Early Stage Of Viral Replication Research Articles

Global siRNA Screen Reveals Critical Human Host Factors of SARS-CoV-2 Multicycle Replication.

Defining the subset of cellular factors governing SARS-CoV-2 replication can provide critical insights into viral pathogenesis and identify targets for host-directed antiviral therapies. While a number of genetic screens have previously reported SARS-CoV-2 host dependency factors, these approaches relied on utilizing pooled genome-scale CRISPR libraries, which are biased towards the discovery of host proteins impacting early stages of viral replication. To identify host factors involved throughout the SARS-CoV-2 infectious cycle, we conducted an arrayed genome-scale siRNA screen. Resulting data were integrated with published datasets to reveal pathways supported by orthogonal datasets, including transcriptional regulation, epigenetic modifications, and MAPK signalling. The identified proviral host factors were mapped into the SARS-CoV-2 infectious cycle, including 27 proteins that were determined to impact assembly and release. Additionally, a subset of proteins were tested across other coronaviruses revealing 17 potential pan-coronavirus targets. Further studies illuminated a role for the heparan sulfate proteoglycan perlecan in SARS-CoV-2 viral entry, and found that inhibition of the non-canonical NF-kB pathway through targeting of BIRC2 restricts SARS-CoV-2 replication both in vitro and in vivo. These studies provide critical insight into the landscape of virus-host interactions driving SARS-CoV-2 replication as well as valuable targets for host-directed antivirals.

The N545S and K717N substitution at the N-glycosylation sites of the S2 subunit of avian infectious bronchitis virus can significantly enhance viral pathogenicity

The S2 subunit of infectious bronchitis virus (IBV) is a heavily glycosylated protein that can impact various characteristics of the virus. It is currently known that N-glycosylation modifications are predominantly located on the S2 subunit. However, the exact role of their N-glycosylation modification remains undisclosed. To elucidate the function of these N-glycosylation sites, we identified 14 common sites distributed on the S2 subunit of the 5 genotypes of IBV in present study. Subsequently, we selected 7 sites to generate mutants and assessed their impact on viral virulence, replication ability, and antigenicity. Our finding revealed that only 2 substitutions, N545S and K717N, increased the viral replication titer and antigenicity, and ultimately the pathogenicity in chicks. To delve into the mechanisms underlying this increased pathogenicity, we discovered that K717N can change the structure of antigenic epitopes. The N545S substitution not only influenced antigenic epitope structure, but also enhanced the ability of the virus to enter CEKs during the early stages of viral replication. These results suggest that the enhanced viral pathogenicity associated with N545S and K717N substitutions is multifaceted, with acceleration of the viral membrane fusion process and alterations in epitope structure representing crucial factors in the capability of N-glycosylation modifications to boost viral virulence. These insights provide valuable guidance for the efficient development of live attenuated vaccines.

Rev Protein Diversity in HIV-1 Group M Clades.

The HIV-1 Rev protein expressed in the early stage of virus replication is involved in the nuclear export of some forms of virus RNA. Naturally occurring polymorphisms in the Rev protein could influence its activity. The association between the genetic features of different virus variants and HIV infection pathogenesis has been discussed for many years. In this study, Rev diversity among HIV-1 group M clades was analyzed to note the signatures that could influence Rev activity and, subsequently, clinical characteristics. From the Los Alamos HIV Sequence Database, 4962 Rev sequences were downloaded and 26 clades in HIV-1 group M were analyzed for amino acid changes, conservation in consensus sequences, and the presence of clade-specific amino acid substitutions (CSSs) and the Wu-Kabat protein variability coefficient (WK). Subtypes G, CRF 02_AG, B, and A1 showed the largest amino acid changes and diversity. The mean conservation of the Rev protein was 80.8%. In consensus sequences, signatures that could influence Rev activity were detected. In 15 out of 26 consensus sequences, an insertion associated with the reduced export activity of the Rev protein, 95QSQGTET96, was identified. A total of 32 CSSs were found in 16 clades, wherein A6 had the 41Q substitution in the functionally significant region of Rev. The high values of WK coefficient in sites 51 and 82, located on the Rev interaction surface, indicate the susceptibility of these positions to evolutionary replacements. Thus, the noted signatures require further investigation.

