Hexestrol, an estrogen receptor agonist, inhibits Lassa virus entry.

Lassa virus (LASV) is the causative agent of human Lassa fever which in severe cases manifests as hemorrhagic fever leading to thousands of deaths annually. However, no approved vaccines or antiviral drugs are currently available. Recently, we screened approximately 2,500 compounds using a recombinant vesicular stomatitis virus (VSV) expressing LASV glycoprotein GP (VSV-LASVGP) and identified a P-glycoprotein inhibitor as a potential LASV entry inhibitor. Here, we show that another identified candidate, hexestrol (HES), an estrogen receptor agonist, is also a LASV entry inhibitor. HES inhibited VSV-LASVGP replication with a 50% inhibitory concentration (IC50) of 0.63 µM. Importantly, HES also inhibited authentic LASV replication with IC50 values of 0.31 µM-0.61 µM. Time-of-addition and cell-based membrane fusion assays suggested that HES inhibits the membrane fusion step during virus entry. Alternative estrogen receptor agonists did not inhibit VSV-LASVGP replication, suggesting that the estrogen receptor itself is unlikely to be involved in the antiviral activity of HES. Generation of a HES-resistant mutant revealed that the phenylalanine at amino acid position 446 (F446) of LASVGP, which is located in the transmembrane region, conferred resistance to HES. Although mutation of F446 enhanced the membrane fusion activity of LASVGP, it exhibited reduced VSV-LASVGP replication, most likely due to the instability of the pre-fusion state of LASVGP. Collectively, our results demonstrated that HES is a promising anti-LASV drug that acts by inhibiting the membrane fusion step of LASV entry. This study also highlights the importance of the LASVGP transmembrane region as a target for anti-LASV drugs.IMPORTANCELassa virus (LASV), the causative agent of Lassa fever, is the most devastating mammarenavirus with respect to its impact on public health in West Africa. However, no approved antiviral drugs or vaccines are currently available. Here, we identified hexestrol (HES), an estrogen receptor agonist, as the potential antiviral candidate drug. We showed that the estrogen receptor itself is not involved in the antiviral activity. HES directly bound to LASVGP and blocked membrane fusion, thereby inhibiting LASV infection. Through the generation of a HES-resistant virus, we found that phenylalanine at position 446 (F446) within the LASVGP transmembrane region plays a crucial role in the antiviral activity of HES. The mutation at F446 caused reduced virus replication, likely due to the instability of the pre-fusion state of LASVGP. These findings highlight the potential of HES as a promising candidate for the development of antiviral compounds targeting LASV.

Capsule-deficient group A Streptococcus evades autophagy-mediated killing in macrophages.

The hyaluronic acid capsule is crucial in protecting group A Streptococcus (GAS) against phagocytic killing. However, there have been reported outbreaks caused by capsule-deficient GAS strains, and the mechanisms underlying their evasion of immune clearance remain unclear. This study demonstrated that the capsule-deficient mutant [Cap(-)] of the emm1 strain increased survival within phagocytic cells compared to the wild-type strain [Cap(+)]. Although both Cap(+) and Cap(-) strains exhibited similar abilities to disrupt the phagosome, only the Cap(+) strain was colocalized with lysosomes and acidified compartments in phagocytic cells, indicating its susceptibility to autophagosome elimination. In contrast, the Cap(-) mutant evaded the recognition of galectin-8 and ubiquitin, impairing selective autophagy-mediated elimination. These findings suggest that a deficiency in the capsule could impair the intracellular elimination of GAS in macrophages, revealing previously unknown aspects of the host's recognition of the GAS capsule in macrophages. IMPORTANCE: Group A Streptococcus (GAS) is a Gram-positive bacterium that causes diseases ranging from mild pharyngitis to severe necrotizing fasciitis. Phagocytic cells serve as the primary defense against bacterial infections, exhibiting remarkable efficiency in eliminating intracellular pathogens. The hyaluronic acid capsule is a critical virulence factor that contributes to the resistance of phagocytosis in GAS. Nevertheless, the outbreaks caused by GAS strains that lack the hyaluronic acid capsule have been reported, and the selective advantage of capsule-deficient strains during infection is not fully understood. This study showed that the autophagic adaptor proteins recognize the capsulated GAS strain but not the capsule-deficient mutant, indicating that the hyaluronic acid capsule could be the autophagic target in macrophages. These findings imply that the hyaluronic acid capsule of GAS actually enhances its elimination within phagocytic cells, subverting the understanding of the capsule in GAS pathogenesis.

