Viral Synthesis Research Articles

Glucose-independent human cytomegalovirus replication is supported by metabolites that feed upper glycolytic branches

Viruses with broad tissue distribution and cell tropism successfully replicate in various nutrient environments in the body. Several viruses reprogram metabolism for viral replication. However, many studies focus on metabolic reprogramming in nutrient-rich conditions that do not recapitulate physiological environments in the body. Here, we investigated how viruses may replicate when a metabolite thought to be essential for replication is limited. We use human cytomegalovirus infection in glucose-free conditions as a model to determine how glucose supports virus replication and how physiologically relevant nutrients contribute to glucose-independent virus production. We find that glucose supports viral genome synthesis, viral protein production and glycosylation, and infectious virus production. Notably, supplement of glucose-free cultures with uridine, ribose, or UDP-GlcNAc-metabolites that feed upper glycolytic branches like the pentose phosphate pathway-results in partially restored virus replication, including low levels of infectious virus production. Supplementing lower glycolysis in glucose-free cultures using pyruvate fails to restore virus replication. These results indicate that nutrients can compensate for glucose via feeding upper glycolytic branches to sustain low levels of virus production. More broadly, our findings suggest that viruses may successfully replicate in diverse metabolic niches, including those in the body with low glucose levels, through alternative nutrient usage.

In vitro one-pot construction of influenza viral genomes for virus particle synthesis based on reverse genetics system.

The reverse genetics system, which allows the generation of influenza viruses from plasmids encoding viral genome, is a powerful tool for basic research on viral infection mechanisms and application research such as vaccine development. However, conventional plasmid construction using Escherichia coli (E.coli) cloning is time-consuming and has difficulties handling DNA encoding genes toxic for E.coli or highly repeated sequences. These limitations hamper rapid virus synthesis. In this study, we establish a very rapid in vitro one-pot plasmid construction (IVOC) based virus synthesis. This method dramatically reduced the time for genome plasmid construction, which was used for virus synthesis, from several days or more to about 8 hours. Moreover, infectious viruses could be synthesized with a similar yield to the conventional E.coli cloning-based method with high accuracy. The applicability of this method was also demonstrated by the generation of recombinant viruses carrying reporter genes from the IVOC products. This method enables the pathogenicity analysis and vaccine development using genetically modified viruses, and it is expected to allow for faster analysis of newly emerging variants than ever before. Furthermore, its application to other RNA viruses is also expected.

Enhanced ER protein processing gene expression increases rAAV yield and full capsid ratio in HEK293 cells

The recombinant adeno-associated virus (rAAV) vector is among the most promising viral vectors in gene therapy. However, the limited manufacturing capacity in human embryonic kidney (HEK) cells is a barrier to rAAV commercialization. We investigated the impact of endoplasmic reticulum (ER) protein processing and apoptotic genes on transient rAAV production in HEK293 cells. We selected four candidate genes based on prior transcriptomic studies: XBP1, GADD34 / PPP1R15A, HSPA6, and BCL2. These genes were stably integrated into HEK293 host cells. Traditional triple-plasmid transient transfection was used to assess the vector production capability and the quality of both the overexpressed stable pools and the parental cells. We show that the overexpression of XBP1, HSPA6, and GADD34 increases rAAV productivity by up to 100% and increases specific rAAV productivity by up to 78% in HEK293T cells. Additionally, more prominent improvement associated with ER protein processing gene overexpression was observed when parental cell productivity was high, but no substantial variation was detected under low-producing conditions. We also confirmed genome titer improvement across different serotypes (AAV2 and AAV8) and different cell lines (HEK293T and HEK293); however, the extent of improvement may vary. This study unveiled the importance of ER protein processing pathways in viral particle synthesis, capsid assembly, and vector production.Key points• Upregulation of endoplasmic reticulum (ER) protein processing (XBP1, HSPA6, and GADD34) leads to a maximum 100% increase in rAAV productivity and a maximum 78% boost in specific rAAV productivity in HEK293T cells• The enhancement in productivity can be validated across different HEK293 cell lines and can be used for the production of various AAV serotypes, although the extent of the enhancement might vary slightly• The more pronounced improvements linked to overexpressing ER protein processing genes were observed when parental cell productivity was high, with minimal variation noted under low-producing conditions

The SAMHD1-MX2 axis restricts HIV-1 infection at postviral DNA synthesis.

