Establishment of an induced pluripotent stem cell (iPSC) line (INNDSUi004-A) from a patient with Congenital Nemaline Myopathy.

We used a non-integrated reprogramming approach to establish a human induced pluripotent stem cell (hiPSC) line (INNDSUi004-A) from the skin fibroblasts of a 13-year-old female individual with Congenital Nemaline Myopath. The cells obtained have typical characteristics of embryonic stem cells, show expression of specific pluripotency markers, and can differentiate into three germ layers in vitro. This iPSC cell line has the genetic information of the patient and is a good model for studying disease mechanisms and developing novel therapies.

Developing nucleoside tailoring strategies against SARS-CoV-2 via ribonuclease targeting chimera.

In response to the urgent need for potent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) therapeutics, this study introduces an innovative nucleoside tailoring strategy leveraging ribonuclease targeting chimeras. By seamlessly integrating ribonuclease L recruiters into nucleosides, we address RNA recognition challenges and effectively inhibit severe acute respiratory syndrome coronavirus 2 replication in human cells. Notably, nucleosides tailored at the ribose 2'-position outperform those modified at the nucleobase. Our in vivo validation using hamster models further bolsters the promise of this nucleoside tailoring approach, positioning it as a valuable asset in the development of innovative antiviral drugs.

Establishment of an induced pluripotent stem cell (iPSC) line (INNDSUi005-A) from a healthy female Chinese Han.

We obtained skin fibroblasts from a 34-year-old healthy woman and established a human induced pluripotent stem cell (hiPSC) line (INDSUi005-A) using a non-integrated reprogramming approach. The obtained cells have typical characteristics of embryonic stem cells, can express specific pluripotency markers and have the ability to differentiate into three germ layers in vitro. This iPSC cell line can be used as an in vitro model for studying disease mechanisms and developing novel therapies.

Virus-host coevolutionary analyses of an Alphabaculovirus with a wide host range.

Baculoviruses are highly host specific, and their host range is usually restricted to a single or a few closely related insect species, except for few virus species, e.g. Alphabaculovirus aucalifonicae and Alphabaculovirus mabrassicae. In this study, two new alphabaculovirus isolates were isolated from the larvae of Mamestra brassicae and Mythimna separata, which were named as Mamestra brassicae multiple nucleopolyhedrovirus isolate QD (MbMNPV-QD) and Mythimna separata multiple nucleopolyhedrovirus isolate Hb (MyseMNPV-Hb), respectively. The Kimura two-parameter values based on the concatenated 38 core genes of baculovirus revealed that MbMNPV (isolates QD/CHb1/K1/CTa), MyseMNPV-Hb, Helicoverpa armigera multiple nucleopolyhedrovirus (HearMNPV) and Mamestra configurata nucleopolyhedrovirus B (MacoNPV-B) were different isolates of a same virus species. A phylogenetic tree of baculoviruses and nudiviruses constructed from their 20 homologous gene sequences, and that of their isolated hosts constructed from 13 protein-coding genes of the insect mitochondrial genomes, were used to analyse the coevolution of baculoviruses with their isolated hosts. The results showed that M. brassicae was the most likely ancestral host of these virus isolates, included MbMNPV isolates, MyseMNPV-Hb, HearMNPV, and MacoNPV-B. Therefore, we concluded that these virus isolates belong to the existing virus species - Alphabaculovirus mabrassicae with M. brassicae as their ancestral host.

A New Cellular Interactome of SARS-CoV-2 Nucleocapsid Protein and Its Biological Implications.