Chlorogenic acid inhibits porcine deltacoronavirus release by targeting apoptosis

Porcine deltacoronavirus (PDCoV), belonging to family Coronaviridae, genus Deltacoronavirus, can cause acute diarrhea in piglets, and also possesses cross-species transmission potential, leading to severe economic losses and threatening public health. However, no approved drug against PDCoV infection is available. Here, we investigated the antiviral effect of chlorogenic acid (CGA), the main active component of Lonicerae Japonicae Flos, against PDCoV infection. The results showed that CGA inhibited the replication of PDCoV significantly both in LLC-PK1 and ST cells, with a selectivity index greater than 80. CGA decreased the synthesis of PDCoV viral RNA and protein, and viral titers in a dose-dependent manner. The results of the time-of-addition assay indicated that CGA mainly affected the early stage of virus replication and viral release. Moreover, CGA significantly reduced apoptosis caused by PDCoV infection, and the application of apoptosis agonist and inhibitor revealed that apoptosis could promote progeny virus release. Further study demonstrated that CGA can inhibit virus release by directly targeting apoptosis caused by PDCoV infection. In conclusion, CGA is an effective agent against PDCoV, which provides a foundation for drug development for the treatment of PDCoV and other coronavirus infections.

A phenothiazine urea derivative broadly inhibits coronavirus replication via viral protease inhibition

Coronavirus (CoV) replication requires efficient cleavage of viral polyproteins into an array of non-structural proteins involved in viral replication, organelle formation, viral RNA synthesis, and host shutoff. Human CoVs (HCoVs) encode two viral cysteine proteases, main protease (Mpro) and papain-like protease (PLpro), that mediate polyprotein cleavage. Using a structure-guided approach, a phenothiazine urea derivative that inhibits both SARS-CoV-2 Mpro and PLpro protease activity was identified. In silico docking studies also predicted the binding of the phenothiazine urea to the active sites of structurally similar Mpro and PLpro proteases from distantly related alphacoronavirus, HCoV-229 E (229 E), and the betacoronavirus, HCoV-OC43 (OC43). The lead phenothiazine urea derivative displayed broad antiviral activity against all three HCoVs tested in cellulo. It was further demonstrated that the compound inhibited 229 E and OC43 at an early stage of viral replication, with diminished formation of viral replication organelles, and the RNAs that are made within them, as expected following viral protease inhibition. These observations suggest that the phenothiazine urea derivative readily inhibits viral replication and may broadly inhibit proteases of diverse coronaviruses.

HSP90AB1 Is a Host Factor Required for Transmissible Gastroenteritis Virus Infection.

Transmissible gastroenteritis virus (TGEV) is an important swine enteric coronavirus causing viral diarrhea in pigs of all ages. Currently, the development of antiviral agents targeting host proteins to combat viral infection has received great attention. The heat shock protein 90 (HSP90) is a critical host factor and has important regulatory effects on the infection of various viruses. However, its roles in porcine coronavirus infection remain unclear. In this study, the effect of HSP90 on TGEV infection was evaluated. In addition, the influence of its inhibitor VER-82576 on proinflammatory cytokine (IL-6, IL-12, TNF-α, CXCL10, and CXCL11) production induced by TGEV infection was further analyzed. The results showed that the knockdown of HSP90AB1 and HSP90 inhibitor VER-82576 treatment resulted in a reduction in TGEV M gene mRNA levels, the N protein level, and virus titers in a dose-dependent manner, while the knockdown of HSP90AA1 and KW-2478 treatment had no significant effect on TGEV infection. A time-of-addition assay indicated that the inhibitory effect of VER-82576 on TGEV infection mainly occurred at the early stage of viral replication. Moreover, the TGEV-induced upregulation of proinflammatory cytokine (IL-6, IL-12, TNF-α, CXCL10, and CXCL11) expression was significantly inhibited by VER-82576. In summary, these findings indicated that HSP90AB1 is a host factor enhancing TGEV infection, and the HSP90 inhibitor VER-82576 could reduce TGEV infection and proinflammatory cytokine production, providing a new perspective for TGEV antiviral drug target design.

The Adenylyl Cyclase Activator Forskolin Increases Influenza Virus Propagation in MDCK Cells by Regulating ERK1/2 Activity.

