin α/β1 pathway. We also report that Atorvastatin (ATV) can modulate the nuclear transport of NS3 protease and NS5 polymerase of DENV-2. On the other hand, we found that IVM and ATV treatments reduce the alteration of nuclear pore complex (NPC) proteins, and an IVM+ATV combination reduced DENV infection both invitro and invivo. Hence, we conclude that ATV transport inhibition is an additional antiviral effect of this drug, suggesting a potential anti-DENV therapy in combination with IVM.

Label-free imaging of nuclear membrane for analysis of nuclear import of viral complexes

HIV-1 enters the nucleus of non-dividing cells through the nuclear pore complex where it integrates into the host genome. The mechanism of HIV-1 nuclear import remains poorly understood. A powerful means to investigate the docking of HIV-1 at the nuclear pore and nuclear import of viral complexes is through single virus tracking in live cells. This approach necessitates fluorescence labeling of HIV-1 particles and the nuclear envelope, which may be challenging, especially in the context of primary cells. Here, we leveraged a deep neural network model for label-free visualization of the nuclear envelope using transmitted light microscopy. A training image set of cells with fluorescently labeled nuclear Lamin B1 (ground truth), along with the corresponding transmitted light images, was acquired and used to train our model to predict the morphology of the nuclear envelope in fixed cells. This protocol yielded accurate predictions of the nuclear membrane and was used in conjunction with virus infection to examine the nuclear entry of fluorescently labeled HIV-1 complexes. Analyses of HIV-1 nuclear import as a function of virus input yielded identical numbers of fluorescent viral complexes per nucleus using the ground truth and predicted nuclear membrane images. We also demonstrate the utility of predicting the nuclear envelope based on transmitted light images for multicolor fluorescence microscopy of infected cells. Importantly, we show that our model can be adapted to predict the nuclear membrane of live cells imaged at 37 °C, making this approach compatible with single virus tracking. Collectively, these findings demonstrate the utility of deep learning approaches for label-free imaging of cellular structures during early stages of virus infection.

Nuclear Delivery of Nanoparticle-Based Drug Delivery Systems by Nuclear Localization Signals.

Nanomedicine 2.0 refers to the next generation of nanotechnology-based medical therapies and diagnostic tools. This field focuses on the development of more sophisticated and precise nanoparticles (NPs) for targeted drug delivery, imaging, and sensing. It has been established that the nuclear delivery of NP-loaded drugs can increase their therapeutic efficacy. To effectively direct the NPs to the nucleus, the attachment of nuclear localization signals (NLSs) to NPs has been employed in many applications. In this review, we will provide an overview of the structure of nuclear pore complexes (NPCs) and the classic nuclear import mechanism. Additionally, we will explore various nanoparticles, including their synthesis, functionalization, drug loading and release mechanisms, nuclear targeting strategies, and potential applications. Finally, we will highlight the challenges associated with developing nucleus-targeted nanoparticle-based drug delivery systems (NDDSs) and provide insights into the future of NDDSs.

Heat shock transcription factors demonstrate a distinct mode of interaction with mitotic chromosomes.

A large number of transcription factors have been shown to bind and interact with mitotic chromosomes, which may promote the efficient reactivation of transcriptional programs following cell division. Although the DNA-binding domain (DBD) contributes strongly to TF behavior, the mitotic behaviors of TFs from the same DBD family may vary. To define the mechanisms governing TF behavior during mitosis in mouse embryonic stem cells, we examined two related TFs: Heat Shock Factor 1 and 2 (HSF1 and HSF2). We found that HSF2 maintains site-specific binding genome-wide during mitosis, whereas HSF1 binding is somewhat decreased. Surprisingly, live-cell imaging shows that both factors appear excluded from mitotic chromosomes to the same degree, and are similarly more dynamic in mitosis than in interphase. Exclusion from mitotic DNA is not due to extrinsic factors like nuclear import and export mechanisms. Rather, we found that the HSF DBDs can coat mitotic chromosomes, and that HSF2 DBD is able to establish site-specific binding. These data further confirm that site-specific binding and chromosome coating are independent properties, and that for some TFs, mitotic behavior is largely determined by the non-DBD regions.