There is still much to uncover regarding the molecular details of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. As the most abundant protein, coronavirus nucleocapsid (N) protein encapsidates viral RNAs, serving as the structural component of ribonucleoprotein and virion, and participates in transcription, replication, and host regulations. Virus-host interaction might give clues to better understand how the virus affects or is affected by its host during infection and identify promising therapeutic candidates. Considering the critical roles of N, we here established a new cellular interactome of SARS-CoV-2 N by using a high-specific affinity purification (S-pulldown) assay coupled with quantitative mass spectrometry and immunoblotting validations, uncovering many N-interacting host proteins unreported previously. Bioinformatics analysis revealed that these host factors are mainly involved in translation regulations, viral transcription, RNA processes, stress responses, protein folding and modification, and inflammatory/immune signaling pathways, in line with the supposed actions of N in viral infection. Existing pharmacological cellular targets and the directing drugs were then mined, generating a drug-host protein network. Accordingly, we experimentally identified several small-molecule compounds as novel inhibitors against SARS-CoV-2 replication. Furthermore, a newly identified host factor, DDX1, was verified to interact and colocalize with N mainly by binding to the N-terminal domain of the viral protein. Importantly, loss/gain/reconstitution-of-function experiments showed that DDX1 acts as a potent anti-SARS-CoV-2 host factor, inhibiting the viral replication and protein expression. The N-targeting and anti-SARS-CoV-2 abilities of DDX1 are consistently independent of its ATPase/helicase activity. Further mechanism studies revealed that DDX1 impedes multiple activities of N, including the N-N interaction, N oligomerization, and N-viral RNA binding, thus likely inhibiting viral propagation. These data provide new clues to better depiction of the N-cell interactions and SARS-CoV-2 infection and may help inform the development of new therapeutic candidates.

Targeting upregulated RNA binding protein RCAN1.1: a promising strategy for neuroprotection in acute ischemic stroke.

AIMS: To explore the expression changes and roles of the RNA-binding protein RCAN1.1 in acute ischemic stroke (AIS), and to preliminarily confirm the medicinal value of the RNA aptamer R1SR13 in AIS by targeting RCAN1.1. METHODS: Two mouse AIS models of middle cerebral artery occlusion (MCAO) and right common carotid artery ligation (R-CCAL) and oxygen glucose deprivation (OGD) model of AIS in primary neurons and SH-SY5Y were performed. The expression pattern of RCAN1.1 was assessed using real-time quantitative PCR (RT-qPCR) and western blotting (WB) in vivo and in vitro. The underlying mechanism for the elevation of RCAN1.1 in the upstream was investigated. Lentiviruses were administrated and the effect of RCAN1.1 in AIS was assessed by ATP level, caspase 3/7 assay, TUNEL and WB. The protective function of R1SR13 in AIS was evaluated both in vivo and in vitro. RESULTS: In two mouse models of AIS, RCAN1.1 mRNA and RCAN1.1 L protein were significantly upregulated in the ischemic brain tissue. The same results were detected in the OGD model of primary neurons and SH-SY5Y. The mechanistic analysis proved that hypoxia-inducible factor-1α (HIF1α) could specifically activate the RCAN1.1 gene promoter through combining with the functional hypoxia-responsive element (HRE) site (-325 to -322 bp). The increased expression of RCAN1.1 L markedly depleted ATP production and aggravated neuronal apoptosis under OGD condition. R1SR13, an antagonizing RNA aptamer of RCAN1.1, was demonstrated to reduce neuronal apoptosis caused by the elevated RCAN1.1 L in the cellular and animal models of AIS. CONCLUSION: RCAN1.1 is a novel target gene of HIF1α and the functional HRE in the RCAN1.1 promoter region is -325 to -322 bp. The marked upregulation of RCAN1.1 in AIS promoted neuronal apoptosis, an effect that could be reversed by its RNA aptamer R1SR13 in vivo and in vitro. Thus, R1SR13 represents a promising strategy for neuroprotection in AIS and our study lays a theoretical foundation for it to become a clinically targeted drug.

Upregulation of RCAN1.4 by HIF1α alleviates OGD-induced inflammatory response in astrocytes.