Micro-patterned culture of iPSC-derived alveolar and airway cells distinguishes SARS-CoV-2 variants.

The emergence of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) variants necessitated a rapid evaluation system for their pathogenesis. Lung epithelial cells are their entry points; however, in addition to their limited source, the culture of human alveolar epithelial cells is especially complicated. Induced pluripotent stem cells (iPSCs) are an alternative source of human primary stem cells. Here, we report a model for distinguishing SARS-CoV-2 variants at high resolution, using separately induced iPSC-derived alveolar and airway cells in micro-patterned culture plates. The position-specific signals induced the apical-out alveolar type 2 and multiciliated airway cells at the periphery and center of the colonies, respectively. The infection studies in each lineage enabled profiling of the pathogenesis of SARS-CoV-2 variants: infection efficiency, tropism to alveolar and airway lineages, and their responses. These results indicate that this culture system is suitable for predicting the pathogenesis of emergent SARS-CoV-2 variants.

The RNA-binding protein Msi2 regulates autophagy during myogenic differentiation.

Skeletal muscle development is a highly ordered process orchestrated transcriptionally by the myogenic regulatory factors. However, the downstream molecular mechanisms of myogenic regulatory factor functions in myogenesis are not fully understood. Here, we identified the RNA-binding protein Musashi2 (Msi2) as a myogenin target gene and a post-transcriptional regulator of myoblast differentiation. Msi2 knockdown in murine myoblasts blocked differentiation without affecting the expression of MyoD or myogenin. Msi2 overexpression was also sufficient to promote myoblast differentiation and myocyte fusion. Msi2 loss attenuated autophagosome formation via down-regulation of the autophagic protein MAPL1LC3/ATG8 (LC3) at the early phase of myoblast differentiation. Moreover, forced activation of autophagy effectively suppressed the differentiation defects incurred by Msi2 loss. Consistent with its functions in myoblasts in vitro, mice deficient for Msi2 exhibited smaller limb skeletal muscles, poorer exercise performance, and muscle fiber-type switching in vivo. Collectively, our study demonstrates that Msi2 is a novel regulator of mammalian myogenesis and establishes a new functional link between muscular development and autophagy regulation.

Pib2 is a cysteine sensor involved in TORC1 activation in Saccharomyces cerevisiae.

Target of rapamycin complex 1 (TORC1) is a master regulator that monitors the availability of various amino acids to promote cell growth in Saccharomyces cerevisiae. It is activated via two distinct upstream pathways: the Gtr pathway, which corresponds to mammalian Rag, and the Pib2 pathway. This study shows that Ser3 was phosphorylated exclusively in a Pib2-dependent manner. Using Ser3 as an indicator of TORC1 activity, together with the established TORC1 substrate Sch9, we investigated which pathways were employed by individual amino acids. Different amino acids exhibited different dependencies on the Gtr and Pib2 pathways. Cysteine was most dependent on the Pib2 pathway and increased the interaction between TORC1 and Pib2 in vivo and in vitro. Moreover, cysteine directly bound to Pib2 via W632 and F635, two critical residues in the T(ail) motif that are necessary to activate TORC1. These results indicate that Pib2 functions as a sensor for cysteine in TORC1 regulation.

