Monkeypox emergency: Urgent questions and perspectives.

Amidst the COVID-19 pandemic, we now face another public health emergency in the form of monkeypox virus. As of August 1, the Centers for Disease Control and Prevention report over 23,000 cases in 80 countries. An inclusive and global collaborative effort to understand the biology, evolution, and spread of the virus as well as commitment to vaccine equity will be critical toward containing this outbreak. We share the voices of leading experts in this space on what they see as the most pressing questions and directions for the community.

Viral growth factor- and STAT3 signaling-dependent elevation of the TCA cycle intermediate levels during vaccinia virus infection.

Metabolism is a crucial frontier of host-virus interaction as viruses rely on their host cells to provide nutrients and energy for propagation. Vaccinia virus (VACV) is the prototype poxvirus. It makes intensive demands for energy and macromolecules in order to build hundreds and thousands of viral particles in a single cell within hours of infection. Our comprehensive metabolic profiling reveals profound reprogramming of cellular metabolism by VACV infection, including increased levels of the intermediates of the tri-carboxylic acid (TCA) cycle independent of glutaminolysis. By investigating the level of citrate, the first metabolite of the TCA cycle, we demonstrate that the elevation of citrate depends on VACV-encoded viral growth factor (VGF), a viral homolog of cellular epidermal growth factor. Further, the upregulation of citrate is dependent on STAT3 signaling, which is activated non-canonically at the serine727 upon VACV infection. The STAT3 activation is dependent on VGF, and VGF-dependent EGFR and MAPK signaling. Together, our study reveals a novel mechanism by which VACV manipulates cellular metabolism through a specific viral factor and by selectively activating a series of cellular signaling pathways.

Poxvirus-encoded decapping enzymes promote selective translation of viral mRNAs.

Cellular decapping enzymes negatively regulate gene expression by removing the methylguanosine cap at the 5' end of eukaryotic mRNA, rendering mRNA susceptible to degradation and repressing mRNA translation. Vaccinia virus (VACV), the prototype poxvirus, encodes two decapping enzymes, D9 and D10, that induce the degradation of both cellular and viral mRNAs. Using a genome-wide survey of translation efficiency, we analyzed vaccinia virus mRNAs in cells infected with wild type VACV and mutant VACVs with inactivated decapping enzymes. We found that VACV decapping enzymes are required for selective translation of viral post-replicative mRNAs (transcribed after viral DNA replication) independent of PKR- and RNase L-mediated translation repression. Further molecular characterization demonstrated that VACV decapping enzymes are necessary for efficient translation of mRNA with a 5'-poly(A) leader, which are present in all viral post-replicative mRNAs. Inactivation of D10 alone in VACV significantly impairs poly(A)-leader-mediated translation. Remarkably, D10 stimulates mRNA translation in the absence of VACV infection with a preference for RNA containing a 5'-poly(A) leader. We further revealed that VACV decapping enzymes are needed for 5'-poly(A) leader-mediated cap-independent translation enhancement during infection. Our findings identified a mechanism by which VACV mRNAs are selectively translated through subverting viral decapping enzymes to stimulate 5'-poly(A) leader-mediated translation.

Rapid and quantitative evaluation of vaccinia virus-induced host shutoff using newly generated cell lines stably expressing secreted Gaussia luciferase.

Host shutoff, characterized by a global decline of cellular protein synthesis, is commonly observed in many viral infections, including vaccinia virus (VACV). Classic methods measuring host shutoff include the use of radioactive or nonradioactive probes to label newly synthesized proteins followed by radioautography or sodium dodecyl-sulfate polyacrylamide gel electrophoresis to resolve the proteins for follow-up detection. Although these are highly reliable methods, they are time- and labor-consuming. Here, we generated two cell lines stably expressing secreted Gaussia luciferase. These reporter cells allow rapid, quantitative, and consecutive monitoring of host shutoff from a single infection sample. We evaluated host shutoff induced by wild-type and various mutant VACVs using the reporter cell lines. The results validated the utilities of the reporter cells and quantitatively characterized VACV-induced host shutoff at different stages of replication. Notably, the results also indicated additional major unidentified VACV shutoff factors. Our study provides new tools to study host shutoff. The reporter cells are also suitable for high throughput settings and rapid testing of clinically isolated viruses. In combination with classical methods, these tools will greatly facilitate understanding of virus-induced host shutoff, and protein synthesis shutoff caused by other physiologically relevant stresses.