OBJECTIVE: Ischemic stroke is a leading cause of human mortality and long-term disability worldwide. As one of the main forms of regulator of calcineurin 1 (RCAN1), the contribution of RCAN1.4 in diverse biological and pathological conditions has been implicated. But the role of RCAN1.4 in ischemic stroke progression remains elusive. This study is to explore the expression changes and roles of RCAN1.4 in ischemic stroke as well as the underlying mechanisms for these changes and effects of RCAN1.4 in ischemic stroke. METHODS: Middle cerebral artery occlusion model in C57BL/6J mice and oxygen-glucose deprivation (OGD) model in primary astrocytes were performed to induce the cerebral ischemic stroke. The expression pattern of RCAN1.4 was assessed using real-time quantitative PCR and western blotting in vivo and in vitro. Mechanistically, the underlying mechanism for the elevation of RCAN1.4 in the upstream was investigated. Lentiviruses were administrated, and the effect of RCAN1.4 in postischemic inflammation was clearly clarified. RESULTS: Here we uncovered that RCAN1.4 was dramatically increased in mouse ischemic brains and OGD-induced primary astrocytes. HIF1α, activated upon OGD, significantly upregulated RCAN1.4 gene expression through specifically binding to the RCAN1.4 promoter region and activating its promoter activity. The functional hypoxia-responsive element (HRE) was located between -254 and -245 bp in the RCAN1.4 promoter region. Moreover, elevated RCAN1.4 alleviated the release of pro-inflammatory cytokines TNFα, IL1ß, IL6 and reduced expression of iNOS, COX2 in primary astrocytes upon OGD, whereas RCAN1.4 silencing has the opposite effect. Of note, RCAN1.4 overexpression inhibited OGD-induced NF-κB activation in primary astrocytes, leading to decreased degradation of IκBα and reduced nuclear translocation of NF-κB/p65. INTERPRETATION: Our results reveal a novel mechanism underscoring the upregulation of RCAN1.4 by HIF1α and the protective effect of RCAN1.4 against postischemic inflammation, suggesting its significance as a promising therapeutic target for ischemic stroke treatment.

Comprehensive Characterization of a Novel E3-Related Gene Signature With Implications in Prognosis and Immunotherapy of Low-Grade Gliomas.

Gliomas, a type of primary brain tumor, have emerged as a threat to global mortality due to their high heterogeneity and mortality. A low-grade glioma (LGG), although less aggressive compared with glioblastoma, still exhibits high recurrence and malignant progression. Ubiquitination is one of the most important posttranslational modifications that contribute to carcinogenesis and cancer recurrence. E3-related genes (E3RGs) play essential roles in the process of ubiquitination. Yet, the biological function and clinical significance of E3RGs in LGGs need further exploration. In this study, differentially expressed genes (DEGs) were screened by three differential expression analyses of LGG samples from The Cancer Genome Atlas (TCGA) database. DEGs with prognostic significance were selected by the univariate Cox regression analysis and log-rank statistical test. The LASSO-COX method was performed to identify an E3-related prognostic signature consisting of seven genes AURKA, PCGF2, MAP3K1, TRIM34, PRKN, TLE3, and TRIM17. The Chinese Glioma Genome Atlas (CGGA) dataset was used as the validation cohort. Kaplan-Meier survival analysis showed that LGG patients in the low-risk group had significantly higher overall survival time than those in the high-risk group in both TCGA and CGGA cohorts. Furthermore, multivariate Cox regression analysis revealed that the E3RG signature could be used as an independent prognostic factor. A nomogram based on the E3RG signature was then established and provided the prediction of the 1-, 3-, and 5-year survival probability of patients with LGGs. Moreover, DEGs were analyzed based on the risk signature, on which function analyses were performed. GO and KEGG analyses uncovered gene enrichment in extracellular matrix-related functions and immune-related biological processes in the high-risk group. GSEA revealed high enrichment in pathways that promote tumorigenesis and progression in the high-risk group. Furthermore, ESTIMATE algorithm analysis showed a significant difference in immune and stroma activity between high- and low-risk groups. Positive correlations between the risk signature and the tumor microenvironment immune cell infiltration and immune checkpoint molecules were also observed, implying that patients with the high-risk score may have better responses to immunotherapy. Overall, our findings might provide potential diagnostic and prognostic markers for LGG patients and offer meaningful insight for individualized treatment.

Vav2 is a novel APP-interacting protein that regulates APP protein level.

Amyloid precursor protein (APP) is a transmembrane protein that plays critical role in the pathogenesis of Alzheimer's disease (AD). It is also involved in many types of cancers. Increasing evidence has shown that the tyrosine phosphorylation site Y682 in the intracellular tail of APP is crucial for APP function. Here, we report that Vav2, a guanine nucleotide exchange factor (GEF) for Rho family GTPase, is a novel interaction partner of APP. We found that Vav2-SH2 domain was able to bind directly to the Y682-phosphorylated intracellular tail of APP through isothermal titration calorimetry and NMR titrating experiments. The crystal structure of Vav2-SH2 in complex with an APP-derived phosphopeptide was determined to understand the structural basis of this recognition specificity. The interaction of APP and Vav2 in a full-length manner was further confirmed in cells by GST pull-down, co-immunoprecipitation and immunofluorescence staining experiments. In addition, we found overexpression of Vav2 could inhibit APP degradation and markedly increase the protein levels of APP and its cleavage productions in 20E2 cells, and this function of Vav2 required a functional SH2 domain.

