Genome-wide identification of sweet potato U-Box E3 ubiquitin ligases and roles of IbPUB52 in negative regulation of drought stress.

The plant U-box (PUB) proteins, a family of ubiquitin ligases (E3) enzymes, are pivotal in orchestrating many biological processes and facilitating plant responses to environmental stressors. Despite their critical roles, exploring the PUB gene family's characteristics and functional diversity in sweet potato (Ipomoea batatas (L.) Lam.) has been notably limited. There were 81 IbPUB genes identified within the sweet potato genome, and they were categorized into eight distinct groups based on domain architecture, revealing a non-uniform distribution across the 15 chromosomes of I. batatas. The investigation of cis-acting elements has shed light on the potential of PUBs to participate in a wide array of biological processes, particularly emphasizing their role in mediating responses to abiotic stresses. Transcriptome profiles revealed that IbPUB genes displayed a wide range of expression levels among different tissues and were regulated by salt or drought stress. IbPUB52 has emerged as a gene of significant interest due to its induction by salt and drought stresses. Localization studies have confirmed the presence of IbPUB52 in both the nucleus and the cytoplasm, and its ubiquitination activity has been validated through rigorous in vitro and in vivo assays. Intriguingly, the heterogeneous expression of IbPUB52 in Arabidopsis resulted in decreased drought tolerance. The virus-induced gene silencing (VIGS) of IbPUB52 in sweet potatoes led to enhanced resistance to drought. This evidence suggests that IbPUB52 negatively regulates the drought tolerance of plants. The findings of this study are instrumental in advancing our comprehension of the functional dynamics of PUB E3 ubiquitin ligases in sweet potatoes.

Diterpene synthases from Leonurus japonicus elucidate epoxy-bridge formation of spiro-labdane diterpenoids.

Spiro-9,13-epoxy-labdane diterpenoids are commonly found in Leonurus species, particularly in Leonurus japonicus Houtt., which is a medicinal herb of long-standing use in Asia and in which such spiro-heterocycles are present in at least 38 diterpenoids. Here, through generation of a transcriptome and functional characterization of six diterpene synthases (diTPSs) from L. japonicus, including three class II diTPSs (LjTPS1, LjTPS3, and LjTPS4) and three class I diTPSs (LjTPS5, LjTPS6, and LjTPS7), formation of the spiro-9,13-epoxy-labdane backbone was elucidated, along with identification of the relevant diTPSs for production of other labdane-related diterpenes. Similar to what has been found with diTPSs from other plant species, while LjTPS3 specifically produces the carbon-9 (C9) hydroxylated bicycle peregrinol diphosphate (PPP), the subsequently acting LjTPS6 yields a mixture of four products, largely labda-13(16),14-dien-9-ol, but with substantial amounts of viteagnusin D and the C13-S/R epimers of 9,13-epoxy-labda-14-ene. Notably, structure-function analysis identified a critical residue in LjTPS6 (I420) in which single site mutations enable specific production of the 13S epimer. Indeed, extensive mutagenesis demonstrated that LjTPS6:I420G reacts with PPP to both specifically and efficiently produce 9,13S-epoxy-labda-14-ene, providing a specialized synthase for further investigation of derived diterpenoid biosynthesis. The results reported here provide a strong foundation for future studies of the intriguing spiro-9,13-epoxy-labdane diterpenoid metabolism found in L. japonicus.

Multi-type feature fusion based on graph neural network for drug-drug interaction prediction.

BACKGROUND: Drug-Drug interactions (DDIs) are a challenging problem in drug research. Drug combination therapy is an effective solution to treat diseases, but it can also cause serious side effects. Therefore, DDIs prediction is critical in pharmacology. Recently, researchers have been using deep learning techniques to predict DDIs. However, these methods only consider single information of the drug and have shortcomings in robustness and scalability. RESULTS: In this paper, we propose a multi-type feature fusion based on graph neural network model (MFFGNN) for DDI prediction, which can effectively fuse the topological information in molecular graphs, the interaction information between drugs and the local chemical context in SMILES sequences. In MFFGNN, to fully learn the topological information of drugs, we propose a novel feature extraction module to capture the global features for the molecular graph and the local features for each atom of the molecular graph. In addition, in the multi-type feature fusion module, we use the gating mechanism in each graph convolution layer to solve the over-smoothing problem during information delivery. We perform extensive experiments on multiple real datasets. The results show that MFFGNN outperforms some state-of-the-art models for DDI prediction. Moreover, the cross-dataset experiment results further show that MFFGNN has good generalization performance. CONCLUSIONS: Our proposed model can efficiently integrate the information from SMILES sequences, molecular graphs and drug-drug interaction networks. We find that a multi-type feature fusion model can accurately predict DDIs. It may contribute to discovering novel DDIs.