Optimal therapeutic conditions for the neural stem cell-based management of ischemic stroke: a systematic review and network meta-analysis based on animal studies.

BACKGROUND: In order to promote the clinical translation of preclinical findings, it is imperative to identify the most optimal therapeutic conditions and adopt them for further animal and human studies. This study aimed to fully explore the optimal conditions for neural stem cell (NSC)-based ischemic stroke treatment based on animal studies. METHODS: The PubMed, Ovid-Embase, and Web of Science databases were searched in December 2021. The screening of search results, extraction of relevant data, and evaluation of study quality were performed independently by two reviewers. RESULTS: In total, 52 studies were included for data analysis. Traditional meta-analysis showed that NSCs significantly reduced the modified neurological severity score (mNSS) and volume of cerebral infarct in animal models of ischemic stroke. Network meta-analysis showed that allogeneic embryonic tissue was the best source of NSCs. Further, intracerebral transplantation was the most optimal route of NSC transplantation, and the acute phase was the most suitable stage for intervention. The optimal number of NSCs for transplantation was 1-5×105 in mouse models and 1×106 or 1.8×106 in rat models. CONCLUSIONS: We systematically explored the therapeutic strategy of NSCs in ischemic stroke, but additional research is required to develop optimal therapeutic strategies based on NSCs. Moreover, it is necessary to further improve and standardize the design, implementation, measuring standards, and reporting of animal-based studies to promote the development of better animal experiments and clinical research.

Molecular characterization and expression analysis of selenoprotein W gene in rainbow trout (Oncorhynchus mykiss) with dietary selenium levels.

Selenium (Se) plays an essential role in the growth of fish and performs its physiological functions mainly through incorporation into selenoproteins. Our previous studies suggested that the selenoprotein W gene (selenow) is sensitive to changes in dietary Se in rainbow trout. However, the molecular characterization and tissue expression pattern of selenow are still unknown. Here, we revealed the molecular characterization, the tissue expression pattern of rainbow trout selenow and analyzed its response to dietary Se. The open reading frame (ORF) of the selenow gene was composed of 393 base pairs (bp) and encodes a 130-amino-acid protein. The 3' untranslated region (UTR) was 372 bp with a selenocysteine insertion sequence (SECIS) element. Remarkably, the rainbow trout selenow gene sequence was longer than those reported for mammals and most other fish. A ß1-α1-ß2-ß3-ß4-α2 pattern made up the secondary structure of SELENOW. Furthermore, multiple sequence alignment revealed that rainbow trout SELENOW showed a high level of identity with SELENOW from Salmo salar. In addition, the selenow gene was ubiquitously distributed in 13 tissues with various abundances and was predominantly expressed in muscle and brain. Interestingly, dietary Se significantly increased selenow mRNA expression in muscle. Our results highlight the vital role of selenow in rainbow trout muscle response to dietary Se levels and provide a theoretical basis for studies of selenow.

Anomalous Size Effect of Pt Ultrathin Nanowires on Oxygen Reduction Reaction.

The classical size effect of Pt particles on oxygen reduction reaction (ORR) suggests that the activity and durability would decrease with reducing the particle size, self-limiting the effectiveness in maximizing the Pt utilization efficiency with the particle-size-reduction strategy. Herein, we discover an anomalous size effect based on Pt nanowires (NWs) with tunable diameters, where the monotonically increasing activity and durability for ORR were observed with decreasing the diameter from 2.4 to 1.1 nm. Our results reveal that the dominant role of increased compressive strain induced by decreasing the diameter of NWs in weakening the adsorption and suppressing the Pt dissolution accounts for this anomalous size effect, where the reduced low-coordinated sites on NWs, the intrinsic structural advantage, is the root. Our findings not only expand the knowledge to the classical size effect but also provide new implications to break through the size limit in the design of Pt-based ORR catalysts.

Intermetallic Compounds: Liquid-Phase Synthesis and Electrocatalytic Applications.

Characterized by long-range atomic ordering, well-defined stoichiometry, and controlled crystal structure, intermetallics have attracted increasing attention in the area of chemical synthesis and catalytic applications. Liquid-phase synthesis of intermetallics has arisen as the promising methodology due to its precise control over size, shape, and resistance toward sintering compared with the traditional metallurgy. This short review tends to provide perspectives on the liquid-phase synthesis of intermetallics in terms of both thermodynamics and methodology, as well as its applications in various catalytic reactions. Specifically, basic thermodynamics and kinetics in the synthesis of intermetallics will be first discussed, followed by discussing the main factors that will affect the formation of intermetallics during synthesis. The application of intermetallics in electrocatalysis will be demonstrated case by case at last. We conclude the review with perspectives on the future developments with respect to both synthesis and catalytic applications.

