Murine Trophoblast Stem Cells and Their Differentiated Cells Attenuate Zika Virus In Vitro by Reducing Glycosylation of the Viral Envelope Protein

Zika virus (ZIKV) infection during pregnancy can cause devastating fetal neuropathological abnormalities, including microcephaly. Most studies of ZIKV infection in pregnancy have focused on post-implantation stage embryos. Currently, we have limited knowledge about how a pre-implantation stage embryo deals with a viral infection. This study investigates ZIKV infection on mouse trophoblast stem cells (TSCs) and their in vitro differentiated TSCs (DTSCs), which resemble the cellular components of the trophectoderm layer of the blastocyst that later develops into the placenta. We demonstrate that TSCs and DTSCs are permissive to ZIKV infection; however, ZIKV propagated in TSCs and DTSCs exhibit substantially lower infectivity, as shown in vitro and in a mouse model compared to ZIKV that was generated in Vero cells or mouse embryonic fibroblasts (MEFs). We further show that the low infectivity of ZIKV propagated in TSCs and DTSCs is associated with a reduced level of glycosylation on the viral envelope (E) proteins, which are essential for ZIKV to establish initial attachment by binding to cell surface glycosaminoglycans (GAGs). The decreased level of glycosylation on ZIKV E is, at least, partially due to the low-level expression of a glycosylation-related gene, Hexa, in TSCs and DTSCs. Furthermore, this finding is not limited to ZIKV since similar observations have been made as to the chikungunya virus (CHIKV) and West Nile virus (WNV) propagated in TSCs and DTSCs. In conclusion, our results reveal a novel phenomenon suggesting that murine TSCs and their differentiated cells may have adapted a cellular glycosylation system that can limit viral infectivity by altering the glycosylation of viral envelope proteins, therefore serving as a unique, innate anti-viral mechanism in the pre-implantation stage embryo.

Rationale for endomyocardial biopsy in the diagnosis of heart disease in children and adults

Endomyocardial biopsy (EMB) is the method of choice for diagnosing a wide range of myocardial diseases.Aim. To assess the rationale for diagnostic EMB in children and adults.Material and methods. Morphological and statistical analysis of 2803 diagnostic EMBs in adults (n=811) and children (n=83), including those in heart transplantation (n=1909), was carried out.Results. In 231 (28%) cases, adults were diagnosed with myocarditis, of which in 6 patients — granulomatous, in 5 — eosinophilic and in 6 — lymphocytic-macrophage myocarditis after coronavirus infection. In children, myocarditis was found in 22 cases (27%). Arrhythmogenic right ventricular dysplasia took the second place in detection rate in children and adults. Immunohistochemical study revealed viral envelope protein 1 (VP1) antigen of enteroviruses in one third of myocarditis cases, and in half — other cardiotropic viruses. Dotted dystrophin expression was observed in myocarditis. A correlation was established between the perforin expression and myocarditis presence (Pearson χ2=27,8; Fisher's exact test=27,3; p=0,01).Conclusion. Analysis of diagnostic EMB results confirmed its rationale in adults and children not only for heart transplantation, but also for identifying cardiac pathology, including for myocarditis diagnosis. It has been shown that immunohistochemical study with antiviral antibodies can be considered as an alternative method for detecting viral infection. An immunohistochemical analysis for perforin and dystrophin can be recommended as additional morphological markers of myocarditis.

One mucosal administration of a live attenuated recombinant COVID-19 vaccine protects non-human primates from SARS-CoV-2

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 global pandemic. Vaccines are needed to control the disease and bring an end to the pandemic. SARS-CoV-2 is an enveloped RNA virus that relies on its trimeric surface glycoprotein, spike, for entry into host cells. Here we describe the COVID-19 vaccine candidate MV-014-212, a live attenuated, recombinant human respiratory syncytial virus (RSV) expressing a chimeric SARS-CoV-2 spike as the only viral envelope protein. MV-014-212 was attenuated and immunogenic in African green monkeys (AGMs). One mucosal administration of MV-014-212 in AGMs protected against SARS-CoV-2 challenge, reducing the peak shedding of SARS-CoV-2 in the nose by more than 200-fold. MV-014-212 elicited mucosal immunity in the nose and neutralizing antibodies in serum that exhibited cross neutralization against two virus variants of concern. Intranasally delivered, live attenuated vaccines such as MV-014-212 entail low-cost manufacturing suitable for global deployment. MV-014-212 is currently in phase I clinical trials as a single-dose intranasal COVID-19 vaccine.