In contrast to most restriction factors that directly bind to viral components to exert their antiviral effects, SAMHD1, the only known deoxynucleotide triphosphate (dNTP) hydrolase in eukaryotes, indirectly inhibits viral replication in quiescent cells by reducing the pool of dNTP substrates available for viral cDNA synthesis. Our study provides a novel perspective on the antiviral functions of SAMHD1. In addition to its role in dNTP hydrolysis, SAMHD1 cooperates with MX2 to inhibit HIV-1 nuclear import. In this process, SAMHD1 acts as a sensor for incoming HIV-1 cores, detecting and binding to them, before subsequently delivering the complex to the molecular trap formed by MX2, thereby immobilizing the virus. This study not only reveals a new antiviral pathway for SAMHD1 but also identifies a unique collaboration and interaction between two distinct restriction factors, establishing a novel line of defense against HIV-1 infection, which challenges the traditional view of restriction factors acting independently. Overall, our findings further indicate the intricate complexity of the host immune defense network and provide potential targets for promoting host antiviral immune defense.

Genomic Variation in Dengue Virus Non-Structural Protein 1 (NS1)

To understand in an improved way how the dengue virus (DENV) spreads, presents, and becomes hazardous, researching its genetic makeup is necessary. The positive sense RNA of DENV encodes three structural proteins and seven non-structural proteins. One of the non-structural proteins that aids in the replication of viral RNA is the non-structural protein 1 (NS1). The objective was to identify the most frequently repeated mutations in the NS1 protein in DENV RNA isolated from dengue patients in the province Punjab, Pakistan. Selection of 120 DENV isolates was done from laboratories of tertiary care hospitals of Punjab for analysis of sequencing of the whole genome. Only 23 samples were sequenced after viral isolation, quantification and cDNA synthesis. A total of 133 different types of mutations were detected along the entire length of NS.1. The most common mutations with the highest frequency were, K324R and K347R (n=7), D278N (n=6), K174R, and F178S (n=4), found at c-terminal of NS.1 protein. Mutations K347R, K174R, and F178S are novel. Future DENV vaccination development research will be especially profited by the mutations found in the current study. During each DENV outbreak in different places, studying genomic variations is crucial for strengthening societal health and developing new policies for future outbreaks.

Tubercidin inhibits PRRSV replication via RIG-I/NF-κB pathways and interrupting viral nsp2 synthesis.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important swine diseases, which causes huge economic loss worldwide. However, there is no effective therapeutic method for PRRS prevention and control. Here, we found that tubercidin, a naturally extracted adenosine analog, exhibited strong anti-porcine reproductive and respiratory syndrome virus (PRRSV) activity. Mechanically, tubercidin inhibited viral binding, replication, and release. Tubercidin suppressed PRRSV non-structural protein 2 expression, which is important for the formation of replication and transcription complex, leading to the block of viral RNA synthesis and PRRSV replication. Moreover, tubercidin could activate retinoic acid-inducible gene I/nuclear factor kappa-light-chain-enhancer of activated B cell innate immune signaling pathway and increased the expression of interferons and proinflammatory cytokines, which was the other way to inhibit PRRSV replication. Our work evaluated the potential value of tubercidin as an antiviral agent on PRRSV replication and provided a new way to prevent PRRSV replication in vitro.

Influenza Virus Host Restriction Factors: The ISGs and Non-ISGs.

Influenza virus has been one of the most prevalent and researched viruses globally. Consequently, there is ample information available about influenza virus lifecycle and pathogenesis. However, there is plenty yet to be known about the determinants of influenza virus pathogenesis and disease severity. Influenza virus exploits host factors to promote each step of its lifecycle. In turn, the host deploys antiviral or restriction factors that inhibit or restrict the influenza virus lifecycle at each of those steps. Two broad categories of host restriction factors can exist in virus-infected cells: (1) encoded by the interferon-stimulated genes (ISGs) and (2) encoded by the constitutively expressed genes that are not stimulated by interferons (non-ISGs). There are hundreds of ISGs known, and many, e.g., Mx, IFITMs, and TRIMs, have been characterized to restrict influenza virus infection at different stages of its lifecycle by (1) blocking viral entry or progeny release, (2) sequestering or degrading viral components and interfering with viral synthesis and assembly, or (3) bolstering host innate defenses. Also, many non-ISGs, e.g., cyclophilins, ncRNAs, and HDACs, have been identified and characterized to restrict influenza virus infection at different lifecycle stages by similar mechanisms. This review provides an overview of those ISGs and non-ISGs and how the influenza virus escapes the restriction imposed by them and aims to improve our understanding of the host restriction mechanisms of the influenza virus.