Asparagine Is a Critical Limiting Metabolite for Vaccinia Virus Protein Synthesis during Glutamine Deprivation.

Viruses actively interact with host metabolism because viral replication relies on host cells to provide nutrients and energy. Vaccinia virus (VACV; the prototype poxvirus) prefers glutamine to glucose for efficient replication to the extent that VACV replication is hindered in glutamine-free medium. Remarkably, our data show that VACV replication can be fully rescued from glutamine depletion by asparagine supplementation. By global metabolic profiling, as well as genetic and chemical manipulation of the asparagine supply, we provide evidence demonstrating that the production of asparagine, which exclusively requires glutamine for biosynthesis, accounts for VACV's preference of glutamine to glucose rather than glutamine's superiority over glucose in feeding the tricarboxylic acid (TCA) cycle. Furthermore, we show that sufficient asparagine supply is required for efficient VACV protein synthesis. Our study highlights that the asparagine supply, the regulation of which has been evolutionarily tailored in mammalian cells, presents a critical barrier to VACV replication due to a high asparagine content of viral proteins and a rapid demand of viral protein synthesis. The identification of asparagine availability as a critical limiting factor for efficient VACV replication suggests a new direction of antiviral strategy development.IMPORTANCE Viruses rely on their infected host cells to provide nutrients and energy for replication. Vaccinia virus, the prototypic member of the poxviruses, which comprise many significant human and animal pathogens, prefers glutamine to glucose for efficient replication. Here, we show that the preference is not because glutamine is superior to glucose as the carbon source to fuel the tricarboxylic acid cycle for vaccinia virus replication. Rather interestingly, the preference is because the asparagine supply for efficient viral protein synthesis becomes limited in the absence of glutamine, which is necessary for asparagine biosynthesis. We provide further genetic and chemical evidence to demonstrate that asparagine availability plays a critical role in efficient vaccinia virus replication. This discovery identifies a weakness of vaccinia virus and suggests a possible direction to intervene in poxvirus infection.

The 5'-poly(A) leader of poxvirus mRNA confers a translational advantage that can be achieved in cells with impaired cap-dependent translation.

The poly(A) leader at the 5'-untranslated region (5'-UTR) is an unusually striking feature of all poxvirus mRNAs transcribed after viral DNA replication (post-replicative mRNAs). These poly(A) leaders are non-templated and of heterogeneous lengths; and their function during poxvirus infection remains a long-standing question. Here, we discovered that a 5'-poly(A) leader conferred a selective translational advantage to mRNA in poxvirus-infected cells. A constitutive and uninterrupted 5'-poly(A) leader with 12 residues was optimal. Because the most frequent lengths of the 5'-poly(A) leaders are 8-12 residues, the result suggests that the poly(A) leader has been evolutionarily optimized to boost poxvirus protein production. A 5'-poly(A) leader also could increase protein production in the bacteriophage T7 promoter-based expression system of vaccinia virus, the prototypic member of poxviruses. Interestingly, although vaccinia virus post-replicative mRNAs do have 5'- methylated guanosine caps and can use cap-dependent translation, in vaccinia virus-infected cells, mRNA with a 5'-poly(A) leader could also be efficiently translated in cells with impaired cap-dependent translation. However, the translation was not mediated through an internal ribosome entry site (IRES). These results point to a fundamental mechanism poxvirus uses to efficiently translate its post-replicative mRNAs.

cAMP/PKA signaling pathway contributes to neuronal apoptosis via regulating IDE expression in a mixed model of type 2 diabetes and Alzheimer's disease.

Type 2 diabetes (T2D) may play a relevant role in the development of Alzheimer's disease (AD), however, the underlying mechanism was not clear yet. We developed an animal model presenting both AD and T2D, morris water maze (MWM) test and recognition task were performed to trace the cognitive function. Fasting plasma glucose (FPG) and oral glucose tolerance test (OGTT) were determined to trace the metabolism evolution. TUNEL assay and apoptosis-related protein levels were analyzed for the detection of neuronal apoptosis. Cyclic adenosine monophosphate (cAMP) agonist bucladesine or protein kinase (PKA) inhibitor H-89 were used to determine the effects of cAMP/PKA signaling pathway on IDE expression and neuronal apoptosis. The results showed that T2D contributes to the AD progress by accelerating and worsening spatial memory and recognition dysfunctions. Metabolic parameters and glucose tolerance were significantly changed in the presence of the AD and T2D. The significantly induced neuronal apoptosis and increased pro-apoptotic proteins in mice with AD and T2D were also observed. We showed the decreased expression level of IDE and the activating of cAMP/PKA signaling pathway in AD and T2D mice. Further studies indicated that cAMP agonist decreased the expression level of IDE and induced the neuronal apoptosis in mice with AD and T2D; whereas PKA inhibitor H-89 treatment showed the completely opposite results. Our study indicated that, in the T2D and AD mice, cAMP/PKA signaling pathway and IDE may participate in the contribute role of T2D in accelerating the pathological process of AD via causing the accumulation of Aß and neuronal apoptosis.