De novo production of versatile oxidized kaurene diterpenes in Escherichia coli.

The oxidized kaurene (Ox-Kau) compounds are the core structures of many important diterpenoids with biological activities and economical values. However, easy access to diverse Ox-Kau products is still limited by low natural abundance, and large-scale manufacture remain challenging due to lack of proper heterologous production. To achieve an abundant source alternative to natural extracts, we here report a highly effective Escherichia coli-based platform for the de novo production of multiple Ox-Kau molecules from simple carbon source. Pathway optimization in prokaryotic cells through modification of transmembrane CYP450 oxidases, cytochrome b5 co-expression and AlphaFold-based protein engineering improved a 50-fold yield of steviol (1.07 g L-1), a key intermediate in the kaurenoid biosynthesis. Combinatorial biosynthetic strategy further led to a series of oxidized derivatives (20-600 mg L-1) with rich oxygenated functional groups on C3, C7, C16 and C19 previously hard to be introduced. Our engineered strains not only laid a foundation for realizing the industrial fermentation of gram-scale ent-kaurene diterpenoids, but also provided a reliable platform for characterization and utilization of kaurene-modifying oxidases, which may generate naturally rare or unnatural ent-kaurenoids with potential bioactivity.

[Accumulation and biosynthetic of curcuminoids and terpenoids in turmeric rhizome in different development periods].

The dynamic accumulation rule of active substances in medicinal plants is of great value not only for medicinal material production and application,but also for the genetic mechanism study on the formation of medicinal ingredients,especially vital to guide medicinal material collection as well as experiment material selection and candidate gene screening in the analysis of biosynthesis pathway. This study investigated the accumulation of curcumins and terpenoids,and the biosynthesis of these metabolites,which are the active metabolites in Curcuma longa,a commonly used traditional Chinese medicine. Rhizoma of C. longa from leaf growing period,rhizome swelling period and dry matter accumulating period were used as experimental materials,to analyze the changes of metabolites and biosynthesis in the three periods by comparative transcriptome and metabolomes analysis.The results indicated that terpenoids accumulation and biosynthesis mainly occurred in leaf growing period,while curcumin accumulation and biosynthesis mainly occurred in dry matter accumulating period. Therefore,we suggested that turmeric rhizomes in leaf growth period were suitable for terpenoids biosynthetic pathway characterization,and rhizome in accumulation of dry matter period was suitable for curcuminoid biosynthesis pathway characterization. This study provides references for medicinal materialproduction and application,as well as biopathway analysis of active compounds for C. longa.

A rapid method for establishment of a reverse genetics system for canine parvovirus.

Canine parvovirus (CPV) is an important and highly prevalent pathogen of dogs that causes acute hemorrhagic enteritis disease. Here, we describe a rapid method for the construction and characterization of a full-length infectious clone (rCPV) of CPV. Feline kidney (F81) cells were transfected with rCPV incorporating an engineered EcoR I site that served as a genetic marker. The rescued virus was indistinguishable from that of wild-type virus in its biological properties.

Genetic characterization of the complete genome of an Aleutian mink disease virus isolated in north China.

The genome of a highly pathogenic strain of Aleutian disease mink virus (AMDV-BJ) isolated from a domestic farm in North China has been determined and compared with other strains. Alignment analysis of the major structural protein VP2 revealed that AMDV-BJ is unique among 17 other AMDV strains. Compared with the nonpathogenic strain ADV-G, the 3' end Y-shaped hairpin was highly conserved, while a 4-base deletion in the 5' U-shaped terminal palindrome resulted in a different unpaired "bubble" group near the NS1-binding region of the 5' end hairpin which may affect replication efficiency in vivo. We also performed a protein analysis of the NS1, NS2, and new-confirmed NS3 of AMDV-BJ with some related AMDV DNA sequence published, providing information on evolution of AMDV genes. This study shows a useful method to obtain the full-length genome of AMDV and some other parvoviruses.