Phosphorylation of the Autographa californica multiple nucleopolyhedrovirus polyhedron envelope protein plays an important role in the formation of the occlusion body envelope.

During the life cycle of a baculovirus, a crystallized protein matrix, formed by polyhedrin (POLH), is produced. The protein matrix is surrounded by a multilayered protein/carbohydrate envelope, and matrix and envelope together form a mature occlusion body (OB). The polyhedron envelope plays an important role in resistance against adverse external environments. The polyhedron envelope protein (PEP) is the main protein that forms the polyhedron envelope, but the mechanism of formation of the polyhedron envelope is unclear. Here, through immunofluorescence localization observations, we found that PEP interacted with both POLH and P10 during formation of the polyhedron envelope in the late stages of infection, and PEP was also required for P10 incorporation on the surface of OBs. In this process, the phosphorylation of PEP played an important role. PEP was determined to be a phosphorylated protein using the Phos-tag technique, and PK1 was determined to be the phosphokinase of PEP by co-immunoprecipitation and in vitro phosphorylation. Immunofluorescence localization revealed that PEP was continuously phosphorylated by PK1 after PEP entered the nucleus until PEP was correctly packaged on the OB surface. Multi-point mutations of PEP conservative potential phosphorylation sites showed that the simultaneous mutation of S85, T86 and Y92 caused changes in the location of PEP and P10 in the late stages of infection, and resulted in an OB surface that lacked the polyhedron envelope. These data suggested that the phosphorylation of PEP at particular sites, i.e. S85, T86 and Y92, plays an important role in the formation of the polyhedron envelope.

Genomic analysis of two Chinese isolates of hyphantria cunea nucleopolyhedrovirus reveals a novel species of alphabaculovirus that infects hyphantria cunea drury (lepidoptera: arctiidae).

BACKGROUND: Baculoviruses act as effective biological control agents against the invasive pest Hyphantria cunea Drury. In this study, two Chinese Hyphantria cunea nucleopolyhedrovirus (HycuNPV) isolates, HycuNPV-BJ and HycuNPV-HB, were deep sequenced and compared with the Japanese isolate, HycuNPV-N9, to determine whole-genome level diversity and evolutionary history. RESULTS: The divergence of the phylogenetic tree and the K2P distances based on 38 core-gene concatenated alignment revealed that two Chinese HycuNPV isolates were a novel species of Alphabaculovirus that infected Hyphantria cunea in China. The gene contents indicated significant differences in the HycuNPV genomes between the Chinese and Japanese isolates. The differences included gene deletions, acquisitions and structural transversions, but the main difference was the high number of single nucleotide polymorphisms (SNPs). In total, 10,393 SNPs, corresponding to approximately 8% of the entire HycuNPV-N9 genome sequence, were detected in the aligned reads. By analyzing non-synonymous variants, we found that hotspot mutation-containing genes had mainly unknown functions and most were early expressing genes. We found that the hycu78 gene which had early and late promoter was under positive selection. Biological activity assays revealed that the infectivity of HycuNPV-HB was greater than that of HycuNPV-BJ, and the killing speed of HycuNPV-HB was faster than that of HycuNPV-BJ. A comparison of molecular genetic characteristics indicated that the virulence differences between the two isolates were affected by SNP and structural variants, especially the homologous repeat regions. CONCLUSIONS: The genomes of the two Chinese HycuNPV isolates were characterized, they belonged to a novel species of Alphabaculovirus that infected Hyphantria cunea in China. We inferred that the loss or gain of genetic material in the HycuNPV-HB and HycuNPV-BJ genomes resulted in new important adaptive capabilities to the H. cunea host. These results extend the current understanding of the genetic diversity of HycuNPV and will be useful for improving the applicability of this virus as a biological control agent.

Let-7c increases BACE2 expression by RNAa and decreases Aß production.