Going against the Tide: Selective Cellular Protein Synthesis during Virally Induced Host Shutoff.

Many viral infections cause host shutoff, a state in which host protein synthesis is globally inhibited. Emerging evidence from vaccinia and influenza A virus infections indicates that subsets of cellular proteins are resistant to host shutoff and continue to be synthesized. Remarkably, the proteins of oxidative phosphorylation, the cellular-energy-generating machinery, are selectively synthesized in both cases. Identifying mechanisms that drive selective protein synthesis should facilitate understanding both viral replication and fundamental cell biology.

Ribosome Profiling Reveals Translational Upregulation of Cellular Oxidative Phosphorylation mRNAs during Vaccinia Virus-Induced Host Shutoff.

Vaccinia virus infection causes a host shutoff that is marked by global inhibition of host protein synthesis. Though the host shutoff may facilitate reallocation of cellular resources for viral replication and evasion of host antiviral immune responses, it poses a challenge for continuous synthesis of cellular proteins that are important for viral replication. It is, however, unclear whether and how certain cellular proteins may be selectively synthesized during the vaccinia virus-induced host shutoff. Using simultaneous RNA sequencing and ribosome profiling, two techniques quantifying genome-wide levels of mRNA and active protein translation, respectively, we analyzed the responses of host cells to vaccinia virus infection at both the transcriptional and translational levels. The analyses showed that cellular mRNA depletion played a dominant role in the shutoff of host protein synthesis. Though the cellular mRNAs were significantly reduced, the relative translation efficiency of a subset of cellular mRNAs increased, particularly those involved in oxidative phosphorylation that are responsible for cellular energy production. Further experiments demonstrated that the protein levels and activities of oxidative phosphorylation increased during vaccinia virus infection, while inhibition of the cellular oxidative phosphorylation function significantly suppressed vaccinia virus replication. Moreover, the short 5' untranslated region of the oxidative phosphorylation mRNAs contributed to the translational upregulation. These results provide evidence of a mechanism that couples translational control and energy metabolism, two processes that all viruses depend on host cells to provide, to support vaccinia virus replication during a host shutoff.IMPORTANCE Many viral infections cause global host protein synthesis shutoff. While host protein synthesis shutoff benefits the virus by relocating cellular resources to viral replication, it also poses a challenge to the maintenance of cellular functions necessary for viral replication if continuous protein synthesis is required. Here we measured the host mRNA translation rate during a vaccinia virus-induced host shutoff by analyzing total and actively translating mRNAs in a genome-wide manner. This study revealed that oxidative phosphorylation mRNAs were translationally upregulated during vaccinia virus-induced host protein synthesis shutoff. Oxidative phosphorylation is the major cellular energy-producing pathway, and we further showed that maintenance of its function is important for vaccinia virus replication. This study highlights the fact that vaccinia virus infection can enhance cellular energy production through translational upregulation in the context of an overall host protein synthesis shutoff to meet energy expenditure.

Enterovirus 71 3C Promotes Apoptosis through Cleavage of PinX1, a Telomere Binding Protein.

