The mRNA-capping enzyme localizes to stress granules in the cytoplasm and maintains cap homeostasis of target mRNAs.

The model of RNA stability has undergone a transformative shift with the revelation of a cytoplasmic capping activity that means a subset of transcripts are recapped autonomously of their nuclear counterparts. The present study demonstrates nucleo-cytoplasmic shuttling of the mRNA-capping enzyme (CE, also known as RNA guanylyltransferase and 5'-phosphatase; RNGTT), traditionally acknowledged for its nuclear localization and functions, elucidating its contribution to cytoplasmic capping activities. A unique nuclear export sequence in CE mediates XPO1-dependent nuclear export of CE. Notably, during sodium arsenite-induced oxidative stress, cytoplasmic CE (cCE) congregates within stress granules (SGs). Through an integrated approach involving molecular docking and subsequent co-immunoprecipitation, we identify eIF3b, a constituent of SGs, as an interactive associate of CE, implying that it has a potential role in guiding cCE to SGs. We measured the cap status of specific mRNA transcripts from U2OS cells that were non-stressed, stressed and recovered from stress, which indicated that cCE-target transcripts lost their caps during stress but remarkably regained cap stability during the recovery phase. This comprehensive study thus uncovers a novel facet of cytoplasmic CE, which facilitates cellular recovery from stress by maintaining cap homeostasis of target mRNAs.

Nucleoporin Nup358 drives the differentiation of myeloid-biased multipotent progenitors by modulating HDAC3 nuclear translocation.

Nucleoporins, the components of nuclear pore complexes (NPCs), can play cell type- and tissue-specific functions. Yet, the physiological roles and mechanisms of action for most NPC components have not yet been established. We report that Nup358, a nucleoporin linked to several myeloid disorders, is required for the developmental progression of early myeloid progenitors. We found that Nup358 ablation in mice results in the loss of myeloid-committed progenitors and mature myeloid cells and the accumulation of myeloid-primed multipotent progenitors (MPPs) in bone marrow. Accumulated MPPs in Nup358 knockout mice are greatly restricted to megakaryocyte/erythrocyte-biased MPP2, which fail to progress into committed myeloid progenitors. Mechanistically, we found that Nup358 is required for histone deacetylase 3 (HDAC3) nuclear import and function in MPP2 cells and established that this nucleoporin regulates HDAC3 nuclear translocation in a SUMOylation-independent manner. Our study identifies a critical function for Nup358 in myeloid-primed MPP2 differentiation and uncovers an unexpected role for NPCs in the early steps of myelopoiesis.

Not just binary: embracing the complexity of nuclear division dynamics.

Cell division presents a challenge for eukaryotic cells: how can chromosomes effectively segregate within the confines of a membranous nuclear compartment? Different organisms have evolved diverse solutions by modulating the degree of nuclear compartmentalization, ranging from complete nuclear envelope breakdown to complete maintenance of nuclear compartmentalization via nuclear envelope expansion. Many intermediate forms exist between these extremes, suggesting that nuclear dynamics during cell division are surprisingly plastic. In this review, we highlight the evolutionary diversity of nuclear divisions, focusing on two defining characteristics: (1) chromosome compartmentalization and (2) nucleocytoplasmic transport. Further, we highlight recent evidence that nuclear behavior during division can vary within different cellular contexts in the same organism. The variation observed within and between organisms underscores the dynamic evolution of nuclear divisions tailored to specific contexts and cellular requirements. In-depth investigation of diverse nuclear divisions will enhance our understanding of the nucleus, both in physiological and pathological states.

The bioenergetics of nucleocytoplasmic transport.

How nucleocytoplasmic transport (NCT) rates change due to cellular physiology-mediated fluctuations in GTP availability remains unclear. In this issue, Scott et al. (https://doi.org/10.1083/jcb.202308152) demonstrate that cell migration, spreading, and nucleocytoskeletal coupling impact GTP levels, thereby regulating NCT, RNA export, and protein synthesis.

Significance of signal recognition particle 9 nuclear translocation: Implications for pancreatic cancer prognosis and functionality.