Vaccination is the most effective method for preventing the spread of the influenza virus. Cell-based influenza vaccines have been developed to overcome the disadvantages of egg-based vaccines and their production efficiency has been previously discussed. In this study, we investigated whether treatment with forskolin (FSK), an adenylyl cyclase activator, affected the output of a cell-based influenza vaccine. We found that FSK increased the propagation of three influenza virus subtypes (A/H1N1/California/4/09, A/H3N2/Mississippi/1/85, and B/Shandong/7/97) in Madin-Darby canine kidney (MDCK) cells. Interestingly, FSK suppressed the growth of MDCK cells. This effect could be a result of protein kinase A (PKA)-Src axis activation, which downregulates extracellular signal-regulated kinase (ERK)1/2 activity and delays cell cycle progression from G1 to S. This delay in cell growth might benefit the binding and entry of the influenza virus in the early stages of viral replication. In contrast, FSK dramatically upregulated ERK1/2 activity via the cAMP-PKA-Raf-1 axis at a late stage of viral replication. Thus, increased ERK1/2 activity might contribute to increased viral ribonucleoprotein export and influenza virus propagation. The increase in viral titer induced by FSK could be explained by the action of cAMP in assisting the entry and binding of the influenza virus. Therefore, FSK addition to cell culture systems could help increase the production efficiency of cell-based vaccines against the influenza virus.

EVALUATION OF INTERACTIONS OF SARS-COV-2 STRUCTURAL PROTEINS WITH SPECIFIC ANTIBODIES BY SPR ASSAY

Background: The World Health Organization admitted that the vaccination against Covid 19 limited the deaths, but not the spread of the disease. This requires a method allowing a specific, rapid and accurate diagnosis of the disease. We report a SPR assay that meets the requirements and can be applied no only for SARS Cov-2 diagnosis but as a tool for early diagnosis of otherinfections. Methods: Surface plasmon resonance (SPR) method was used to identify the binding of S/N protein to monoclonal antibodies. N-protein monoclonal antibody (NP mAb), S-protein monoclonal antibody (SP mAb), and receptor bind domain (RBD) antibody were used as recognition molecules. Ligands were deposited by the matrix-assisted laser evaporation (MAPLE) method, which guarantees maximum interaction specificity. Results: We registered S/N protein binding to the corresponding mAbs and S protein to RBD antibody with high sensitivity: the interactions were observed at protein concentration about 130 femtomoles (fM). A very good specificity was observed: the measured S protein binding activity to NP mAb was below the limit of detection (LOD). The same was noticed for N protein binding to SP mAb. Conclusions: The presented SPR assay possesses high sensitivity and selectivity and provides quantitative analysis. This makes it applicable for following the evolution of acute SARS-CoV-2 infection, especially at the early stages of viral replication which can be clinically useful.

N-dihydrogalactochitosan reduces mortality in a lethal mouse model of SARS-CoV-2.

The rapid emergence and global dissemination of SARS-CoV-2 that causes COVID-19 continues to cause an unprecedented global health burden resulting in nearly 7 million deaths. While multiple vaccine countermeasures have been approved for emergency use, additional treatments are still needed due to sluggish vaccine rollout, vaccine hesitancy, and inefficient vaccine-mediated protection. Immunoadjuvant compounds delivered intranasally can guide non-specific innate immune responses during the critical early stages of viral replication, reducing morbidity and mortality. N-dihydrogalactochitosan (GC) is a novel mucoadhesive immunostimulatory polymer of β-0-4-linked N-acetylglucosamine that is solubilized by the conjugation of galactose glycans with current applications as a cancer immunotherapeutic. We tested GC as a potential countermeasure for COVID-19. GC was well-tolerated and did not produce histopathologic lesions in the mouse lung. GC administered intranasally before and after SARS-CoV-2 exposure diminished morbidity and mortality in humanized ACE2 receptor expressing mice by up to 75% and reduced infectious virus levels in the upper airway. Fluorescent labeling of GC shows that it is confined to the lumen or superficial mucosa of the nasal cavity, without involvement of adjacent or deeper tissues. Our findings demonstrate a new application for soluble immunoadjuvants such as GC for preventing disease associated with SARS-CoV-2 and may be particularly attractive to persons who are needle-averse.

BMS-265246, a Cyclin-Dependent Kinase Inhibitor, Inhibits the Infection of Herpes Simplex Virus Type 1.