Unraveling the mystery of viroid nuclear import.

Viroids are closed-circular infectious RNAs that are known to infect plants. Despite their small noncoding genome, they have the ability to cause disease. The nuclear import mechanism of nucleus replicating viroids is not well understood. Ma et al. have recently highlighted the route of viroid entry into the nucleus.

AR-V7 exhibits non-canonical mechanisms of nuclear import and chromatin engagement in castrate-resistant prostate cancer.

Expression of the AR splice variant, androgen receptor variant 7 (AR-V7), in prostate cancer is correlated with poor patient survival and resistance to AR targeted therapies and taxanes. Currently, there is no specific inhibitor of AR-V7, while the molecular mechanisms regulating its biological function are not well elucidated. Here, we report that AR-V7 has unique biological features that functionally differentiate it from canonical AR-fl or from the second most prevalent variant, AR-v567. First, AR-V7 exhibits fast nuclear import kinetics via a pathway distinct from the nuclear localization signal dependent importin-α/β pathway used by AR-fl and AR-v567. We also show that the dimerization box domain, known to mediate AR dimerization and transactivation, is required for AR-V7 nuclear import but not for AR-fl. Once in the nucleus, AR-V7 is transcriptionally active, yet exhibits unusually high intranuclear mobility and transient chromatin interactions, unlike the stable chromatin association of liganded AR-fl. The high intranuclear mobility of AR-V7 together with its high transcriptional output, suggest a Hit-and-Run mode of transcription. Our findings reveal unique mechanisms regulating AR-V7 activity, offering the opportunity to develop selective therapeutic interventions.

Abstract 3946: Development of selective small molecule AR-V7 inhibitors for prostate cancer treatment

Abstract Castration resistance prostate cancer (CRPC) is a lethal disease in which the expression of ligand-independent androgen receptor (AR) splice variants (AR-Vs) is associated with worse clinical outcomes. AR-V7 is the most prevalent variant in CRPC, it lacks the ligand binding domain and is constitutively active in the nucleus. We and others have showed that, AR-V7 expression confers resistance to the AR signaling inhibitors (abiraterone/enzalutamide) and to taxanes in vivo and in patients with metastatic CRPC. Since abiraterone/enzalutamide and taxanes represent the two most effective therapeutic modalities for men with CRPC, the development of selective AR-V7 inhibitors is a high priority, clinically unmet need. AR-V7 shares with full-length AR (AR-fl), high sequence homology, largely overlapping cistromes and gene transactivation profiles. To develop selective AR-V7 inhibitors, we sought to identify unique biological features of AR-V7, that differentiate it from AR-fl and therapeutically exploit them. Mechanistic studies showed that AR-V7 utilizes a unique nuclear import pathway, not shared by AR-fl. Using fluorescently tagged-AR-fl or AR-V7 proteins in conjunction with live cell imaging, FRAP assays and pathway inhibitors, we showed that AR-V7 exhibits fast nuclear import kinetics partially mediated by the dimerization D-box domain, independently of microtubules and importin α/β. Taken together, these data suggest that AR-V7 nuclear import mechanism is distinct providing a window of therapeutic opportunity to selectively target it. To identify AR-V7 selective inhibitors, we designed and performed a high throughput enzyme complementation screening (HTS) assay using nuclear AR-V7 as a surrogate for AR-V7 activity. We screened a chemical library of ~170K compounds and identified hit compounds inhibiting AR-V7 by proteasomal degradation. Among the degraders, we observed 2 main modes of action: I. compounds selectively degrading AR-V7 and II. compounds degrading both AR-fl and AR-V7. Ongoing efforts include medicinal chemistry for lead compound optimization and target validation experiments.In conclusion, we identified first-in-class selective AR-V7 inhibitors, with the potential to be clinically combined with existing, but mechanistically unrelated AR signaling inhibitors. Further mechanistic studies will elucidate their potential for future clinical development. Citation Format: CheukMan Cherie Au, Seaho Kim, Prerna Vatsa, Mohd Azrin Bin Jamalruddin, Michael Miller, Peter T. Meinke, Paraskevi Giannakakou. Development of selective small molecule AR-V7 inhibitors for prostate cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3946.