Signal recognition particles (SRPs) are essential for regulating intracellular protein transport and secretion. Patients with tumors with high SRP9 expression tend to have a poorer overall survival. However, to the best of our knowledge, no reports have described the relationship between SRP9 localization and prognosis in pancreatic cancer. Thus, the present study aimed to investigate this relationship. Immunohistochemical staining for SRP9 using excised specimens from pancreatic cancer surgery cases without preoperative chemotherapy or radiotherapy showed that SRP9 was preferentially expressed in the nucleus of the cancerous regions in some cases, which was hardly detected in other cases, indicating that SRP9 was transported to the nucleus in the former cases. To compare the prognosis of patients with SRP9 nuclear translocation, patients were divided into two groups: Those with a nuclear translocation rate of >50% and those with a nuclear translocation rate of ≤50%. The nuclear translocation rate of >50% group had a significantly better recurrence­free survival than the nuclear translocation rate of ≤50% group (P=0.037). Subsequent in vitro experiments were conducted; notably, the nuclear translocation rate of SRP9 was reduced under amino acid­deficient conditions, suggesting that multiple factors are involved in this phenomenon. To further study the function of SRP9 nuclear translocation, in vitro experiments were performed by introducing SRP9 splicing variants (v1 and v2) and their deletion mutants lacking C­terminal regions into MiaPaCa pancreatic cancer cells. The results demonstrated that both splicing variants showed nuclear translocation regardless of the C­terminal deletions, suggesting the role of the N­terminal regions. Given that SRP9 is an RNA­binding protein, the study of RNA immunoprecipitation revealed that signaling pathways involved in cancer progression and protein translation were downregulated in nuclear­translocated v1 and v2. Undoubtedly, further studies of the nuclear translocation of SRP9 will open an avenue to optimize the precise evaluation and therapeutic control of pancreatic cancer.

Regions of Bovine Adenovirus-3 Protein VII Involved in Interactions with Viral and Cellular Proteins.

The L 1 region of bovine adenovirus (BAdV)-3 encodes a multifunctional protein named protein VII. Anti-protein VII sera detected a protein of 26 kDa in transfected or BAdV-3-infected cells, which localizes to nucleus and nucleolus of infected/transfected cells. Analysis of mutant protein VII identified four redundant overlapping nuclear/nucleolar localization signals as deletion of all four potential nuclear/nucleolar localization signals localizes protein VII predominantly to the cytoplasm. The nuclear import of protein VII appears to use importin α (α-1), importin-ß (ß-1) and transportin-3 nuclear transport receptors. In addition, different nuclear transport receptors also require part of protein VII outside nuclear localization sequences for efficient interaction. Proteomic analysis of protein complexes purified from recombinant BAdV-3 expressing protein VII containing Strep Tag II identified potential viral and cellular proteins interacting with protein VII. Here, we confirm that protein VII interacts with IVa2 and protein VIII in BAdV-3-infected cells. Moreover, amino acids 91-101 and 126-137, parts of non-conserved region of protein VII, are required for interaction with IVa2 and protein VIII, respectively.

The nuclear export protein exportin-1 in solid malignant tumours: From biology to clinical trials.

BACKGROUND: Exportin-1 (XPO1), a crucial protein regulating nuclear-cytoplasmic transport, is frequently overexpressed in various cancers, driving tumor progression and drug resistance. This makes XPO1 an attractive therapeutic target. Over the past few decades, the number of available nuclear export-selective inhibitors has been increasing. Only KPT-330 (selinexor) has been successfully used for treating haematological malignancies, and KPT-8602 (eltanexor) has been used for treating haematologic tumours in clinical trials. However, the use of nuclear export-selective inhibitors for the inhibition of XPO1 expression has yet to be thoroughly investigated in clinical studies and therapeutic outcomes for solid tumours. METHODS: We collected numerous literatures to explain the efficacy of XPO1 Inhibitors in preclinical and clinical studies of a wide range of solid tumours. RESULTS: In this review, we focus on the nuclear export function of XPO1 and results from clinical trials of its inhibitors in solid malignant tumours. We summarized the mechanism of action and therapeutic potential of XPO1 inhibitors, as well as adverse effects and response biomarkers. CONCLUSION: XPO1 inhibition has emerged as a promising therapeutic strategy in the fight against cancer, offering a novel approach to targeting tumorigenic processes and overcoming drug resistance. SINE compounds have demonstrated efficacy in a wide range of solid tumours, and ongoing research is focused on optimizing their use, identifying response biomarkers, and developing effective combination therapies. KEY POINTS: Exportin-1 (XPO1) plays a critical role in mediating nucleocytoplasmic transport and cell cycle. XPO1 dysfunction promotes tumourigenesis and drug resistance within solid tumours. The therapeutic potential and ongoing researches on XPO1 inhibitors in the treatment of solid tumours. Additional researches are essential to address safety concerns and identify biomarkers for predicting patient response to XPO1 inhibitors.