Comparison of biological and genomic characteristics between a newly isolated mink enteritis parvovirus MEV-LHV and an attenuated strain MEV-L.

A virus isolated from mink showing clinical signs of enteritis was identified as a high virulent mink enteritis parvovirus (MEV) based on its biological characteristics in vivo and in vitro. Mink, challenged with this strain named MEV-LHV, exhibited severe pathological lesions as compared to those challenged with attenuated strain MEV-L. MEV-LHV also showed higher infection and replication efficiencies in vitro than MEV-L. Sequence of the complete genome of MEV-LHV was determined and analyzed in comparison with those in GenBank, which revealed that MEV-LHV shared high homology with virulent strain MEV SD12/01, whereas MEV-L was closely related to Abashiri and vaccine strain MEVB, and belonged to a different branch of the phylogenetic tree. The genomes of the two strains differed by insertions and deletions in their palindromic termini and specific unique mutations (especially VP2 300) in coding sequences which may be involved in viral replication and pathogenicity. The results of this study provide a better understanding of the biological and genomic characteristics of MEV and identify certain regions and sites that may be involved in viral replication and pathogenicity.

A recombinant canine distemper virus expressing a modified rabies virus glycoprotein induces immune responses in mice.

Canine distemper virus (CDV) and rabies virus (RV) are two important pathogens of the dog. CDV, a member of the morbillivirus genus, has shown promise as an expression vector. The glycoprotein from RV is a main contributor to protective immunity and capable of eliciting the production of virus-neutralizing antibodies. In this study, we recovered an attenuated strain of canine distemper virus and constructed a recombinant virus, rCDV-RV-G, expressing a modified (R333Q) rabies virus glycoprotein (RV-G) of RV Flury strain LEP. RV-G expression by the recombinant viruses was confirmed. Furthermore, G was proved to be incorporated into the surface of CDV particles. While replication of the recombinant virus was slightly reduced compared with the parental CDV, it stably expressed the RV-G over ten serial passages. Inoculation of mice induced specific neutralizing antibodies against both RV-G and CDV. Therefore, the rCDV-RV-G has the potential as a vaccine that may be used to control rabies virus infection in dogs and other animals.

MicroRNA miR-320a and miR-140 inhibit mink enteritis virus infection by repression of its receptor, feline transferrin receptor.

Mink enteritis virus (MEV) is one of the most important pathogens in the mink industry. Recent studies have shed light into the role of microRNAs (miRNAs), small noncoding RNAs of length ranging from 18-23 nucleotides (nt), as critical modulators in the host-pathogen interaction networks. We previously showed that miRNA miR-181b can inhibit MEV replication by repression of viral non-structural protein 1 expression. Here, we report that two other miRNAs (miR-320a and miR-140) inhibit MEV entry into feline kidney (F81) cells by downregulating its receptor, transferrin receptor (TfR), by targeting the 3' untranslated region (UTR) of TfR mRNA, while being themselves upregulated.

Combination of specific single chain antibody variable fragment and siRNA has a synergistic inhibitory effect on the propagation of avian influenza virus H5N1 in chicken cells.

BACKGROUND: The avian influenza virus (AIV) causes frequent disease with high morbidity and mortality. RNA interference (RNAi) has been shown to provide an effective antiviral defense in animals, and several studies have focused on harnessing small interfering RNAs (siRNAs) to inhibit viral infections. In addition, single chain variable fragments (scFvs) contain the complete antigen binding site, and specific scFvs can bind to and neutralize viruses. RESULTS: Fourteen positive scFvs were selected by the yeast two-hybrid system. Using molecular docking technology, we selected the three highest affinity scFvs for further functional validation. Results of indirect ELISA and IFA showed that all three scFvs could bind to FJ13 strain and had neutralizing activity, decreasing the viral infectivity markedly. Chicken fibroblastic DF-1 cells were transfected with scFvs in combination with siRNA-NP604 (an siRNA of anti-AIV NP protein previously reported). Following infection with FJ13 virus, copy numbers of the virus were significantly reduced from 12 h to at least 60 h post-infection compared to that achieved in cells transfected with scFv or siRNA-NP604 separately. CONCLUSIONS: A novel combination of antiviral siRNAs expressed in chicken cells and chicken antibody single-chain variable fragments (scFvs) secreted from the cells has a synergistic inhibitory effect on the avian influenza viral proliferation in vitro. Intracellular application of scFvs and anti-viral siRNA may provide a new approach to influenza prevention and treatment.