Herpes simplex virus type 1 (HSV-1) infections are prevalent illnesses that can cause mucocutaneous ulcerative disease, keratitis, and genital herpes. In patients with compromised immune systems, the infection can lead to serious problems, such as encephalitis. Additionally, neonatal infections can cause brain problems and even death. Current first-line antiviral drugs are nucleoside analog inhibitors that target viral polymerase, and resistant strains have emerged. As a result, new drugs with distinct action modes are needed. Recent research indicates that cyclin-dependent kinases (CDKs) are prospective antiviral targets. Thus, CDK inhibitors may be effective antiviral agents against HSV-1 infection. In this study, we examined a panel of CDK inhibitors that target CDKs in the present study. BMS-265246 (BMS), a CDK 1/2 inhibitor, was found to effectively limit HSV-1 multiplication in Vero, HepG2, and Hela cells. A mechanism of action study suggested that BMS inhibits the early stages of viral replication when added early in the viral infection. The suppression of multiple steps in viral replication by BMS was revealed when HSV-1 infected cells were treated at different time periods in the viral life cycle. Our results suggest that BMS is a potent anti-HSV-1 agent and unique in that it may interfere with multiple steps in HSV-1 replication.

Synthesis and pharmacodynamic evaluation of naphthalene derivatives against influenza A virus in vitro and in vivo

Influenza A virus is a highly mutable pathogenic pathogen that could cause a global pandemic. It is necessary to find new anti-influenza drugs to resist influenza epidemics due to the seasonal popularity of a certain area every year. Naphthalene derivatives had potential antiviral activity. A series of naphthalene derivatives were synthesized via the metal-free intramolecular hydroarylation reactions of alkynes. Evaluation of their biological efficacy showed that compound 2-aminonaphthalene 4d had better antiviral activity in vitro than ribavirin. By studying the mechanism of action of 2-aminonaphthalene 4din vivo and in vitro, we found that 4d had antiviral activity to three different subtype influenza viruses of A/Weiss/43 (H1N1), A/Virginia/ATCC2/2009 (H1N1) and A/California/2/2014 (H3N2). Compound 4d had the best effect after viral adsorption, and mainly played in the early stage of virus replication. 2-Aminonaphthalene 4d could reduce the replication of virus by inhibiting the NP and M proteins of virus. Compound 4d cut down ROS accumulation, autophagy and apoptosis induced by influenza virus. Inflammatory response mediated by RIG-1 pathway were suppressed in the cell and mice. In addition, the pathological changes of lung tissue and virus titer in mice were reduced by the administration of 4d. Therefore, naphthalene derivative 4d is a potential drug for the treatment of influenza A virus infection.

Synthesis and evaluation of alkoxy-substituted enamides against influenza A virus in vitro and in vivo

Alkoxy-substituted enamides are often used as synthetic intermediates due to their special reactivity. To the best our knowledge, the biological activity of alkoxy-substituted amines has never been reported so far. We have synthesized a series of alkoxy-substituted enamides to study their anti-influenza A virus activity in vitro and in vivo. Among these compounds, compound E-2o had the best antiviral activity (EC50 = 2.76 ± 0.67 μM) and low cytotoxicity (CC50 = 662.87 ± 24.85 μM). The mechanism of action of this compound was preliminarily explored by us. It alleviated the cytopathic effects and cell death caused by different subtypes of influenza A virus. Different drug delivery methods and timed dosing experiments had shown that E-2o had the best therapeutic effect and mainly played a role in the early stages of virus replication. The expansion of influenza viruses in cells was inhibited by reducing ROS accumulation, cell apoptosis, and autophagy. Alkoxy-substituted enamide E-2o reduced the production of interferon and other pro-inflammatory factors in the RIG-Ⅰ pathway and its downstream NF-κB was induced by influenza A virus in vitro and in vivo. It avoided damage in the mice which was caused by excessive inflammatory factors. In addition, the weight loss and lung lesion damage in mice caused by influenza virus were improved by compound E-2o. Therefore, Alkoxy-substituted enamide E-2o could inhibit the replication of influenza viruses in vivo and in vitro, and has the potential to be developed into a drug for treating influenza.