MicroRNAs (miRNAs) are highly conserved, non-coding transcripts that regulate gene expression in various ways. Evidence suggests that miRNAs may be a contributory factor in neurodegeneration, including Alzheimer's disease (AD), Parkinson's disease (PD), and triplet repeat disorders. In order to further understand the potential roles of miRNAs in the pathogenesis of AD, we analyzed Down syndrome (DS), a special model of AD, by using a TaqMan microRNA array and found that miRNA let-7c was up-regulated in both DS and AD. ELISA assay showed that let-7c reduced the expression level of Aß significantly. Real-time quantitative-polymerase chain reaction (RT-qPCR) was conducted to reveal that the expression level of let-7c increased dramatically in DS cells, patients with DS and mice with AD compared with normal ones respectively. Additionally, western blotting illustrated that let-7c suppressed the expression of Aß by inducing BACE2 to cut C99 and increase the content of C83/80. BACE2 expression was inhibited by let-7c and luciferase reporter gene assay revealed that let-7c increased the activity of wild-type BACE2 promoter but not 3'UTR. Furthermore, promoter analysis of BACE2 confirmed that let-7c could bind to BACE2 in the sequence between -1368 and -1347. In addition, immunoblotting assay demonstrated that let-7c induced BACE2 expression by RNAa. To the best of our knowledge, our study revealed for the first time that let-7c up-regulated BACE2 expression and decreased Aß production.

Cleavage of Kv2.1 by BACE1 decreases potassium current and reduces neuronal apoptosis.

As an aspartic protease, ß-site APP cleaving enzyme 1 (BACE1) can efficiently cleave amyloid precursor protein (APP) to produce amyloid beta (Aß), a chief constituent of senile plaques in Alzheimer's disease. Thus, BACE1 inhibitor is identified as a therapeutic candidate for AD. However, recent failures of clinical trials using BACE1 inhibitors emphasized that comprehensively understanding of BACE1 function is particularly important. Kv2.1, a potassium channel, modulates potassium current in cortical neurons and potassium efflux is a requisite event in the process of cell apoptosis. Previously we showed that BACE2 cleaves Kv2.1 and reduces neuronal apoptosis. Our study here showed that BACE1 cleaves Kv2.1, and results in decreased Ik of Kv2.1. Furthermore, we demonstrated that the BACE1-cleaved Kv2.1 reduces neuronal apoptosis and BACE1 inhibitor markedly increases neuronal apoptosis. Our work indicates that BACE1 plays a neuroprotective role to reduce potassium efflux by cleavage of Kv2.1, implying inhibition of BACE1 may be neurotoxic.

Heat shock protein 70, glutamate dehydrogenase, and angiotensin-converting enzyme of Bombyx mori mediate the cell attachment of Cypovirus 1.

Dendrolimus punctatus causes great damage to pine forests worldwide. Dendrolimus punctatus cypovirus 1 (DpCPV-1) is an important pathogen of D. punctatus. However, the mechanism of DpCPV-1 cell entry has not been elucidated. In this study, we revealed that both GTase and MTase domains of VP3 (B-spike) and VP4 (A-spike) of DpCPV-1 interacted with the midgut proteins of Bombyx mori. Binding and competition assays revealed that GTase, MTase and VP4 played roles as viral attachment proteins. Far-Western blotting and LC-MS/MS analyses identified that heat shock protein 70 (BmHSP70), glutamate dehydrogenase (BmGDH), and angiotensin-converting enzyme (BmACE) in the midgut proteins as ligand candidates of the viral attachment proteins, and this was further verified by co-immunoprecipitation and fluorescence co-localization assays. Viral binding to the host midgut in vitro was inhibited by pre-treating B. mori midgut proteins with anti-BmHSP70, anti-BmGDH, anti-BmACE antibodies singly and in combination. Incubating DpCPV-1 virions with prokaryotically expressed BmHSP70, BmGDH, and BmACE also decreased viral attachment to the host midgut. In vivo bioassays revealed that viral infection in Helicoverpa armigera was partially neutralized by BmHSP70, BmGDH, and BmACE. Taking together, we concluded that HSP70, GDH, and ACE mediate DpCPV attachment and entry via binding to the viral attachment proteins, VP3 and VP4. The findings provide foundation for further understanding the entry mechanisms of cypoviruses.

Immune evasion of SARS-CoV-2 from interferon antiviral system.