Overexpression of Banna mini-pig inbred line fatty acid binding protein 3 promotes adipogenesis in 3T3-L1 preadipocytes.

Fatty acid binding protein 3 (H-FABP, FABP3) has been significantly associated with intramuscular fat (IMF) content in pigs, which is positively correlated with palatability of pork. However, its underlying function is not fully elucidated. We have investigated the effects of overexpression of the FABP3 gene on differentiation and adipogenesis of 3T3-L1 preadipocytes in the fat Banna mini-pig inbred line (fBMIL). Eukaryotic vectors that expressed the FABP3 protein were constructed, and stably established in the 3T3-L1 preadipocytes cell line. Cells were induced in a standard differentiation cocktail. Morphological changes and the degree of adipogenesis were measured by Oil Red O staining assay and triacylglycerol content measurement, respectively. mRNA expression levels of triacylglycerol metabolism-related genes were measured by qPCR. FABP3 significantly promoted differentiation of 3T3-L1 cells and enhanced triacylglycerol levels (P < 0.05). mRNA of the peroxisome proliferator-activated receptor γ (PPARγ), adipocyte fatty acid binding protein (422/aP2) and glycerol-3-phosphate dehydrogenase (GPDH) gene increased markedly (P < 0.05). In conclusion, expression of the FABP3 gene enhances adipogenesis in 3T3-L1 preadipocytes primarily by upregulating lipogenic PPARγ, 422/aP2 and GPDH genes.

Prenatal BPA exposure disrupts male reproductive functions by interfering with DNA methylation and GDNF expression in the testes of male offspring rats.

BPA is a ubiquitous environmental endocrine-disrupting chemical, and maternal exposure to BPA is associated with impaired male reproductive functions; however, the mechanisms remain to be elucidated. Glial cell line-derived neurotrophic factor (GDNF) plays an important role in maintaining normal spermatogenesis and fertility. However, the effect of prenatal BPA exposure on GDNF expression and its mechanism in the testis has not been reported. In this study, pregnant Sprague-Dawley rats were respectively exposed to 0, 0.05, 0.5, 5, and 50 mg/kg/day BPA via oral gavage from gestational day (GD) 5 to GD 19, with 6 pregnant rats in each group. ELISA, histochemistry, real-time PCR, western blot, and methylation-specific PCR (MSP) were used to detect the sex hormone levels, testicular histopathology, mRNA and protein expression of DNA methyltransferases (DNMTs) and GDNF, and the promoter methylation of Gdnf in the testes of male offspring at postnatal day (PND) 21 and PND 56. Prenatal BPA exposure increased body weight; decreased sperm counts and serum levels of testosterone (T), follicle-stimulating hormone (FSH), and luteinizing hormone (LH); and induced testicular histological damage, which indicated the damage of male reproductive function. Prenatal BPA exposure also upregulated Dnmt1 in 5 mg/kg group and Dnmt3b in 0.5 mg/kg group, but down-regulated Dnmt1 in 50 mg/kg group at PND 21. At PND 56, Dnmt1 was significantly increased in 0.05 mg/kg group but decreased in 0.5, 5, and 50 mg/kg groups, Dnmt3a was decreased, and Dnmt3b was markedly increased in 0.05 and 0.5 mg/kg groups but decreased in 5 and 50 mg/kg groups. The mRNA and protein expression levels of Gdnf were decreased markedly in 0.5 and 50 mg/kg groups at PND 21. And the methylation level of Gdnf promoter was significantly increased in 0.5 mg/kg group, but decreased in 5 and 50 mg/kg groups at PND 21. In conclusion, our study indicates that prenatal BPA exposure disrupts male reproductive functions, interferes with the expression of DNMTs, and decreases Gdnf expression in the testes of male offspring. Gdnf expression may be regulated by DNA methylation; however, the detailed mechanism needs to be further investigated.

A chromosome-level genome assembly reveals that tandem-duplicated CYP706V oxidase genes control oridonin biosynthesis in the shoot apex of Isodon rubescens.