Transforming acidic coiled-coil containing protein 3 suppresses influenza A virus replication by impeding viral endosomal trafficking and nuclear import

Transforming acidic coiled-coil containing protein 3 (TACC3) is a motor spindle protein that plays an essential role in stabilization of the mitotic spindle. In this study, we show that the overexpression of TACC3 reduces the viral titers of multiple influenza A viruses (IAVs). In contrast, the downregulation of TACC3 increases IAVs propagation. Next, we map the target steps of TACC3 requirement to the early stages of viral replication. By confocal microscopy and nuclear plasma separation experiment, we reveal that overexpression of TACC3 results in a substantial decrease of IAV NP accumulation in the nuclei of infected cells. We further show that viral attachment and internalization are not affected by TACC3 overexpression and detect that the early and late endosomal trafficking of IAV in TACC3 overexpression cells is slower than negative control cells. These results suggest that TACC3 exerts an impaired effect on the endosomal trafficking and nuclear import of vRNP, thereby negatively regulating IAV replication. Moreover, the infection of different IAV subtypes decreases the expression level of TACC3 in turn. Consequently, we speculate that IAV ensures the generation of offspring virions by antagonizing the expression of inhibitory factor TACC3. Collectively, our results establish TACC3 as an important inhibitory factor for replication of the IAV, suggesting that TACC3 could be a potential target for the development of future antiviral compounds.

Capabilities of Double-Resonance LPG and SPR Methods for Hypersensitive Detection of SARS-CoV-2 Structural Proteins: A Comparative Study.

The danger of the emergence of new viral diseases and their rapid spread demands apparatuses for continuous rapid monitoring in real time. This requires the creation of new bioanalytical methods that overcome the shortcomings of existing ones and are applicable for point-of-care diagnostics. For this purpose, a variety of biosensors have been developed and tested in proof-of-concept studies, but none of them have been introduced for commercial use so far. Given the importance of the problem, in this study, long-period grating (LPG) and surface plasmon resonance (SPR) biosensors, based on antibody detection, were examined, and their capabilities for SARS-CoV-2 structural proteins detection were established. Supersensitive detections of structural proteins in the order of several femtomoles were achieved by the LPG method, while the SPR method demonstrated a sensitivity of about one hundred femtomoles. The studied biosensors are compatible in sensitivity with ELISA and rapid antigen tests but, in contrast, they are quantitative, which makes them applicable for acute SARS-CoV-2 infection detection, especially during the early stages of viral replication.

ABHD16A Negatively Regulates the Palmitoylation and Antiviral Function of IFITM Proteins.

Interferon-inducible transmembrane (IFITM) proteins are small homologous proteins that are encoded by the interferon-stimulated genes (ISGs), which can be strongly induced by interferon (IFN) and provide resistance to invasion by a variety of viral pathogens. However, the exact molecular mechanisms underlying this function have remained elusive. The antiviral activity of IFITMs from different species depends on S-palmitoylation at conserved cysteine residues. However, specific enzymes involved in the dynamic palmitoylation cycle of IFITMs, especially depalmitoylase, have not yet been reported. Here, we demonstrate that α/-hydrolase domain-containing 16A (ABHD16A) is a depalmitoylase and a negative regulator of IFITM protein that can catalyze the depalmitoyl reaction of S-palmitoylated IFITM proteins, thereby decreasing their antiviral activities on RNA viruses. Using the acyl-PEGyl exchange gel shift (APEGS) assay, we identified ABHD16A proteins from humans, pigs, and mice that can directly participate in the palmitoylation/depalmitoylation cycles of IFITMs in the constructed abhd16a-/- cells and ABHD16A-overexpressing cells. Furthermore, we showed that ABHD16A functions as a regulator of subcellular localization of IFITM proteins and is related to the immune system. It is tempting to suggest that pharmacological intervention in IFITMs and ABHD16A can be achieved either through controlling their expression or regulating their activity, thereby providing a broad-spectrum therapeutic strategy for animal viral diseases. IMPORTANCE IFITM protein is the cells first line of antiviral defense that blocks early stages of viral replication; the underlying mechanism might be associated with the proper distribution in cells. The palmitoylation/depalmitoylation cycle can dynamically regulate protein localization, stability, and function. This work is the first one that found the critical enzyme that participates in the palmitoylation/depalmitoylation cycle of IFITM, and this type of palmitoyl loss may be an essential regulation mode for balancing the antiviral functions of the IFN pathway. These findings imply that the pharmacological intervention in IFITM and ABHD16A, either through controlling their expression or regulating their activities, could provide a broad-spectrum therapeutic strategy for animal viral diseases and complications linked to interferon elevation.