Proof-of-concept for multiple AON delivery by a single U7snRNA vector to restore splicing defects in ABCA4

The high allelic heterogeneity in Stargardt disease (STGD1) complicates the design of intervention strategies. A significant proportion of pathogenic intronic ABCA4 variants alters thepre-mRNA splicing process. Antisense oligonucleotides (AONs) are an attractive yet mutation-specific therapeutic strategy to restore these splicing defects. In this study, we experimentally assessed the potential of a splicing modulation therapy to target multiple intronic ABCA4 variants. AONs were inserted into U7snRNA gene cassettes and tested in midigene-based splice assays. Five potent antisense sequences were selected to generate a multiple U7snRNA cassette construct, and this combination vector showed substantial rescue of all of the splicing defects. Therefore, the combination cassette was used for viral synthesis and assessment in patient-derived photoreceptor precursor cells (PPCs). Simultaneous delivery of several modified U7snRNAs through a single AAV, however, did not show substantial splicing correction, probably due to suboptimal transduction efficiency in PPCs and/or a heterogeneous viral population containing incomplete AAV genomes. Overall, these data demonstrate the potential of the U7snRNA system to rescue multiple splicing defects, but also suggest that AAV-associated challenges are still a limiting step, underscoring the need for further optimization before implementing this strategy as a potential treatment for STGD1.

ZNF283, a Krüppel-associated box zinc finger protein, inhibits RNA synthesis of porcine reproductive and respiratory syndrome virus by interacting with Nsp9 and Nsp10

Porcine reproductive and respiratory syndrome virus (PRRSV) is a viral pathogen with substantial economic implications for the global swine industry. The existing vaccination strategies and antiviral drugs offer limited protection. Replication of the viral RNA genome encompasses a complex series of steps, wherein a replication complex is assembled from various components derived from both viral and cellular sources, as well as from the viral genomic RNA template. In this study, we found that ZNF283, a Krüppel-associated box (KRAB) containing zinc finger protein, was upregulated in PRRSV-infected Marc-145 cells and porcine alveolar macrophages and that ZNF283 inhibited PRRSV replication and RNA synthesis. We also found that ZNF283 interacts with the viral proteins Nsp9, an RNA-dependent RNA polymerase, and Nsp10, a helicase. The main regions involved in the interaction between ZNF283 and Nsp9 were determined to be the KRAB domain of ZNF283 and amino acids 178–449 of Nsp9. The KRAB domain of ZNF283 plays a role in facilitating Nsp10 binding. In addition, ZNF283 may have an affinity for the 3' untranslated region of PRRSV. These findings suggest that ZNF283 is an antiviral factor that inhibits PRRSV infection and extend our understanding of the interactions between KRAB-containing zinc finger proteins and viruses.

Decay of HCoV-OC43 infectivity is lower in cell debris-containing media than in fresh culture media.

In the quantitative description of viral dynamics within cell cultures and, more broadly, in modeling within-host viral infections, a question that commonly arises is whether the degradation of a fraction of the virus could be disregarded in comparison with the massive synthesis of new viral particles. Surprisingly, quantitative data on the synthesis and degradation rates of RNA viruses in cell cultures are scarce. In this study, we investigated the decay of the human betacoronavirus OC43 (HCoV-OC43) infectivity in cell culture lysates and in fresh media. Our findings revealed a significantly slower viral decay rate in the medium containing lysate cells compared to the fresh medium. This observation suggests that the presence of cellular debris from lysed cells may offer protection or stabilize virions, slowing down their degradation. Moreover, the growth rate of HCoV-OC43 infectivity is significantly higher than degradation as long as there are productive cells in the medium, suggesting that, as a first approximation, degradation can be neglected during early infection.

Alphavirus-based replicons demonstrate different interactions with host cells and can be optimized to increase protein expression.