HIV envelope antigen valency on peptide nanofibers modulates antibody magnitude and binding breadth

A major challenge in developing an effective vaccine against HIV-1 is the genetic diversity of its viral envelope. Because of the broad range of sequences exhibited by HIV-1 strains, protective antibodies must be able to bind and neutralize a widely mutated viral envelope protein. No vaccine has yet been designed which induces broadly neutralizing or protective immune responses against HIV in humans. Nanomaterial-based vaccines have shown the ability to generate antibody and cellular immune responses of increased breadth and neutralization potency. Thus, we have developed supramolecular nanofiber-based immunogens bearing the HIV gp120 envelope glycoprotein. These immunogens generated antibody responses that had increased magnitude and binding breadth compared to soluble gp120. By varying gp120 density on nanofibers, we determined that increased antigen valency was associated with increased antibody magnitude and germinal center responses. This study presents a proof-of-concept for a nanofiber vaccine platform generating broad, high binding antibody responses against the HIV-1 envelope glycoprotein.

Glyceraldehyde 3 phosphate dehydrogenase restricted in cytoplasmic location by viral GP5 facilitates PRRSV replication via its glycolytic activity

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important endemic swine pathogens, causing enormous losses in the global swine industry. Commercially available vaccines only partially prevent or counteract the virus infection and correlated losses. PRRSV replication mechanism has not been well understood. In this study, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was screened to bind with the viral major envelope glycoprotein 5 (GP5) after PRRSV infection. The interacting sites are located within a 13 amino acid region (93 105 aa) of GP5 and at Lys227 of GAPDH. Interestingly, viral GP5 restricts the translocation of GAPDH from the cytoplasm to the nucleus. Moreover, cytoplasmic GAPDH facilitates PRRSV replication by virtue of its glycolytic activity. The results suggest that PRRSV GP5 restricts GAPDH into the nucleus and exploits its glycolytic activity to stimulate virus replication. The data provide insight into the role of GAPDH in PRRSV replication and reveal a potential target for controlling viral infection. Importance PRRSV poses a severe economic threat to the pig industry. PRRSV GP5, the major viral envelope protein, plays an important role in viral infection, pathogenicity, and immunity. However, interactions between GP5 and host proteins have not yet been well studied. Here, we show that GAPDH interacts with GP5 through binding a 13-amino-acid sequence (93–105 aa) in GP5, while GP5 interacts with GAPDH at the K277 amino acid residue of GAPDH. We demonstrate that GP5 interacts with GAPDH in the cytoplasm during PPRSV infection, inhibiting GAPDH entry into the nucleus. PRRSV exploits the glycolytic activity of GAPDH to promote viral replication. These results enrich our understanding of PRRSV infection and pathogenesis, and open a new avenue for antiviral prevention and PRRSV treatment strategies.

SARS-CoV-2 envelope-protein corruption of homeostatic signaling mechanisms in mammalian cells