The on-going pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to unprecedented medical and socioeconomic crises. Although the viral pathogenesis remains elusive, deficiency of effective antiviral interferon (IFN) responses upon SARS-CoV-2 infection has been recognized as a hallmark of COVID-19 contributing to the disease pathology and progress. Recently, multiple proteins encoded by SARS-CoV-2 have been shown to act as potential IFN antagonists with diverse possible mechanisms. Here, we summarize and discuss the strategies of SARS-CoV-2 for evasion of innate immunity (particularly the antiviral IFN responses), understanding of which will facilitate not only the elucidation of SARS-CoV-2 infection and pathogenesis but also the development of antiviral intervention therapies.

DYRK1A activates NFATC1 to increase glioblastoma migration.

Glioblastoma (GBM) is the most aggressive glioma, and is prone to develop resistance to chemotherapy and radiotherapy; hence, patients with glioblastoma have a high recurrence rate and a low 1-year survival rate. In addition, the pathogenesis of glioblastoma is complex and largely unknown, and the available treatments are limited. Here, we uncovered a fundamental role of DYRK1A in regulating NFATC1 in GBMs. We found that DYRK1A was highly expressed in glioma and glioblastoma cells, and its expression was positively correlated with that of NFATC1. Moreover, inhibition of DYRK1A promoted NFATC1 degradation in GBM cells and sharply reduced the transactivation of NFATC1, not only by decreasing the expression of NFATC1-targeted genes, but also by reducing the luciferase activity, and vice versa. However, DYRK1A had the opposite effect on NFATC2. Most importantly, our data suggest that DYRK1A inhibition reduces glioblastoma migration. Polypeptides derived from the DYRK1A-targeted motif of NFATC1, by competitively blocking DYRK1A kinase activity on NFATC1, clearly destabilized NFATC1 protein and impaired glioblastoma migration. We propose that the recovery of NFATC1 stability is a key oncogenic event in a large proportion of gliomas, and pharmacological inhibition of DYRK1A by polypeptides could represent a promising therapeutic intervention for GBM.

DYRK1A phosphorylates MEF2D and decreases its transcriptional activity.

Myocyte enhancer factor 2D (MEF2D) is predominantly expressed in the nucleus and associated with cell growth, differentiation, survival and apoptosis. Previous studies verified that phosphorylation at different amino acids determined MEF2's transcriptional activity which was essential in regulating downstream target genes expression. What regulates phosphorylation of MEF2D and affects its function has not been fully elucidated. Here, we uncovered that dual-specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A), a kinase critical in Down's syndrome pathogenesis, directly bound to and phosphorylated MEF2D at Ser251 in vitro. Phosphorylation of MEF2D by DYRK1A significantly increased MEF2D protein level but attenuated its transcriptional activity, which resulted in decreased transcriptions of MEF2D target genes. Phosphorylation mutated Ser251A MEF2D exhibited enhanced transcriptional activity compared with wild type MEF2D. MEF2D and DYRK1A were observed co-localized in HEK293 and U87MG cells. Moreover, DYRK1A-mediated MEF2D phosphorylation in vitro might influence its nuclear export upon subcellular fractionation, which partially explained the reduction of MEF2D transcriptional activity by DYRK1A. Our results indicated that DYRK1A might be a regulator of MEF2D transcriptional activity and indirectly get involved in regulation of MEF2D target genes.

A Conserved Phenylalanine Residue of Autographa Californica Multiple Nucleopolyhedrovirus AC75 Protein Is Required for Occlusion Body Formation.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf75 (ac75) is a highly conserved gene that is essential for AcMNPV propagation. However, the key domains or residues of the AC75 protein that play a role in viral propagation have not been identified. In this study, sequence alignment revealed that residues Phe-54 and Gln-81 of AC75 were highly conserved among alphabaculoviruses and betabaculoviurses. Thus, Phe-54 and Gln-81 AC75 mutation bacmids were constructed. We found that Gln-81 was not required for viral propagation, whereas mutating Phe-54 reduced budded virus production by 10-fold and impaired occlusion body formation when compared with that of the wild-type AcMNPV. Electron microscopy observations showed that the Phe-54 mutation affected polyhedrin assembly and also occlusion-derived virus embedding, whereas western blot analysis revealed that mutating Phe-54 reduced the amount of AC75 but did not affect the localization of AC75 in infected cells. A protein stability assay showed that the Phe-54 mutation affected AC75 stability. Taken together, Phe-54 was identified as an important residue of AC75, and ac75 is a pivotal gene in budding virus production and occlusion body formation.