Gene alterations in the nuclear transport receptor superfamily: A study of head and neck cancer.

In cancer cells, the nuclear transport system is often disrupted, leading to abnormal localization of nuclear proteins and altered gene expression. This disruption can arise from various mechanisms such as mutations in genes that regulate nuclear transport, altered expression of transport proteins, and changes in nuclear envelope structure. Oncogenic protein build-up in the nucleus due to the disturbance in nuclear transport can also boost tumor growth and cell proliferation. In this study, we performed bioinformatic analyses of 23 key nuclear transport receptors using genomic and transcriptomic data from pancancer and head and neck squamous cell carcinoma (HNSCC) datasets from The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia and found that the total alteration frequency of 23 nuclear transport receptors in 2691 samples of the PCAWG Consortium was 42.1% and a high levels of genetic alterations was significantly associated with poor overall survival. Amplification was the most common type of genetic alterations, and results in the overexpression of nuclear transport receptors in HNSCC compared to normal tissues. Furthermore, our study revealed that seven out of eight cell cycle genes (CDK1, CDK2, CDK4, CDK6, CCNA1, CCNB1, and CCNE2) were significantly and positively correlated with nuclear transport receptor genes in TCGA pancancer and CCLE datasets. Additionally, functional enrichment analysis showed that nuclear transport receptor genes were mainly enriched in the adhesion junction, cell cycle, ERBB, MAPK, MTOR and WNT signaling pathways.

Monitoring HIV-1 Nuclear Import Kinetics Using a Chemically Induced Nuclear Pore Blockade Assay.

To integrate with host chromatin and establish a productive infection, HIV-1 must translocate the viral Ribonucleoprotein (RNP) complex through the nuclear pore complex (NPC). Current assay to measure HIV-1 nuclear import relies on a transient byproduct of HIV-1 integration failure called 2-LTR circles. However, 2-LTR circles require complete or near-complete reverse transcription and association with the non-homologous end joining (NHEJ) machinery in the nucleus, which can complicate interpretation of 2-LTR circle formation as a measure of nuclear import kinetics. Here, we describe an approach to measure nuclear import of infectious HIV-1 particles. This involves chemically induced dimerization of Nup62, a central FG containing nucleoporin. Using this technique, nuclear import of infectious particles can be monitored in both primary and cell culture models. In response to host factor depletion or restriction factors, changes in HIV-1 nuclear import can be effectively measured using the nuclear import kinetics (NIK) assay.

Nuclear export is a limiting factor in eukaryotic mRNA metabolism.

The eukaryotic mRNA life cycle includes transcription, nuclear mRNA export and degradation. To quantify all these processes simultaneously, we perform thiol-linked alkylation after metabolic labeling of RNA with 4-thiouridine (4sU), followed by sequencing of RNA (SLAM-seq) in the nuclear and cytosolic compartments of human cancer cells. We develop a model that reliably quantifies mRNA-specific synthesis, nuclear export, and nuclear and cytosolic degradation rates on a genome-wide scale. We find that nuclear degradation of polyadenylated mRNA is negligible and nuclear mRNA export is slow, while cytosolic mRNA degradation is comparatively fast. Consequently, an mRNA molecule generally spends most of its life in the nucleus. We also observe large differences in the nuclear export rates of different 3'UTR transcript isoforms. Furthermore, we identify genes whose expression is abruptly induced upon metabolic labeling. These transcripts are exported substantially faster than average mRNAs, suggesting the existence of alternative export pathways. Our results highlight nuclear mRNA export as a limiting factor in mRNA metabolism and gene regulation.

Parental experiences orchestrate locust egg hatching synchrony by regulating nuclear export of precursor miRNA.