The ent-kaurenoids (e.g., oridonin and enmein) from the Isodon genus (Lamiaceae) are one class of diterpenoids with rich structural diversity and intriguing pharmaceutical activity. In contrast to the well-established gibberellin pathway, oxidative modifications diversifying the ent-kaurene skeleton in Isodon have remained undetermined for half a century. Here we report a chromosome-level genome assembly of I. rubescens, a well-recognized oridonin producer long favored by Asian people as a traditional herb with antitumor effects. The shoot apex was confirmed to be the actual region actively producing ent-kaurene diterpenoids. Through comparative genomics and phylogenetic analyses, we discovered a cluster of tandem-duplicated CYP706V oxygenase-encoding genes located on an ancient genomic block widely distributed in eudicots, whereas almost exclusively emerged in Isodon plants. In the shoot apex, IrCYP706V2 and IrCYP706V7 oxidized the ent-kaurene core in the initial stage of oridonin biosynthesis. Loss of CYP706Vs in other Lamiaceae plants offered an explanation for the specific kaurenoid production in Isodon plants. Moreover, we found that the Isodon genomes encode multiple diterpenoid synthases that are potentially involved in generating diterpenoid diversity. These findings provided new insights into the evolution of the lineage-specific diterpenoid pathway and laid a foundation for improving production of bioactive ent-kaurene-type diterpenoids by molecular breeding and synthetic biology approaches.

Dodder-transmitted mobile systemic signals activate a salt-stress response characterized by a transcriptome change in Citrus sinensis.

Citrus is an essential horticultural fruit whose yield and quality are affected by salinity all over the world. The recognition and adaptive regulation of citrus against salt stress are important areas for cultivar improvement, but the vascular system signal transduction mechanism of the plant response to salt stress remains elusive. In this study, we constructed a dodder (Cuscuta spp.) linked Hamlin sweet orange (Citrus sinensis) plant community in which deliver a vascular signal through the dodder in response to salt stress. RNA-seq technology was used to analyze the gene expression profile of citrus leaves after salt treatment. The results showed that a vascular signal was transmitted to a dodder-linked host plant, triggering a transcriptional response to salt stress. However, the phenotypic and transudative ability of the dodder changed after 24 h. The salt treatment group (Group S) and the dodder-linked group (Group D) respectively contained 1,472 and 557 differentially expressed genes (DEGs). 454 of which were common to both groups. The results of our analysis revealed that the gene expression categories in Group D represented a highly consistent trend compared to the group S plants, indicating that the dodder-bridged vascular signals activated the stress-response of citrus leaves for transcriptomic reconfiguration. The KEGG pathway database and an analysis of key drivers revealed that phenylpropanoid biosynthesis, photosynthesis-antenna proteins, starch and sucrose metabolism, plant hormone signal transduction, circadian rhythm, and MAPK signaling pathways were significantly enriched as the critical genes during salt stress. A systemic signal in the dodder-bridged host significantly regulated abiotic stress-related secondary metabolic pathways, including those for phenylpropanoids, lignin, and lignans. The physiological indexes of photosynthetic intensity, respiration, and attractiveness among communities supported the transcriptional changes. Thus, our results indicate that salt stress-induced vascular system signals can be transmitted through the vascular system of a dodder linking citrus plants, revealing the genetic regulation and physiological changes of citrus leaves responding to plant stress signal transmission.

Whole-Cell Biocatalyst for Rubusoside Production in Saccharomyces cerevisiae.

Rubusoside (Rub) is a highly sweet diterpene glycoside mainly isolated from the leaves of Rubus suavissimus (Rosaceae). It has been used as a low-calorie natural sweetener for decades and was recently found to be a potential drug lead. In this study, we designed a whole-cell biocatalyst to achieve the glycosylation of steviol to Rub in Saccharomyces cerevisiae. The sucrose synthases were applied to construct a uridine diphosphate glucose regeneration system, which were coupled with optimal combinations of different uridine diphosphate (UDP) glycosyltransferases from multiple plant species. After optimization of reaction conditions, the residues in SrUGT74G1 probably influencing glycosylation efficiency were subjected to site-directed mutagenesis. Double mutations of S84A/E87A reduced the accumulation of intermediates, finally glucosylating 1.27 g/L steviol to 0.45 ± 0.06 g/L steviolmonoside (conversion rate = 23.3%) and 1.92 ± 0.17 g/L Rub (conversion rate = 74.9%). A high efficiency of Rub biosynthesis could be achieved without supply of additional UDPG. This work provided the first example of multi-step glycosylation reactions in whole-cell biocatalysis, which laid a foundation of scalable production of the value-added diterpene sweetener in the future.