ACE2 knockout hinders SARS-CoV-2 propagation in iPS cell-derived airway and alveolar epithelial cells.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, continues to spread around the world with serious cases and deaths. It has also been suggested that different genetic variants in the human genome affect both the susceptibility to infection and severity of disease in COVID-19 patients. Angiotensin-converting enzyme 2 (ACE2) has been identified as a cell surface receptor for SARS-CoV and SARS-CoV-2 entry into cells. The construction of an experimental model system using human iPS cells would enable further studies of the association between viral characteristics and genetic variants. Airway and alveolar epithelial cells are cell types of the lung that express high levels of ACE2 and are suitable for in vitro infection experiments. Here, we show that human iPS cell-derived airway and alveolar epithelial cells are highly susceptible to viral infection of SARS-CoV-2. Using gene knockout with CRISPR-Cas9 in human iPS cells we demonstrate that ACE2 plays an essential role in the airway and alveolar epithelial cell entry of SARS-CoV-2 in vitro. Replication of SARS-CoV-2 was strongly suppressed in ACE2 knockout (KO) lung cells. Our model system based on human iPS cell-derived lung cells may be applied to understand the molecular biology regulating viral respiratory infection leading to potential therapeutic developments for COVID-19 and the prevention of future pandemics.

Structural insights into the agonists binding and receptor selectivity of human histamine H4 receptor.

Histamine is a biogenic amine that participates in allergic and inflammatory processes by stimulating histamine receptors. The histamine H4 receptor (H4R) is a potential therapeutic target for chronic inflammatory diseases such as asthma and atopic dermatitis. Here, we show the cryo-electron microscopy structures of the H4R-Gq complex bound with an endogenous agonist histamine or the selective agonist imetit bound in the orthosteric binding pocket. The structures demonstrate binding mode of histamine agonists and that the subtype-selective agonist binding causes conformational changes in Phe3447.39, which, in turn, form the "aromatic slot". The results provide insights into the molecular underpinnings of the agonism of H4R and subtype selectivity of histamine receptors, and show that the H4R structures may be valuable in rational drug design of drugs targeting the H4R.

Rab32 and Rab38 maintain bone homeostasis by regulating intracellular traffic in osteoclasts.

Osteoclasts play a crucial role in bone homeostasis by forming resorption pits on bone surfaces, resulting in bone resorption. The osteoclast expression of Rab38 protein is highly induced during differentiation from macrophages. Here we generated mice with double knockout (DKO) of Rab38 and its paralogue, Rab32, to investigate the roles of these proteins in osteoclasts. Bone marrow-derived macrophages from Rab32/38 DKO mice differentiated normally into osteoclasts in vitro. However, DKO osteoclasts showed reduced bone resorption activity. These osteoclasts also demonstrated defective secretion of tartrate-resistant acid phosphatase and cathepsin K into culture medium. Furthermore, the plasma membrane localization of a3, an osteoclast-specific a subunit of V-ATPase, was abrogated in DKO mice, substantiating the reduced resorption activity. In vivo, Rab32- and Rab38-positive cells were attached to the bone surface. Eight-week-old DKO mice showed significantly thickened trabecular bones in micro-CT and histomorphometry analysis, as well as reduced serum levels of cross-linked C-telopeptide of type I collagen, indicating diminished bone resorption in vivo. In DKO male mice over 10 weeks of age, hyperostosis appeared at the talofibular syndesmosis, the distal junction of the tibia and fibula. Furthermore, middle-aged mice (10 to 12 months of age) exhibited kyphosis, which is not usually observed in wild-type male mice until around 24 months of age. These results indicate that Rab32 and Rab38 contribute to osteoclast function by supporting intracellular traffic, thereby maintaining normal bone homeostasis.Key words: Rab32, Rab38, osteoclast, lysosome-related organelle, secretory lysosome.

Left-Right Reversal Recurrently Evolved Regardless of Diaphanous-Related Formin Gene Duplication or Loss in Snails.