Parental experiences can affect the phenotypic plasticity of offspring. In locusts, the population density that adults experience regulates the number and hatching synchrony of their eggs, contributing to locust outbreaks. However, the pathway of signal transmission from parents to offspring remains unclear. Here, we find that transcription factor Forkhead box protein N1 (FOXN1) responds to high population density and activates the polypyrimidine tract-binding protein 1 (Ptbp1) in locusts. FOXN1-PTBP1 serves as an upstream regulator of miR-276, a miRNA to control egg-hatching synchrony. PTBP1 boosts the nucleo-cytoplasmic transport of pre-miR-276 in a "CU motif"-dependent manner, by collaborating with the primary exportin protein exportin 5 (XPO5). Enhanced nuclear export of pre-miR-276 elevates miR-276 expression in terminal oocytes, where FOXN1 activates Ptbp1 and leads to egg-hatching synchrony in response to high population density. Additionally, PTBP1-prompted nuclear export of pre-miR-276 is conserved in insects, implying a ubiquitous mechanism to mediate transgenerational effects.

Inhibition of mRNA nuclear export promotes SARS-CoV-2 pathogenesis.

The nonstructural protein 1 (Nsp1) of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a virulence factor that targets multiple cellular pathways to inhibit host gene expression and antiviral response. However, the underlying mechanisms of the various Nsp1-mediated functions and their contributions to SARS-CoV-2 virulence remain unclear. Among the targets of Nsp1 is the mRNA (messenger ribonucleic acid) export receptor NXF1-NXT1, which mediates nuclear export of mRNAs from the nucleus to the cytoplasm. Based on Nsp1 crystal structure, we generated mutants on Nsp1 surfaces and identified an acidic N-terminal patch that is critical for interaction with NXF1-NXT1. Photoactivatable Nsp1 probe reveals the RNA Recognition Motif (RRM) domain of NXF1 as an Nsp1 N-terminal binding site. By mutating the Nsp1 N-terminal acidic patch, we identified a separation-of-function mutant of Nsp1 that retains its translation inhibitory function but substantially loses its interaction with NXF1 and reverts Nsp1-mediated mRNA export inhibition. We then generated a recombinant (r)SARS-CoV-2 mutant on the Nsp1 N-terminal acidic patch and found that this surface is key to promote NXF1 binding and inhibition of host mRNA nuclear export, viral replication, and pathogenicity in vivo. Thus, these findings provide a mechanistic understanding of Nsp1-mediated mRNA export inhibition and establish the importance of this pathway in the virulence of SARS-CoV-2.

Exportin XPO6 upregulation activates the TLR2/MyD88/NF-κB signaling by facilitating TLR2 mRNA nuclear export in COPD pulmonary monocytes.

Chronic obstructive pulmonary disease (COPD) poses a significant health threat characterized by lung inflammation primarily triggered by pulmonary monocytes. Despite the centrality of inflammation in COPD, the regulatory mechanisms governing this response remain elusive, presenting a challenge for anti-inflammatory interventions. In this study, we assessed the expression of exportins in COPD mouse models, revealing a notable upregulation of XPO6 in the mouse lung (P = 0.0011). Intriguingly, we observed a consistent upregulation of XPO6 in pulmonary monocytes from both human and mouse COPD subjects (P < 0.0001). Furthermore, in human lung tissue, XPO6 expression exhibited a positive correlation with TLR2 expression (P = 0). In vitro investigations demonstrated that XPO6 enhances TLR2 expression, activating the MyD88/NF-κB inflammatory signaling pathway. This activation, in turn, promotes the secretion of pro-inflammatory cytokines such as TNFα, IL-6, and IL-1ß in monocytes. Mechanistically, XPO6 facilitates the nuclear export of TLR2 mRNA, ensuring its stability and subsequent protein expression in monocytes. In conclusion, our findings unveil that the upregulation of XPO6 in COPD pulmonary monocytes activates the MyD88/NF-κB inflammatory signaling pathway by facilitating the nuclear export of TLR2 mRNA, thereby identifying XPO6 as a promising therapeutic target for anti-inflammatory interventions in COPD.

CLK2 mediates IκBα-independent early termination of NF-κB activation by inducing cytoplasmic redistribution and degradation.