Alphavirus replicons are being developed as self-amplifying RNAs aimed at improving the efficacy of mRNA vaccines. These replicons are convenient for genetic manipulations and can express heterologous genetic information more efficiently and for a longer time than standard mRNAs. However, replicons mimic many aspects of viral replication in terms of induction of innate immune response, modification of cellular transcription and translation, and expression of nonstructural viral genes. Moreover, all replicons used in this study demonstrated expression of heterologous genes in cell- and replicon's origin-specific modes. Thus, many aspects of the interactions between replicons and the host remain insufficiently investigated, and further studies are needed to understand the biology of the replicons and their applicability for designing a new generation of mRNA vaccines. On the other hand, our data show that replicons are very flexible expression systems, and additional modifications may have strong positive impacts on protein expression.

A DNA Plasmid-Based Approach for Efficient Synthesis of Sacbrood Virus Infectious Clones within Host Cells.

RNA viruses are often cited as a significant factor affecting the populations of both domestic honey bees and wild pollinators. To expedite the development of effective countermeasures against these viruses, a more comprehensive understanding of virus biology necessitates extensive collaboration among scientists from diverse research fields. While the infectious virus clone is a robust tool for studying virus diseases, the current methods for synthesizing infectious clones of bee-infecting RNA viruses entail the in vitro transcription of the viral genome RNA in 8-10 kb, presenting challenges in reproducibility and distribution. This article reports on the synthesis of an infectious clone of the Chinese variant sacbrood virus (SBV) using a DNA plasmid containing an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) immediate-early protein (IE1) promoter to trigger transcription of the downstream viral genome within hosts. The results demonstrate that the IE1-SBV plasmid can synthesize SBV clones in a widely used lepidopteran immortal cell line (Sf9) and honey bee pupae. Furthermore, the negative strand of the clone was detected in both Sf9 cells and honey bee pupae, indicating active infection and replication. However, the transfection of Sf9 cells was observed in only a limited proportion (less than 10%) of the cells, and the infection did not appear to spread to adjacent cells or form infective virions. The injection of honey bee pupae with 2500 ng of the IE1-SBV plasmid resulted in high infection rates in Apis cerana pupae but low rates in A. mellifera pupae, although the dosage was comparatively high compared with other studies using in vitro transcribed viral RNA. Our findings suggest that the synthesis of bee-infecting RNA viruses using DNA plasmids is feasible, albeit requiring additional optimization. However, this method holds substantial potential for facilitating the production of clones with various sequence modifications, enabling the exploration of viral gene functions and biology. The ease of distributing infectious clones in DNA plasmid form may foster collaboration among scientists in applying the clone to bee biology, ecology, and behavior, ultimately offering a comprehensive approach to managing virus diseases in the future.

Rational Design of Highly Potent SARS-CoV-2 nsp14 Methyltransferase Inhibitors.

The search for new drugs against COVID-19 and its causative agent, SARS-CoV-2, is one of the major trends in the current medicinal chemistry. Targeting capping machinery could be one of the therapeutic concepts based on a unique mechanism of action. Viral RNA cap synthesis involves two methylation steps, the first of which is mediated by the nsp14 protein. Here, we rationally designed and synthesized a series of compounds capable of binding to both the S-adenosyl-l-methionine and the RNA-binding site of SARS-CoV-2 nsp14 N7-methyltransferase. These hybrid molecules showed excellent potency, high selectivity toward various human methyltransferases, nontoxicity, and high cell permeability. Despite the outstanding activity against the enzyme, our compounds showed poor antiviral performance in vitro. This suggests that the activity of this viral methyltransferase has no significant effect on virus transcription and replication at the cellular level. Therefore, our compounds represent unique tools to further explore the role of the SARS-CoV-2 nsp14 methyltransferase in the viral life cycle and the pathogenesis of COVID-19.