Bilateria exhibit whole-body handedness in internal structure. This left-right polarity is evolutionarily conserved with virtually no reversed extant lineage, except in molluscan Gastropoda. Phylogenetically independent snail groups contain both clockwise-coiled (dextral) and counterclockwise-coiled (sinistral) taxa that are reversed from each other in bilateral handedness as well as in coiling direction. Within freshwater Hygrophila, Lymnaea with derived dextrality have diaphanous related formin (diaph) gene duplicates, while basal sinistral groups possess one diaph gene. In terrestrial Stylommatophora, dextral Bradybaena also have diaph duplicates. Defective maternal expression of one of those duplicates gives rise to sinistral hatchlings in Lymnaea and handedness-mixed broods in Bradybaena, through polarity change in spiral cleavage of embryos. These findings led to the hypothesis that diaph duplication was crucial for the evolution of dextrality by reversal. The present study discovered that diaph duplication independently occurred four times and its duplicate became lost twice in gastropods. The dextrality of Bradybaena represents the ancestral handedness conserved across gastropods, unlike the derived dextrality of Lymnaea. Sinistral lineages recurrently evolved by reversal regardless of whether diaph had been duplicated. Amongst the seven formin gene subfamilies, diaph has most thoroughly been conserved across eukaryotes of the 14 metazoan phyla and choanoflagellate. Severe embryonic mortalities resulting from insufficient expression of the duplicate in both of Bradybaena and Lymnaea also support that diaph duplicates bare general roles for cytoskeletal dynamics other than controlling spiralian handedness. Our study rules out the possibility that diaph duplication or loss played a primary role for reversal evolution.

Opposing roles of RUBCN isoforms in autophagy and memory B cell generation.

RUBCN (also known as Rubicon) was originally identified as a negative regulator of autophagy, a process by which cells degrade and recycle damaged components or organelles and that requires the activity of the class III PI3K VPS34 and the mTORC1 protein complex. Here, we characterized the role of a shorter isoform, RUBCN100, as an autophagy-promoting factor in B cells. RUBCN100 was translated from alternative translation initiation sites and lacked the RUN domain of the longer, previously characterized RUBCN130 isoform. Specific deficiency of RUBCN130 in B cells enhanced autophagy, which promoted memory B cell generation. In contrast to RUBCN130, which is localized in late endosomes and lysosomes and suppresses the enzymatic activity of VPS34, an effect thought to mediated by its RUN domain, RUBCN100 was preferentially located in early endosomes and enhanced VPS34 activity, presumably because of the absence of the RUN domain. Furthermore, RUBCN100, but not RUBCN130, enhanced autophagy and suppressed mTORC1 activation. Our findings reveal that the opposing roles of two RUBCN isoforms are critical for autophagy regulation and memory B cell differentiation.

Characterization of Rab32- and Rab38-positive lysosome-related organelles in osteoclasts and macrophages.

Both the biogenesis and functions of osteoclasts and macrophages involves dynamic membrane traffic. We screened transcript levels for Rab family small GTPases related to osteoclasts and identified Rab38. Rab38 expression is upregulated during osteoclast differentiation and maturation. In osteoclasts, both Rab38 and its paralog, Rab32, colocalize to lysosome-related organelles (LROs). In macrophages, Rab32 is also found in LROs. LROs are part of the endocytic pathway but are distinct from lysosomes. After receptor activator of NF-κB ligand stimulation, LROs contain cathepsin K and tartrate-resistant acid phosphatase inside and help both proteins to accumulate around bone resorption pits. After osteoclast maturation, these enzymes are hardly found within LROs. In macrophages derived from Rab32 and Rab38 double knockout mice, both acidification and V-ATPase a3 localization were severely compromised. Both the double knockout macrophage and bafilomycin-treated wildtype macrophage show an increase in Lamp1-positive organelles, implying that biogenesis of lysosomes and LROs are related. These results indicate that Rab32 and Rab38 both play a crucial role in LRO biogenesis in macrophages and in osteoclasts.