Anaplasma phagocytophilum Ankyrin A Protein (AnkA) Enters the Nucleus Using an Importin-β-, RanGTP-Dependent Mechanism.

Anaplasma phagocytophilum, a tick-borne obligately intracellular bacterium of neutrophils, causes human granulocytic anaplasmosis. Ankyrin A (AnkA), an effector protein with multiple ankyrin repeats (AR) is injected via type IV-secretion into the host neutrophil to gain access to the nucleus where it modifies the epigenome to promote microbial fitness and propagation. AR proteins transported into the host cell nucleus must use at least one of two known eukaryotic pathways, the classical importin β-dependent pathway, and/or the RanGDP- and AR (ankyrin-repeat)-dependent importin β-independent (RaDAR) pathway. Truncation of the first four AnkA N-terminal ARs (AR1-4), but not other regions, prevents AnkA nuclear accumulation. To investigate the mechanism of nuclear import, we created point mutations of AnkA N-terminal ARs, predicted to interfere with RaDAR protein import, and used importazole, a specific inhibitor of the importin α/β, RanGTP-dependent pathway. Nuclear colocalization analysis shows that nuclear localization of AnkA is unaffected by single AR1-4 mutations but is significantly reduced by single mutations in consecutive ARs suggesting RaDAR protein nuclear import. However, AnkA nuclear localization was also decreased with importazole, and with GTPγS. Furthermore, A. phagocytophilum growth in HL-60 cells was completely suppressed with importazole, indicating that A. phagocytophilum propagation requires a β-importin-dependent pathway. A typical classical NLS overlapping AR4 was subsequently identified suggesting the primacy of the importin-α/β system in AnkA nuclear localization. Whether the mutational studies of putative key residues support RaDAR NLS function or simply reflect structural changes that diminish engagement of an AR-NLS-importin pathway needs to be resolved through careful structure-function studies.

Nuclear Entry of DNA Tumor Viruses: Finding the LINC in Nuclear Transport

DNA viruses that replicate in the nuclei of infected cells must transport to and deliver their viral genomes into the nucleus to cause infection. How polyomavirus (PyV), a tumor virus that causes debilitating disease in humans, accomplishes this essential step in infection is not well‐understood. During entry, PyV sorts from the cell surface to the endoplasmic reticulum (ER) where it penetrates the ER membrane to reach the cytosol. From there, the virus is transported to the nucleus for entry through the narrow nuclear pore complex (NPC). The incoming viral genome is harbored within an icosahedral capsid that is approximately 45 nm in diameter and larger than the NPC cargo limit. Using the archetype PyV, simian virus 40 (SV40), which shares both structural and genetic organization with human PyVs as well as the same infectious life cycle, we recently reported that the virus is disassembled in the cytosol by the bicaudal D2 (BICD2) dynein cargo adaptor to generate a subviral particle competent for transport through the NPC. How this disassembled particle is subsequently targeted to the nuclear membrane as well as the exact mechanism of nuclear import is unclear. In mammalian cells, intracellular transport to the nucleus is facilitated largely by the cytoplasmic dynein motor, which moves cargo along microtubules towards the center of the cell. Interestingly, BICD2 was shown to mediate the recruitment of dynein to the nuclear membrane through its association with either the linker of nucleoskeleton and cytoskeleton (LINC) complex component Nesprin‐2 or the NPC cytoplasmic filament RanBP2. Our preliminary experiments reveal that loss of Nesprin‐2 (and not RanBP2) blocks nuclear entry, suggesting that SV40 is recruited to the nucleus by a dynein‐BICD2‐Nesprin‐2 nexus. Moreover, SV40 associates with Nesprin‐2 and this interaction appears to mediate a second, previously unreported, disassembly step at the nuclear membrane. We are currently investigating the role of BICD2/Nesprin‐2 in nuclear arrival of SV40 as well as the functional relationship between Nesprin‐2 and the NPC during nuclear import. Our studies will clarify the mechanism responsible for driving these final steps of viral entry, and more broadly, further our understanding of host cellular nucleocytoplasmic transport.