Oncolytic virotherapy with intratumoral injection of vaccinia virus TG6002 and 5-fluorocytosine administration in dogs with malignant tumors

TG6002 is an oncolytic vaccinia virus expressing FCU1 protein, which converts 5-fluorocytosine into 5-fluorouracil. The studyobjectives were to assess tolerance, viral replication, 5-fluorouracil synthesis, and tumor microenvironment modifications to treatment in dogs with spontaneous malignant tumors. Thirteen dogs received one to three weekly intratumoral injections of TG6002 and 5-fluorocytosine. The viral genome was assessed in blood and tumor biopsies by qPCR. 5-Fluorouracil concentrations were measured in serum and tumor biopsies by liquid chromatography or high-resolution mass spectrometry. Histological and immunohistochemical analyses were performed. The viral genome was detected in blood (7/13) and tumor biopsies (4/11). Viral replication was suspected in 6/13 dogs. The median intratumoral concentration of 5-fluorouracil was 314 pg/mg. 5-Fluorouracil was not detected in the blood. An increase in necrosis (6/9) and a downregulation of intratumoral regulatory T lymphocytes (6/6) were observed. Viral replication, 5-fluorouracil synthesis, and tumor microenvironment changes were more frequently observed with higher TG6002 doses. This study confirmed the replicative properties, targeted chemotherapy synthesis, and reversion of the immunosuppressive tumor microenvironment in dogs with spontaneous malignant tumors treated with TG6002 and 5-fluorocytosine.

Glycolytic interference blocks influenza A virus propagation by impairing viral polymerase-driven synthesis of genomic vRNA.

Influenza A virus (IAV), like any other virus, provokes considerable modifications of its host cell's metabolism. This includes a substantial increase in the uptake as well as the metabolization of glucose. Although it is known for quite some time that suppression of glucose metabolism restricts virus replication, the exact molecular impact on the viral life cycle remained enigmatic so far. Using 2-deoxy-d-glucose (2-DG) we examined how well inhibition of glycolysis is tolerated by host cells and which step of the IAV life cycle is affected. We observed that effects induced by 2-DG are reversible and that cells can cope with relatively high concentrations of the inhibitor by compensating the loss of glycolytic activity by upregulating other metabolic pathways. Moreover, mass spectrometry data provided information on various metabolic modifications induced by either the virus or agents interfering with glycolysis. In the presence of 2-DG viral titers were significantly reduced in a dose-dependent manner. The supplementation of direct or indirect glycolysis metabolites led to a partial or almost complete reversion of the inhibitory effect of 2-DG on viral growth and demonstrated that indeed the inhibition of glycolysis and not of N-linked glycosylation was responsible for the observed phenotype. Importantly, we could show via conventional and strand-specific qPCR that the treatment with 2-DG led to a prolonged phase of viral mRNA synthesis while the accumulation of genomic vRNA was strongly reduced. At the same time, minigenome assays showed no signs of a general reduction of replicative capacity of the viral polymerase. Therefore, our data suggest that the significant reduction in IAV replication by glycolytic interference occurs mainly due to an impairment of the dynamic regulation of the viral polymerase which conveys the transition of the enzyme's function from transcription to replication.

Bergamottin Inhibits PRRSV Replication by Blocking Viral Non-Structural Proteins Expression and Viral RNA Synthesis.

The porcine reproductive and respiratory syndrome virus (PRRSV) causes economic losses in the swine industry worldwide. However, current vaccines cannot provide effective protection against PRRSV, and PRRSV-specific treatments for infected herds are still unavailable. In this study, we found that bergamottin showed strong inhibitory effects against PRRSV replication. Bergamottin inhibited PRRSV at the stage of the replication cycle. Mechanically, bergamottin promoted the activation of IRF3 and NF-κB signaling, leading to the increased expression of proinflammatory cytokines and interferon, which inhibited viral replication to some extent. In addition, bergamottion could reduce the expression of the non-structural proteins (Nsps), leading to the interruption of replication and transcription complex (RTC) formation and viral dsRNA synthesis, ultimately restraining PRRSV replication. Our study identified that bergamottin possesses potential value as an antiviral agent against PRRSV in vitro.