Cryoelectron microscopic structure of the nucleoprotein-RNA complex of the European filovirus, Lloviu virus.

Lloviu virus (LLOV) is a novel filovirus detected in Schreiber's bats in Europe. The isolation of the infectious LLOV from bats has raised public health concerns. However, the virological and molecular characteristics of LLOV remain largely unknown. The nucleoprotein (NP) of LLOV encapsidates the viral genomic RNA to form a helical NP-RNA complex, which acts as a scaffold for nucleocapsid formation and de novo viral RNA synthesis. In this study, using single-particle cryoelectron microscopy, we determined two structures of the LLOV NP-RNA helical complex, comprising a full-length and a C-terminally truncated NP. The two helical structures were identical, demonstrating that the N-terminal region determines the helical arrangement of the NP. The LLOV NP-RNA protomers displayed a structure similar to that in the Ebola and Marburg virus, but the spatial arrangements in the helix differed. Structure-based mutational analysis identified amino acids involved in the helical assembly and viral RNA synthesis. These structures advance our understanding of the filovirus nucleocapsid formation and provide a structural basis for the development of antifiloviral therapeutics.

Elucidation of the liver pathophysiology of COVID-19 patients using liver-on-a-chips.

SARS-CoV-2 induces severe organ damage not only in the lung but also in the liver, heart, kidney, and intestine. It is known that COVID-19 severity correlates with liver dysfunction, but few studies have investigated the liver pathophysiology in COVID-19 patients. Here, we elucidated liver pathophysiology in COVID-19 patients using organs-on-a-chip technology and clinical analyses. First, we developed liver-on-a-chip (LoC) which recapitulating hepatic functions around the intrahepatic bile duct and blood vessel. We found that hepatic dysfunctions, but not hepatobiliary diseases, were strongly induced by SARS-CoV-2 infection. Next, we evaluated the therapeutic effects of COVID-19 drugs to inhibit viral replication and recover hepatic dysfunctions, and found that the combination of anti-viral and immunosuppressive drugs (Remdesivir and Baricitinib) is effective to treat hepatic dysfunctions caused by SARS-CoV-2 infection. Finally, we analyzed the sera obtained from COVID-19 patients, and revealed that COVID-19 patients, who were positive for serum viral RNA, are likely to become severe and develop hepatic dysfunctions, as compared with COVID-19 patients who were negative for serum viral RNA. We succeeded in modeling the liver pathophysiology of COVID-19 patients using LoC technology and clinical samples.

Evaluation of Broad Anti-Coronavirus Activity of Autophagy-Related Compounds Using Human Airway Organoids.

To deal with the broad spectrum of coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that threaten human health, it is essential to not only drugs develop that target viral proteins but also consider drugs that target host proteins/cellular processes to protect them from being hijacked for viral infection and replication. To this end, it has been reported that autophagy is deeply involved in coronavirus infection. In this study, we used airway organoids to screen a chemical library of autophagic modulators to identify compounds that could potentially be used to fight against infections by a broad range of coronaviruses. Among the 80 autophagy-related compounds tested, cycloheximide and thapsigargin reduced SARS-CoV-2 infection efficiency in a dose-dependent manner. Cycloheximide treatment reduced the infection efficiency of not only six SARS-CoV-2 variants but also human coronavirus (HCoV)-229E and HCoV-OC43. Cycloheximide treatment also reversed viral infection-induced innate immune responses. However, even low-dose (1 µM) cycloheximide treatment altered the expression profile of ribosomal RNAs; thus, side effects such as inhibition of protein synthesis in host cells must be considered. These results suggest that cycloheximide has broad-spectrum anti-coronavirus activity in vitro and warrants further investigation.

Filovirus helical nucleocapsid structures.