Localization and translocation of mature miRNAs

The scientific review shows the ways of nuclear import and export of miRNAs in the cell. The authors present a clear and accessible scheme of microRNA translocation in the cell. The article shows that the main site of localization in the cytoplasm of cells of the RISC complex and its components, including miRNAs, are processing P-cells. The authors cite the fact that Argonaute proteins — signature components of the effector complex of RISC RNA interference — are localized in mammalian P-bodies. It is shown that proteins of the karyopherin family mediate the translocation of miRISC into the cell nucleus. These proteins recognize nuclear localization sequences (NLS) in the amino acid sequences of proteins and actively transport these proteins through the pores of the cell’s nuclear membrane. It is emphasized that in addition to non-selective mechanisms of nuclear import of miRNAs, there are transport mechanisms that carry certain miRNAs across the cell membrane. Some miRNAs are presented, which are mainly localized in the nucleus of a certain type of cell. Scientists believe that much of the nucleus miRNA is concentrated in polysomes. Export of nuclear pool microRNA into the cytoplasm of the cell occurs with the help of export 1. Thus, in the cytoplasm of the cell, mature forms of microRNA accumulate, some of which are translocated to the cell nucleus or the extracellular space. Assembly of the miRISC complex is carried out in the cytoplasm of the cell, and only after the formation of the complex, it is imported into the cell nucleus. The spectrum of exosome-associated miRNAs can be a highly important diagnostic criterion for some nosologies, and exosomes containing certain miRNAs can be used for targeted therapy of specific diseases. To write the article, information was searched using databases Scopus, Web of Science, MedLine, PubMed, Google Scholar, EMBASE, Global Health, The Cochrane Library, CyberLeninka.

A viral ubiquitination switch attenuates innate immunity and triggers nuclear import of virion DNA and infection.

Antiviral defense and virus exclusion from the cell nucleus restrict foreign nucleic acid influx and infection. How the genomes of DNA viruses evade cytosolic pattern recognition and cross the nuclear envelope is incompletely understood. Here, we show that the virion protein V of adenovirus functions as a linchpin between the genome and the capsid, thereby securing particle integrity. Absence of protein V destabilizes cytoplasmic particles and promotes premature genome release, raising cytokine levels through the DNA sensor cGAS. Non-ubiquitinable V yields stable virions, genome misdelivery to the cytoplasm, and increased cytokine levels. In contrast, normal protein V is ubiquitinated at the nuclear pore complex, dissociates from the virion depending on the E3 ubiquitin ligase Mib1 and the proteasome, and allows genome delivery into the nucleus for infection. Our data uncover previously unknown cellular and viral mechanisms of viral DNA nuclear import in pathogenesis, vaccination, gene therapy, and synthetic biology.

Nuclear Import of HIV-1.

The delivery of the HIV-1 genome into the nucleus is an indispensable step in retroviral infection of non-dividing cells, but the mechanism of HIV-1 nuclear import has been a longstanding debate due to controversial experimental evidence. It was commonly believed that the HIV-1 capsid would need to disassemble (uncoat) in the cytosol before nuclear import because the capsid is larger than the central channel of nuclear pore complexes (NPCs); however, increasing evidence demonstrates that intact, or nearly intact, HIV-1 capsid passes through the NPC to enter the nucleus. With the protection of the capsid, the HIV-1 core completes reverse transcription in the nucleus and is translocated to the integration site. Uncoating occurs while, or after, the viral genome is released near the integration site. These independent discoveries reveal a compelling new paradigm of this important step of the HIV-1 life cycle. In this review, we summarize the recent studies related to HIV-1 nuclear import, highlighting the spatial–temporal relationship between the nuclear entry of the virus core, reverse transcription, and capsid uncoating.

Near-UV Light Induced ROS Production Initiates Spatial Ca2+ Spiking to Fire NFATc3 Translocation.