Antiviral activity of Inonotusin A an active compound isolated from Boletus bellinii and Boletus subtomentosus.

Mushrooms produce various classes of secondary metabolites that could be used as antivirals in the future. The aim of this study was to determine the antiviral activity of methanolic extracts obtained from two edible mushrooms, Boletus bellinii (B. bellinii) and Boletus subtomentosus (B. subtomentosus), collected from the north forests of Tunisia, against Herpes Simplex Virus type 2 and Coxsackie Virus B type 3. In vitro micro-inhibition assays and cytotoxicity screening were performed on Vero cells. The tested Boletus methanolic extracts were found to be non-cytotoxic at high doses (50% cytotoxic concentration - CC50 > 1mg/mL) and exhibited relevant viral inhibition with 50% inhibitory concentration, i.e., IC50 of 3.60 ± 0.66µg/mL and 35.70 ± 7.42µg/mL for B. bellinii, and 5.67 ± 1.02µg/mL and 56.88 ± 9.56µg/mL for B. subtomentosus, against HSV-2 and CVB-3, respectively. Interestingly, Boletus methanolic extracts showed high selectivity index (SI) values against both viruses, with the highest values against HSV-2 (SI > 800). Both viral strains were inhibited when treated with extracts during the early stages of virus replication. Inonotusin A was isolated and identified as the compound responsible for these activities. The latter is a novel antiviral agent that may have clinical utility or serve as a lead compound for further development. This study is the first attempt to investigate the antiviral activity of inonotusin A, isolated from the genus Boletus. The information from the present work should be a valuable reference for future studies on the antiviral activity of inonotusin A.

Development of an Effective Double Antigen Sandwich ELISA Based on p30 Protein to Detect Antibodies against African Swine Fever Virus.

African swine fever (ASF), the highly lethal swine infectious disease caused by the African swine fever virus (ASFV), is a great threat to the swine industry. There is no effective vaccine or diagnostic method to prevent and control this disease currently. The p30 protein of ASFV is an important target for serological diagnosis, expressed in the early stage of viral replication and has high immunogenicity and sequence conservatism. Here, the CP204L gene was cloned into the expression vector pET-30a (+), and the soluble p30 protein was successfully expressed in the E. coli prokaryotic expression system and then labeled with horseradish peroxidase (HRP) to be the enzyme-labeled antigen. Using the purified recombinant p30 protein, a double-antigen sandwich ELISA for ASFV antibody detection was developed. This method exhibits excellent specificity, sensitivity and reproducibility in clinical sample detection with lower cost and shorter production cycles. Taken together, this study provides technical support for antibody detection for ASFV.

Styrylpyrone Derivative (SPD) Extracted from Goniothalamus umbrosus Binds to Dengue Virus Serotype-2 Envelope Protein and Inhibits Early Stage of Virus Replication.

A study was conducted to investigate the anti-viral effect of a styrylpyrone derivative (SPD) called goniothalamin and the effects on the dengue virus serotype 2 (DENV-2) replication cycle. The SPD was prepared from the root of Goniothalamus umbrosus after purification with petroleum ether. The isolated SPD was then subjected to gas chromatography–mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) analyses for structure validation. The cytotoxicity of the SPD was evaluated using a cell viability assay, while the anti-viral activity of the SPD towards DENV-2 was confirmed by conducting a foci reduction assay which involved virus yield reduction, time-of-addition, and time removal assays. Transcriptomic analysis via quantitative real-time polymerase chain reaction (qRT-PCR) using the DENV-2 E gene was conducted to investigate the level of gene transcript. Immunocytochemistry analysis was used to investigate the effects of SPD treatment on protein E expression. Finally, software molecular docking of the SPD and E protein was also performed. The cytotoxicity assay confirmed that the SPD was not toxic to Vero cells, even at the highest concentration tested. In the time-of-addition assay, more than 80% foci reduction was observed when SPDs were administered at 2 h post-infection (hpi), and the reduction percentage then dropped with the delay of the treatment time, suggesting the inhibition of the early replication cycle. However, the time removal assay showed that more than 80% reduction could only be observed after 96 h post-treatment with the SPD. Treatment with the SPD reduced the progeny infectivity when treated for 24 h and was dose-dependent. The result showed that transcript level of the E gene in infected cells treated with the SPD was reduced compared to infected cells without treatment. In immunocytochemistry analysis, the DENV-2 E protein exhibited similar expression trends, shown by the gene transcription level. Molecular docking showed that the SPD can interact with E protein through hydrogen bonds and other interactions. Overall, this study showed that SPDs have the potential to be anti-DENV-2 via a reduction in viral progeny infectivity and a reduction in the expression of the DENV-2 E gene and protein at different phases of viral replication. SPDs should be further researched to be developed into an effective anti-viral treatment, particularly for early-phase dengue viral infection.