Filoviruses are filamentous enveloped viruses belonging to the family Filoviridae, in the order Mononegavirales. Some filovirus members, such as Ebola virus and Marburg virus, cause severe hemorrhagic fever in humans and non-human primates. The filovirus ribonucleoprotein complex, called the nucleocapsid, forms a double-layered helical structure in which a non-segmented, single-stranded, negative-sense RNA genome is encapsidated by the nucleoprotein (NP), viral protein 35 (VP35), VP24, VP30 and RNA-dependent RNA polymerase (L). The inner layer consists of the helical NP-RNA complex, acting as a scaffold for the binding of VP35 and VP24 that constitute the outer layer. Recent structural studies using cryo-electron microscopy have advanced our understanding of the molecular mechanism of filovirus nucleocapsid formation. Here, we review the key characteristics of the Ebola virus and Marburg virus nucleocapsid structures, highlighting the similarities and differences between the two viruses. In particular, we focus on the structure of the helical NP-RNA complex, the RNA binding mechanism and the NP-NP interactions in the helix. The structural analyses reveal a possible mechanism of nucleocapsid assembly and provide potential targets for the anti-filovirus drug design.

Compact wide-field femtoliter-chamber imaging system for high-speed and accurate digital bioanalysis.

The femtoliter-chamber array is a bioanalytical platform that enables highly sensitive and quantitative analysis of biological reactions at the single-molecule level. This feature has been considered a key technology for "digital bioanalysis" in the biomedical field; however, its versatility is limited by the need for a large and expensive setup such as a fluorescence microscope, which requires a long time to acquire the entire image of a femtoliter-chamber array. To address these issues, we developed a compact and inexpensive wide-field imaging system (COWFISH) that can acquire fluorescence images with a large field of view (11.8 mm × 7.9 mm) and a high spatial resolution of ∼ 3 µm, enabling high-speed analysis of sub-million femtoliter chambers in 20 s. Using COWFISH, we demonstrated a CRISPR-Cas13a-based digital detection of viral RNA of SARS-CoV-2 with an equivalent detection sensitivity (limit of detection: 480 aM) and a 10-fold reduction in total imaging time, as compared to confocal fluorescence microscopy. In addition, we demonstrated the feasibility of COWFISH to discriminate between SARS-CoV-2-positive and -negative clinical specimens with 95% accuracy, showing its application in COVID-19 diagnosis. Therefore, COWFISH can serve as a compact and inexpensive imaging system for high-speed and accurate digital bioanalysis, paving a way for various biomedical applications, such as diagnosis of viral infections.

A circulating subset of iNKT cells mediates antitumor and antiviral immunity.

Invariant natural killer T (iNKT) cells are a group of innate-like T lymphocytes that recognize lipid antigens. They are supposed to be tissue resident and important for systemic and local immune regulation. To investigate the heterogeneity of iNKT cells, we recharacterized iNKT cells in the thymus and peripheral tissues. iNKT cells in the thymus were divided into three subpopulations by the expression of the natural killer cell receptor CD244 and the chemokine receptor CXCR6 and designated as C0 (CD244-CXCR6-), C1 (CD244-CXCR6+), or C2 (CD244+CXCR6+) iNKT cells. The development and maturation of C2 iNKT cells from C0 iNKT cells strictly depended on IL-15 produced by thymic epithelial cells. C2 iNKT cells expressed high levels of IFN-γ and granzymes and exhibited more NK cell-like features, whereas C1 iNKT cells showed more T cell-like characteristics. C2 iNKT cells were influenced by the microbiome and aging and suppressed the expression of the autoimmune regulator AIRE in the thymus. In peripheral tissues, C2 iNKT cells were circulating that were distinct from conventional tissue-resident C1 iNKT cells. Functionally, C2 iNKT cells protected mice from the tumor metastasis of melanoma cells by enhancing antitumor immunity and promoted antiviral immune responses against influenza virus infection. Furthermore, we identified human CD244+CXCR6+ iNKT cells with high cytotoxic properties as a counterpart of mouse C2 iNKT cells. Thus, this study reveals a circulating subset of iNKT cells with NK cell-like properties distinct from conventional tissue-resident iNKT cells.