Enterovirus 71 (EV71) is an emerging pathogen causing hand, foot, and mouth disease (HFMD) and fatal neurological diseases in infants and young children due to their underdeveloped immunocompetence. EV71 infection can induce cellular apoptosis through a variety of pathways, which promotes EV71 release. The viral protease 3C plays an important role in EV71-induced apoptosis. However, the molecular mechanism responsible for 3C-triggered apoptosis remains elusive. Here, we found that EV71 3C directly interacted with PinX1, a telomere binding protein. Furthermore, 3C cleaved PinX1 at the site of Q50-G51 pair through its protease activity. Overexpression of PinX1 reduced the level of EV71-induced apoptosis and EV71 release, whereas depletion of PinX1 by small interfering RNA promoted apoptosis induced by etoposide and increased EV71 release. Taken together, our study uncovered a mechanism that EV71 utilizes to promote host cell apoptosis through cleavage of cellular protein PinX1 by 3C. IMPORTANCE: EV71 3C plays an important role in processing viral proteins and interacting with host cells. In this study, we showed that 3C promoted apoptosis through cleaving PinX1, a telomere binding protein, and that this cleavage facilitated EV71 release. Our study demonstrated that PinX1 plays an important role in EV71 release and revealed a novel mechanism that EV71 utilizes to induce apoptosis. This finding is important in understanding EV71-host cell interactions and has potential impact on understanding other enterovirus-host cell interactions.

RPFdb: a database for genome wide information of translated mRNA generated from ribosome profiling.

Translational control is crucial in the regulation of gene expression and deregulation of translation is associated with a wide range of cancers and human diseases. Ribosome profiling is a technique that provides genome wide information of mRNA in translation based on deep sequencing of ribosome protected mRNA fragments (RPF). RPFdb is a comprehensive resource for hosting, analyzing and visualizing RPF data, available at www.rpfdb.org or http://sysbio.sysu.edu.cn/rpfdb/index.html. The current version of database contains 777 samples from 82 studies in 8 species, processed and reanalyzed by a unified pipeline. There are two ways to query the database: by keywords of studies or by genes. The outputs are presented in three levels. (i) Study level: including meta information of studies and reprocessed data for gene expression of translated mRNAs; (ii) Sample level: including global perspective of translated mRNA and a list of the most translated mRNA of each sample from a study; (iii) Gene level: including normalized sequence counts of translated mRNA on different genomic location of a gene from multiple samples and studies. To explore rich information provided by RPF, RPFdb also provides a genome browser to query and visualize context-specific translated mRNA. Overall our database provides a simple way to search, analyze, compare, visualize and download RPF data sets.

Molluscum Contagiosum Virus Transcriptome in Abortively Infected Cultured Cells and a Human Skin Lesion.

UNLABELLED: Molluscum contagiosum virus (MOCV), the only circulating human-specific poxvirus, has a worldwide distribution and causes benign skin lesions that may persist for months in young children and severe infections in immunosuppressed adults. Studies of MOCV are restricted by the lack of an efficient animal model or a cell culture replication system. We used next-generation sequencing to analyze and compare polyadenylated RNAs from abortive MOCV infections of several cell lines and a human skin lesion. Viral RNAs were detected for 14 days after MOCV infection of cultured cells; however, there was little change in the RNA species during this time and a similar pattern occurred in the presence of an inhibitor of protein synthesis, indicating a block preventing postreplicative gene expression. Moreover, a considerable number of MOCV RNAs mapped to homologs of orthopoxvirus early genes, but few did so to homologs of intermediate or late genes. The RNAs made during in vitro infections represent a subset of RNAs detected in human skin lesions which mapped to homologs of numerous postreplicative as well as early orthopoxvirus genes. Transfection experiments using fluorescent protein and luciferase reporters demonstrated that vaccinia virus recognized MOCV intermediate and late promoters, indicating similar gene regulation. The specific recognition of the intermediate promoter in MOCV-infected cells provided evidence for the synthesis of intermediate transcription factors, which are products of early genes, but not for late transcription factors. Transcriptome sequencing (RNA-seq) and reporter gene assays may be useful for testing engineered cell lines and conditions that ultimately could provide an in vitro replication system. IMPORTANCE: The inability to propagate molluscum contagiosum virus, which causes benign skin lesions in young children and more extensive infections in immunosuppressed adults, has constrained our understanding of the biology of this human-specific virus. In the present study, we characterized the RNAs synthesized in abortively infected cultured cells and a human skin lesion by next-generation sequencing. These studies provided an initial transcription map of the MOCV genome, suggested temporal regulation of gene expression, and indicated that the in vitro replication block occurs prior to intermediate and late gene expression. RNA-seq and reporter assays, as described here, may help to further evaluate MOCV gene expression and define conditions that could enable MOCV replication in vitro.

[Prevalence of erectile dysfunction in men with pre-diabetes: An investigation in Lanzhou].