N6-methyladenosine is required for efficient RNA synthesis of Ebola virus and other haemorrhagic fever viruses

ABSTRACT N6-methyladenosine (m6A) is one of the most abundant modifications of cellular RNA, where it serves various functions. m6A methylation of many viral RNA species has also been described; however, little is known about the m6A epitranscriptome of haemorrhagic fever-causing viruses like Ebola virus (EBOV). Here, we analysed the importance of the methyltransferase METTL3 for the life cycle of this virus. We found that METTL3 interacts with the EBOV nucleoprotein and the transcriptional activator VP30 to support viral RNA synthesis, and that METTL3 is recruited into EBOV inclusions bodies, where viral RNA synthesis occurs. Analysis of the m6A methylation pattern of EBOV mRNAs showed that they are methylated by METTL3. Further studies revealed that METTL3 interaction with the viral nucleoprotein, as well as its importance for RNA synthesis and protein expression, is also observed for other haemorrhagic fever viruses such as Junín virus (JUNV) and Crimean-Congo haemorrhagic fever virus (CCHFV). The negative effects on viral RNA synthesis due to loss of m6A methylation are independent of innate immune sensing, as METTL3 knockout did not affect type I interferon induction in response to viral RNA synthesis or infection. Our results suggest a novel function for m6A that is conserved among diverse haemorrhagic fever-causing viruses (i.e. EBOV, JUNV and CCHFV), making METTL3 a promising target for broadly-acting antivirals.

Human cytomegalovirus UL138 interaction with USP1 activates STAT1 in infection.

Innate immune responses are crucial for limiting virus infection. However, viruses often hijack our best defenses for viral objectives. Human Cytomegalovirus (HCMV) is a beta herpesvirus which establishes a life-long latent infection. Defining the virus-host interactions controlling latency and reactivation is vital to the control of viral disease risk posed by virus reactivation. We defined an interaction between UL138, a pro-latency HCMV gene, and the host deubiquitinating complex, UAF1-USP1. UAF1 is a scaffold protein pivotal for the activity of ubiquitin specific peptidases (USP), including USP1. UAF1-USP1 sustains an innate immune response through the phosphorylation and activation of signal transducer and activator of transcription-1 (pSTAT1), as well as regulates the DNA damage response. After the onset of viral DNA synthesis, pSTAT1 levels are elevated in infection and this depends upon UL138 and USP1. pSTAT1 localizes to viral centers of replication, binds to the viral genome, and influences UL138 expression. Inhibition of USP1 results in a failure to establish latency, marked by increased viral genome replication and production of viral progeny. Inhibition of Jak-STAT signaling also results in increased viral genome synthesis in hematopoietic cells, consistent with a role for USP1-mediated regulation of STAT1 signaling in the establishment of latency. These findings demonstrate the importance of the UL138-UAF1-USP1 virus-host interaction in regulating HCMV latency establishment through the control of innate immune signaling. It will be important going forward to distinguish roles of UAF1-USP1 in regulating pSTAT1 relative to its role in the DNA damage response in HCMV infection.

In silico screening, ADMET analysis and MD simulations of phytochemicals of Onosma bracteata Wall. as SARS CoV-2 inhibitors.

Being attracted with their cardiotonic, antidiabetic, cough relieving activity, treatment of fever, absorbent, anti-asthmatic, etc. activities reported in ancient Ayurvedic literature, phytochemicals of Onosma bracteata wall should be evaluated for their activity against SARS-CoV-2 virus. The main objective of this study is to identify a hit molecule for the inhibition of entry, replication, and protein synthesis of SARS CoV-2 virus into the host. To achieve given objective, computational virtual screening of phytochemicals of Onosma bracteata wall has been performed against three main viral targets: spike, RdRp, and Mpro. Further, the analysis of Lipinski's Ro5 and their estimation of ADMET profiles were performed using computational tools. The MD simulations studies of top hits against each viral target have also been performed for 20ns to ensure their stability. The analysis of results revealed that Pulmonarioside C (9) and other plant compounds showed better binding affinity towards targets than existing antiviral compounds, making them probable lead compounds against SARS-CoV-2. Structural modifications and studies through in silico analysis provided the founding stone for the establishment of SARS CoV-2 inhibitory potential of phytoconstitutents of Onosma bracteata wall.

Synthesis and Inhibition of Influenza H1N1 Virus by Propargylaminoalkyl Derivative of Lithocholic Acid

In the current study, the conjugate of 3-oxo-lithocholic acid with N-methylpiperazine and paraform was synthesized using the Mannich reaction and evaluated for antiviral activity. This modification resulted in a dramatic increase of antiviral activity combined with a two-fold decrease of toxicity. Together, these effects led to a strong increase of selectivity of compound (SI = 40 vs. 3 for 3 and 2, correspondingly).