SARS-CoV-2 disrupts respiratory vascular barriers by suppressing Claudin-5 expression.

In the initial process of coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects respiratory epithelial cells and then transfers to other organs the blood vessels. It is believed that SARS-CoV-2 can pass the vascular wall by altering the endothelial barrier using an unknown mechanism. In this study, we investigated the effect of SARS-CoV-2 on the endothelial barrier using an airway-on-a-chip that mimics respiratory organs and found that SARS-CoV-2 produced from infected epithelial cells disrupts the barrier by decreasing Claudin-5 (CLDN5), a tight junction protein, and disrupting vascular endothelial cadherin-mediated adherens junctions. Consistently, the gene and protein expression levels of CLDN5 in the lungs of a patient with COVID-19 were decreased. CLDN5 overexpression or Fluvastatin treatment rescued the SARS-CoV-2-induced respiratory endothelial barrier disruption. We concluded that the down-regulation of CLDN5 expression is a pivotal mechanism for SARS-CoV-2-induced endothelial barrier disruption in respiratory organs and that inducing CLDN5 expression is a therapeutic strategy against COVID-19.

Role of VP30 Phosphorylation in Ebola Virus Nucleocapsid Assembly and Transport.

Ebola virus (EBOV) VP30 regulates viral genome transcription and replication by switching its phosphorylation status. However, the importance of VP30 phosphorylation and dephosphorylation in other viral replication processes such as nucleocapsid and virion assembly is unclear. Interestingly, VP30 is predominantly dephosphorylated by cellular phosphatases in viral inclusions, while it is phosphorylated in the released virions. Thus, uncertainties regarding how VP30 phosphorylation in nucleocapsids is achieved and whether VP30 phosphorylation provides any advantages in later steps in viral replication have arisen. In the present study, to characterize the roles of VP30 phosphorylation in nucleocapsid formation, we used electron microscopic analyses and live cell imaging systems. We identified VP30 localized to the surface of protrusions surrounding nucleoprotein (NP)-forming helical structures in the nucleocapsid, suggesting the involvement in assembly and transport of nucleocapsids. Interestingly, VP30 phosphorylation facilitated its association with nucleocapsid-like structures (NCLSs). On the contrary, VP30 phosphorylation does not influence the transport characteristics and NCLS number leaving from and coming back into viral inclusions, indicating that the phosphorylation status of VP30 is not a prerequisite for NCLS departure. Moreover, the phosphorylation status of VP30 did not cause major differences in nucleocapsid transport in authentic EBOV-infected cells. In the following budding step, the association of VP30 and its phosphorylation status did not influence the budding efficiency of virus-like particles. Taken together, it is plausible that EBOV may utilize the phosphorylation of VP30 for its selective association with nucleocapsids, without affecting nucleocapsid transport and virion budding processes. IMPORTANCE Ebola virus (EBOV) causes severe fevers with unusually high case fatality rates. The nucleocapsid provides the template for viral genome transcription and replication. Thus, understanding the regulatory mechanism behind its formation is important for the development of novel therapeutic approaches. Previously, we established a live-cell imaging system based on the ectopic expression of viral fluorescent fusion proteins, allowing the visualization and characterization of intracytoplasmic transport of nucleocapsid-like structures. EBOV VP30 is an essential transcriptional factor for viral genome synthesis, and, although its role in viral genome transcription and replication is well understood, the functional importance of VP30 phosphorylation in assembly of nucleocapsids is still unclear. Our work determines the localization of VP30 at the surface of ruffled nucleocapsids, which differs from the localization of polymerase in EBOV-infected cells. This study sheds light on the novel role of VP30 phosphorylation in nucleocapsid assembly, which is an important prerequisite for virion formation.