OBJECTIVE: To investigate the prevalence of erectile dysfunction (ED) in men with pre-diabetes. METHODS: This study included 500 men with impaired fasting glycaemia (IFG), 500 with impaired glucose tolerance (IGT), and another 500 with normal blood glucose (NBG), all from Lanzhou. We conducted a questionnaire investigation among the subjects using the International Index of Erectile Dysfunction 5 (IIEF-5). RESULTS: The prevalence rates of ED in the IFG, IGT, and NBG groups were 14.8%, 29.2%, and 33.2%, respectively. After controlling for age, nationality, occupation, education, income, obesity, and blood pressure, the incidence rate was markedly higher in the IFG and IGT than in the NBG group (29.2% and 33.2% vs 14.8%, P <0.05), but showed no statistically significant difference between the IFG and IGT groups (P >0.05). CONCLUSIONS: The prevalence of ED is higher in men with pre-diabetes than in those with normal blood glucose in Lanzhou.

Deciphering poxvirus gene expression by RNA sequencing and ribosome profiling.

UNLABELLED: The more than 200 closely spaced annotated open reading frames, extensive transcriptional read-through, and numerous unpredicted RNA start sites have made the analysis of vaccinia virus gene expression challenging. Genome-wide ribosome profiling provided an unprecedented assessment of poxvirus gene expression. By 4 h after infection, approximately 80% of the ribosome-associated mRNA was viral. Ribosome-associated mRNAs were detected for most annotated early genes at 2 h and for most intermediate and late genes at 4 and 8 h. Cluster analysis identified a subset of early mRNAs that continued to be translated at the later times. At 2 h, there was excellent correlation between the abundance of individual mRNAs and the numbers of associated ribosomes, indicating that expression was primarily transcriptionally regulated. However, extensive transcriptional read-through invalidated similar correlations at later times. The mRNAs with the highest density of ribosomes had host response, DNA replication, and transcription roles at early times and were virion components at late times. Translation inhibitors were used to map initiation sites at single-nucleotide resolution at the start of most annotated open reading frames although in some cases a downstream methionine was used instead. Additional putative translational initiation sites with AUG or alternative codons occurred mostly within open reading frames, and fewer occurred in untranslated leader sequences, antisense strands, and intergenic regions. However, most open reading frames associated with these additional translation initiation sites were short, raising questions regarding their biological roles. The data were used to construct a high-resolution genome-wide map of the vaccinia virus translatome. IMPORTANCE: This report contains the first genome-wide, high-resolution analysis of poxvirus gene expression at both transcriptional and translational levels. The study was made possible by recent methodological advances allowing examination of the translated regions of mRNAs including start sites at single-nucleotide resolution. Vaccinia virus ribosome-associated mRNA sequences were detected for most annotated early genes at 2 h and for most intermediate and late genes at 4 and 8 h after infection. The ribosome profiling approach was particularly valuable for poxviruses because of the close spacing of approximately 200 open reading frames and extensive transcriptional read-through resulting in overlapping mRNAs. The expression of intermediate and late genes, in particular, was visualized with unprecedented clarity and quantitation. We also identified novel putative translation initiation sites that were mostly associated with short protein coding sequences. The results provide a framework for further studies of poxvirus gene expression.

Role of vaccinia virus growth factor in stimulating the mTORC1-CAD axis of the de novo pyrimidine pathway under different nutritional cues.

Vaccinia virus (VACV), the prototype poxvirus, actively reprograms host cell metabolism upon infection. However, the nature and molecular mechanisms remain largely elusive. Given the diverse nutritional exposures of cells in different physiological contexts, it is essential to understand how VACV may alter various metabolic pathways in different nutritional conditions. In this study, we established the importance of de novo pyrimidine biosynthesis in VACV infection. We elucidated the significance of vaccinia growth factor (VGF), a viral early protein and a homolog of cellular epidermal growth factor, in enabling VACV to phosphorylate the key enzyme CAD of the de novo pyrimidine pathway at serine 1859, a site known to positively regulate CAD activity. While nutrient-poor conditions typically inhibit mTORC1 activation, VACV activates CAD via mTORC1-S6K1 signaling axis, in conditions where glutamine and asparagine are absent. However, unlike its cellular homolog, epidermal growth factor (EGF), VGF peptide alone in the absence of VACV infection has minimal ability to activate CAD, suggestive of the involvement of other viral factor(s) and differential functions to EGF acquired during poxvirus evolution. Our research provides a foundation for understanding the regulation of a significant metabolic pathway, namely, de novo pyrimidine synthesis during VACV infection, shedding new light on viral regulation under distinct nutritional environments. This study not only has the potential to contribute to the advancement of antiviral treatments but also improve the development of VACV as an oncolytic agent and vaccine vector.