Ca2+-dependent gene regulation controls several functions to determine the fate of the cells. Proteins of the nuclear factor of activated T-cells (NFAT) family are Ca2+ sensitive transcription factors that control the cell growth, proliferation and insulin secretion in β-cells. Translocation of NFAT proteins to the nucleus occurs in a sequence of events that starts with activating calmodulin-dependent phosphatase calcineurin in a Ca2+-dependent manner, which dephosphorylates the NFAT proteins and leads to their translocation to the nucleus. Here, we examined the role of IP3-generating agonists and near-UV light in the induction of NFATc3 migration to the nucleus in the pancreatic β-cell line INS-1. Our results show that IP3 generation yields cytosolic Ca2+ rise and NFATc3 translocation. Moreover, near-UV light exposure generates reactive oxygen species (ROS), resulting in cytosolic Ca2+ spiking via the L-type Ca2+ channel and triggers NFATc3 translocation to the nucleus. Using the mitochondria as a Ca2+ buffering tool, we showed that ROS-induced cytosolic Ca2+ spiking, not the ROS themselves, was the triggering mechanism of nuclear import of NFATc3. Collectively, this study reveals the mechanism of near-UV light induced NFATc3 migration.

Subcellular Localization of Epstein-Barr Virus BLLF2 and Its Underlying Mechanisms.

Epstein–Barr virus (EBV), the pathogen of several human malignancies, encodes many proteins required to be transported into the nucleus for viral DNA reproduction and nucleocapsids assembly in the lytic replication cycle. Here, fluorescence microscope, mutation analysis, interspecies heterokaryon assays, co-immunoprecipitation assay, RNA interference, and Western blot were performed to explore the nuclear import mechanism of EBV encoded BLLF2 protein. BLLF2 was shown to be a nucleocytoplasmic shuttling protein neither by a chromosomal region maintenance 1 (CRM1)- nor by a transporter associated with antigen processing (TAP)-dependent pathway. Yet, BLLF2’s two functional nuclear localization signals (NLSs), NLS1 (16KRQALETVPHPQNRGR31) and NLS2 (44RRPRPPVAKRRRFPR58), were identified, whereas the predicted NES was nonfunctional. Finally, BLLF2 was proven to transport into the nucleus via a Ran-dependent and importin β1-dependent pathway. This mechanism may contribute to a more extensive insight into the assembly and synthesis of EBV virions in the nucleus, thus affording a new direction for the treatment of viruses.

Spodoptera frugiperda mRNA export factor interacts with and mediates the nuclear import of Autographa californica multiple nucleopolyhedrovirus ORF34 (Ac34)

Autographa californica multiple nucleopolyhedrovirus orf34 (ac34) is one of the unique genes of alphabaculoviruses. For successful alphabaculovirus replication, viral proteins must be transported to the nucleus. Our previous study showed that the nuclear localization of Ac34 was required for optimal production of budded virions. To investigate the mechanism of Ac34 nuclear import, mass spectrometric analysis was performed to identify potential proteins that may be involved in the nuclear import of Ac34. The result indicated that Spodoptera frugiperda mRNA export factor (SfMEF) may interact with Ac34 during baculovirus infection. Co-immunoprecipitation assays confirmed that Ac34 could interact with SfMEF in the absence of other baculovirus proteins. The deletion of ac34 did not affect the subcellular localization of SfMEF; however, knocking down Sfmef prevented the nuclear import of Ac34 in virus-infected cells. The mutations of C116 or C119 in a potential CCCH zinc finger motif (C116-X2-C119-X8-C128-X2-H131) of Ac34 led to an exclusive cytoplasmic distribution of Ac34, in consistent with our previous finding of mutations of C128 or H131 in this motif. Co-immunoprecipitation analysis showed that the above mutations in the potential zinc finger motif disrupted the interaction between Ac34 and SfMEF, and the loss of the interaction resulted in decreased BV production. Our findings demonstrated that SfMEF interacts with and mediates the nuclear import of Ac34, which is a new nucleocytoplasmic transport pathway used by alphabaculovirus to achieve successful viral replication within the nucleus of the infected cells.