Activation of the NF-κB pathway is strictly regulated to prevent excessive inflammatory and immune responses. In a well-known negative feedback model, IκBα-dependent NF-κB termination is a delayed response pattern in the later stage of activation, and the mechanisms mediating the rapid termination of active NF-κB remain unclear. Here, we showed IκBα-independent rapid termination of nuclear NF-κB mediated by CLK2, which negatively regulated active NF-κB by phosphorylating the RelA/p65 subunit of NF-κB at Ser180 in the nucleus to limit its transcriptional activation through degradation and nuclear export. Depletion of CLK2 increased the production of inflammatory cytokines, reduced viral replication and increased the survival of the mice. Mechanistically, CLK2 phosphorylated RelA/p65 at Ser180 in the nucleus, leading to ubiquitin‒proteasome-mediated degradation and cytoplasmic redistribution. Importantly, a CLK2 inhibitor promoted cytokine production, reduced viral replication, and accelerated murine psoriasis. This study revealed an IκBα-independent mechanism of early-stage termination of NF-κB in which phosphorylated Ser180 RelA/p65 turned off posttranslational modifications associated with transcriptional activation, ultimately resulting in the degradation and nuclear export of RelA/p65 to inhibit excessive inflammatory activation. Our findings showed that the phosphorylation of RelA/p65 at Ser180 in the nucleus inhibits early-stage NF-κB activation, thereby mediating the negative regulation of NF-κB.

p37 regulates VCP/p97 shuttling and functions in the nucleus and cytosol.

The AAA+-ATPase valosin-containing protein (VCP; also called p97 or Cdc48), a major protein unfolding machinery with a variety of essential functions, localizes to different subcellular compartments where it has different functions. However, the processes regulating the distribution of VCP between the cytosol and nucleus are not understood. Here, we identified p37 (also called UBXN2B) as a major factor regulating VCP nucleocytoplasmic shuttling. p37-dependent VCP localization was crucial for local cytosolic VCP functions, such as autophagy, and nuclear functions in DNA damage repair. Mutations in VCP causing multisystem proteinopathy enhanced its association with p37, leading to decreased nuclear localization of VCP, which enhanced susceptibility to DNA damage accumulation. Both VCP localization and DNA damage susceptibility in cells with such mutations were normalized by lowering p37 levels. Thus, we uncovered a mechanism by which VCP nucleocytoplasmic distribution is fine-tuned, providing a means for VCP to respond appropriately to local needs.

Identifying cellular RNA-binding proteins during infection uncovers a role for MKRN2 in influenza mRNA trafficking.

Utilisation of RNA-binding proteins (RBPs) is an important aspect of post-transcriptional regulation of viral RNA. Viruses such as influenza A viruses (IAV) interact with RBPs to regulate processes including splicing, nuclear export and trafficking, while also encoding RBPs within their genomes, such as NP and NS1. But with almost 1000 RBPs encoded within the human genome it is still unclear what role, if any, many of these proteins play during viral replication. Using the RNA interactome capture (RIC) technique, we isolated RBPs from IAV infected cells to unravel the RBPome of mRNAs from IAV infected human cells. This led to the identification of one particular RBP, MKRN2, that associates with and positively regulates IAV mRNA. Through further validation, we determined that MKRN2 is involved in the nuclear-cytoplasmic trafficking of IAV mRNA potentially through an association with the RNA export mediator GLE1. In the absence of MKRN2, IAV mRNAs accumulate in the nucleus of infected cells, which may lead to their degradation by the nuclear RNA exosome complex. MKRN2, therefore, appears to be required for the efficient nuclear export of IAV mRNAs in human cells.

Imaging HIV-1 Nuclear Import, Uncoating, and Proviral Transcription.

Live-cell imaging has become a powerful tool for dissecting the behavior of viral complexes during HIV-1 infection with high temporal and spatial resolution. Very few HIV-1 particles in a viral population are infectious and successfully complete replication (~1/50). Single-particle live-cell imaging enables the study of these rare infectious viral particles, which cannot be accomplished in biochemical assays that measure the average property of the entire viral population, most of which are not infectious. The timing and location of many events in the early stage of the HIV-1 life cycle, including nuclear import, uncoating, and integration, have only recently been elucidated. Live-cell imaging also provides a valuable approach to study interactions of viral and host factors in distinct cellular compartments and at specific stages of viral replication. Successful live-cell imaging experiments require careful consideration of the fluorescent labeling method used and avoid or minimize its potential impact on normal viral replication and produce misleading results. Ideally, it is beneficial to utilize multiple virus labeling strategies and compare the results to ensure that the virion labeling did not adversely influence the viral replication step that is under investigation. Another potential benefit of using different labeling strategies is that they can provide information about the state of the viral complexes. Here, we describe our methods that utilize multiple fluorescent protein labeling approaches to visualize and quantify important events in the HIV-1 life cycle, including docking HIV-1 particles with the nuclear envelope (NE) and their nuclear import, uncoating, and proviral transcription.