Hypericin blocks the function of HSV-1 alkaline nuclease and suppresses viral replication

Ethnopharmacological relevanceHypericum perforatum L. has a long history in many countries of being used as a herbal medicine. It is also widely used in Chinese herbal medicine for the treatment of infections. Hypericin, a main component extracted from Hypericum perforatum L., has attracted the attention of many researchers for its remarkable antiviral, antitumor and antidepressant effects. Aim of the studyTo find plant molecules that inhibit the alkaline nuclease (AN) of herpes simplex virus type 1 (HSV-1) and suppress viral replication. Materials and methodsBioinformatics methods were used to determine which compounds from a variety of natural compounds in our laboratory interact with AN. By this means we predicted that hypericin may interact with AN and suppress HSV-1 replication. Experiments were then carried out to verify whether hypericin inhibits the bioactivity of AN. The Pichia pastoris expression system was used to obtain recombinant AN. The exonuclease and endonuclease activity of AN treated with hypericin were tested by electrophoresis. Immunohistochemical staining of the HSV-1 nucleocapsids was used to find out whether hypericin inhibits the intracellular function of AN. Real-time PCR and western blotting analysis were performed to test viral gene expression and viral protein synthesis. The extent of viral replication inhibited by hypericin was determined by a plaque assay and a time of addition assay. ResultsRecombinant AN was obtained by Pichia pastoris expression system. The exonuclease and endonuclease activity of recombinant AN were inhibited by hypericin in the electrophoresis assay. Hypericin showed no inhibitory effect on BeyoZonase™ Super Nuclease or DNase I. T5 Exonuclease activity was inhibited partially by10 μM hypericin, and was completely suppressed by 50 μM hypericin. Hind Ⅲ was inhibited by hypericin at concentrations greater than 100 μM, but EcoR I, BamH I, and Sal I were not inhibited by hypericin. HSV-1 nucleocapsids gathered in the nucleus when the viruses were treated with hypericin. Plaque formation was significantly reduced by hypericin (EC50 against HSV-1 F is 2.59 ± 0.08 μM and EC50 against HSV-1 SM44 is 2.94 ± 0.10 μM). UL12, ICP27, ICP8, gD, and UL53 gene expression (P < 0.01, 4.0 μM hypericin treated group vs control group) and ICP4 (P < 0.05, 6.0 μM hypericin treated group vs control group), ICP8 and gD (P < 0.05, 2.0 μM hypericin treated group vs control group) protein synthesis were inhibited by hypericin. In the time of addition assay, HSV-1 was suppressed by hypericin in the early stages of viral replication. Hypericin exhibits potent virucidal activity against HSV-1 and inhibits the adsorption and penetration of HSV-1. ConclusionHypericin inhibits the bioactivity of AN and suppresses HSV-1 replication. The data revealed a novel mechanism of the antiherpetic effect of hypericin.

Borneol Ester Derivatives as Entry Inhibitors of a Wide Spectrum of SARS-CoV-2 Viruses.

In the present work we studied the antiviral activity of the home library of monoterpenoid derivatives using the pseudoviral systems of our development, which have glycoproteins of the SARS-CoV-2 virus strains Wuhan and Delta on their surface. We found that borneol derivatives with a tertiary nitrogen atom can exhibit activity at the early stages of viral replication. In order to search for potential binding sites of ligands with glycoprotein, we carried out additional biological tests to study the inhibition of the re-receptor-binding domain of protein S. For the compounds that showed activity on the pseudoviral system, a study using three strains of the infectious SARS-CoV-2 virus was carried out. As a result, two leader compounds were found that showed activity on the Wuhan, Delta, and Omicron strains. Based on the biological results, we searched for the potential binding site of the leader compounds using molecular dynamics and molecular docking methods. We suggested that the compounds can bind in conserved regions of the central helices and/or heptad repeats of glycoprotein S of SARS-CoV-2 viruses.