Functional Integration of Two CYP450 Genes Involved in Biosynthesis of Tanshinones for Improved Diterpenoid Production by Synthetic Biology.

Cytochrome P450s (CYPs) are important enzymes in the secondary metabolism of plants and have been recognized as key players in bioengineering and synthetic biology. Previously reported CYP76AH1 and CYP76AH3, having greater than 80% sequence homology, played a continuous catalytic role in the biosynthesis of tanshinones in Salvia miltiorrhiza. Homology modeling indicates that four sites might be responsible for differences in catalytic activity between the two enzymes. A series of modeling-based mutational variants of CYP76AH1 were designed to integrate the functions of the two CYPs. The mutant CYP76AH1D301E,V479F, which integrated the functions of CYP76AH1 and CYP76AH3, was found to efficiently catalyze C11 and C12 hydroxylation and C7 oxidation of miltiradiene substrates. Integration and utilization of CYP76AH1D301E,V479F by synthetic biology methods allowed the robust production of 11-hydroxy ferruginol, sugiol, and 11-hydroxy sugiol in yeast. The functionally integrated CYP gene after active site modifications improves catalytic efficiency by reducing the transfer of intermediate metabolites between component proteins. This provides a synthetic biology reference for improving the catalytic efficiencies of systems that produce plant natural products in microorganisms.

Interaction between CYP1A1/CYP17A1 polymorphisms and parental risk factors in the risk of hypospadias in a Chinese population.

Hypospadias (HS) is a common congenital malformation of the genitourinary tract in males and its etiology is viewed as multifactorial, and studies about gene-environment interaction in the etiology of HS are rare. A total of 152 cases and 151 controls were selected in the present study. Information before and during pregnancy from questionnaires finished by mothers of subjects were extracted, and the relating data were analyzed to determine the risk factors of HS. Meanwhile, maternal genomic DNA was genotyped for the single nucleotide polymorphisms (SNPs) of CYP1A1 rs1048943 and CYP17A1 rs4919686. Results of multivariable logistic regression analyses showed that several factors were associated with hypospadias risk. Analysis of the distributions of SNPs in CYP1A1 and CYP17A1 genes showed that the mutant genotype CC (OR = 4.87) of CYP1A1 rs1048943, and mutant genotype CC (OR = 5.82), recessive genotype AC + CC (OR = 2.17) and allele C (OR = 1.77) of CYP17A1 rs4919686 significantly increased the risk of HS. In addition, the additive gene-environment interactions were also found in several models. Several maternal risk factors that are associated with HS risk can interact with CYP1A1/CYP17A1 polymorphisms, which lead to infants vulnerable to occurrence of HS in Chinese populations.

Induction and suppression of type I interferon responses by mink enteritis virus in CRFK cells.

Mink enteritis virus (MEV) is one of the most important viral pathogens causing serious disease in mink. Type I interferon (IFN) plays a critical role in antiviral innate immunity and, for successful infection, many viruses have evolved evasive strategies against it. Here, we show that MEV infection does not evoke IFN or interferon-stimulated genes (ISGs) responses in feline kidney (CRFK) cells, and that MEV suppresses IFN production in both poly I:C-stimulated and untreated cells. In CRFK cells pre-exposure to IFN, show that infection with, and replication of, MEV remain unaffected. This inhibition appears to be mediated by the MEV nonstructural protein (NS1) with its ORI-binding domain playing a major role.

The phosphorylation of Ser221 in VP2 of mink enteritis virus and its roles in virus amplification.

Recent reports have indicated that phosphorylation of capsid proteins plays an important role in virion assemblage. Autonomous parvoviruses are among the smallest known viruses with an ssDNA genome enclosed within an icosahedral capsid. Here, we demonstrate that a structural protein (VP2) of one member, mink enteritis virus (MEV), is phosphorylated at serine-221 (Ser221) in vivo. Mutant viruses containing an S221A non-phosphorylatable alanine substitution, or an S221E glutamic acid substitution to mimic serine phosphorylation, were able to express VP2 but had either limited ability or were unable to propagate in feline F81 cells. We propose a new mechanism whereby VP2 phosphorylation plays an essential role in amplification during MEV infection.