Detection of CPSF6 in Biomolecular Condensates as a Reporter of HIV-1 Nuclear Import.

The initial stages of HIV-1 infection involve the transport of the viral core into the nuclear compartment. The presence of the HIV-1 core in the nucleus triggers the translocation of CPSF6/CPSF5 from paraspeckles into nuclear speckles, forming puncta-like structures. While this phenomenon is well-documented, the efficiency of CPSF6 translocation to nuclear speckles upon HIV-1 infection varies depending on the type of cell used. In some human cell lines, only 1-2% of the cells translocate CPSF6 to nuclear speckles when exposed to a 95% infection rate. To address the issue that only 1-2% of cells translocate CPSF6 to nuclear speckles when a 95% infection rate is achieved, we screened several human cell lines and identified a human a cell line in which approximately 85% of the cells translocate CPSF6 to nuclear speckles when 95% infection rate is achieved. This cellular system has enabled the development of a robust fluorescence microscopy method to quantify the translocation of CPSF6 into nuclear speckles following HIV-1 infection. This assay holds the potential to support studies aimed at understanding the role of CPSF6 translocation to nuclear speckles in HIV-1 infection. Additionally, since the translocation of CPSF6 into nuclear speckles depends on the physical presence of the viral core in the nucleus, our method also serves as a reporter of HIV-1 nuclear import.

PI3K/HSCB axis facilitates FOG1 nuclear translocation to promote erythropoiesis and megakaryopoiesis.

Erythropoiesis and megakaryopoiesis are stringently regulated by signaling pathways. However, the precise molecular mechanisms through which signaling pathways regulate key transcription factors controlling erythropoiesis and megakaryopoiesis remain partially understood. Herein, we identified heat shock cognate B (HSCB), which is well known for its iron-sulfur cluster delivery function, as an indispensable protein for friend of GATA 1 (FOG1) nuclear translocation during erythropoiesis of K562 human erythroleukemia cells and cord-blood-derived human CD34+CD90+hematopoietic stem cells (HSCs), as well as during megakaryopoiesis of the CD34+CD90+HSCs. Mechanistically, HSCB could be phosphorylated by phosphoinositol-3-kinase (PI3K) to bind with and mediate the proteasomal degradation of transforming acidic coiled-coil containing protein 3 (TACC3), which otherwise detained FOG1 in the cytoplasm, thereby facilitating FOG1 nuclear translocation. Given that PI3K is activated during both erythropoiesis and megakaryopoiesis, and that FOG1 is a key transcription factor for these processes, our findings elucidate an important, previously unrecognized iron-sulfur cluster delivery independent function of HSCB in erythropoiesis and megakaryopoiesis.

Vaccinia virus subverts xenophagy through phosphorylation and nuclear targeting of p62.

Autophagy is an essential degradation program required for cell homeostasis. Among its functions is the engulfment and destruction of cytosolic pathogens, termed xenophagy. Not surprisingly, many pathogens use various strategies to circumvent or co-opt autophagic degradation. For poxviruses, it is known that infection activates autophagy, which however is not required for successful replication. Even though these complex viruses replicate exclusively in the cytoplasm, autophagy-mediated control of poxvirus infection has not been extensively explored. Using the prototypic poxvirus, vaccinia virus (VACV), we show that overexpression of the xenophagy receptors p62, NDP52, and Tax1Bp1 restricts poxvirus infection. While NDP52 and Tax1Bp1 were degraded, p62 initially targeted cytoplasmic virions before being shunted to the nucleus. Nuclear translocation of p62 was dependent upon p62 NLS2 and correlated with VACV kinase mediated phosphorylation of p62 T269/S272. This suggests that VACV targets p62 during the early stages of infection to avoid destruction and further implies that poxviruses exhibit multi-layered control of autophagy to facilitate cytoplasmic replication.