During a SARS-CoV2 infection, host cells produce large amounts of the viral envelope protein (Ep-CoV2). Ep-CoV2 is partially inserted into the membrane of nascent viral particles and into cellular membranes. To mimic the pathophysiological impact of the cellular protein fraction, Ep-CoV2 was overexpressed in mammalian cells and effects on key signaling parameters were monitored. By tagging with green fluorescent protein (GFP), we found that Ep-CoV2 protein is mostly present in the endoplasmic reticulum with additional trace amounts in the plasma membrane. We observed that wild-type Ep-CoV2 and, to a lesser extent, its mutants (N15A, V25F) corrupted some of the most important homeostatic mechanisms in cells. The same was observed with isolated transmembrane domains of the protein. The Ep-CoV2-evoked elevation of intracellular Ca2+ and pH as well as the induced membrane depolarization produced by the presence of the protein interfere with major signal transduction cascades in host cells. These functions of Ep-CoV2, which likely contribute to the pathogenesis of the viral protein, result from the ion-channel activity of the viral protein. Two independent assays, a functional reconstitution of Ep-CoV2 protein in artificial membranes and a rescue of K+-deficient yeast mutants, confirm that Ep-CoV2 generates a cation-conducting channel with a low unitary conductance and a complex ion selectivity. The data presented here suggest that specific channel function inhibitors of Ep-CoV2 can provide cell protection and virostatic effects.

Macrocyclic peptides exhibit antiviral effects against influenza virus HA and prevent pneumonia in animal models

Most anti-influenza drugs currently used, such as oseltamivir and zanamivir, inhibit the enzymatic activity of neuraminidase. However, neuraminidase inhibitor-resistant viruses have already been identified from various influenza virus isolates. Here, we report the development of a class of macrocyclic peptides that bind the influenza viral envelope protein hemagglutinin, named iHA. Of 28 iHAs examined, iHA-24 and iHA-100 have inhibitory effects on the in vitro replication of a wide range of Group 1 influenza viruses. In particular, iHA-100 bifunctionally inhibits hemagglutinin-mediated adsorption and membrane fusion through binding to the stalk domain of hemagglutinin. Moreover, iHA-100 shows powerful efficacy in inhibiting the growth of highly pathogenic influenza viruses and preventing severe pneumonia at later stages of infection in mouse and non-human primate cynomolgus macaque models. This study shows the potential for developing cyclic peptides that can be produced more efficiently than antibodies and have multiple functions as next-generation, mid-sized biomolecules.

Sinapic Acid Suppresses SARS CoV-2 Replication by Targeting Its Envelope Protein

SARS CoV-2 is still considered a global health issue, and its threat keeps growing with the emergence of newly evolved strains. Despite the success in developing some vaccines as a protective measure, finding cost-effective treatments is urgent. Accordingly, we screened a number of phenolic natural compounds for their in vitro anti-SARS CoV-2 activity. We found sinapic acid (SA) selectively inhibited the viral replication in vitro with an half-maximal inhibitory concentration (IC50) value of 2.69 µg/mL with significantly low cytotoxicity (CC50 = 189.3 µg/mL). Subsequently, we virtually screened all currently available molecular targets using a multistep in silico protocol to find out the most probable molecular target that mediates this compound’s antiviral activity. As a result, the viral envelope protein (E-protein) was suggested as the most possible hit for SA. Further in-depth molecular dynamic simulation-based investigation revealed the essential structural features of SA antiviral activity and its binding mode with E-protein. The structural and experimental results presented in this study strongly recommend SA as a promising structural motif for anti-SARS CoV-2 agent development.

Altered O-glycosylation Level of SARS-CoV-2 Spike Protein by Host O-glycosyltransferase Strengthens Its Trimeric Structure

SummaryThe trimeric spike protein (S) mediates host-cell entry and membrane fusion of SARS-CoV-2. S protein is highly glycosylated, whereas its O-glycosylation is still poorly understood. Herein, we site-specifically examine the O-glycosylation of S protein through a mass spectrometric approach with HCD-triggered-ETD model. We identify 15 high-confidence O-glycosites and at least 10 distinct O-glycan structures on S protein. Peptide microarray assays prove that human ppGalNAc-T6 actively participates in O-glycosylation of S protein. Importantly, the upregulation of ppGalNAc-T6 expression can profoundly enhance the O-glycosylation level by generating new O-glycosites and increasing both O-glycan heterogeneity and intensities. Further molecular dynamics simulations reveal that the O-glycosylation on the protomer-interface regions, which are mainly modified by ppGalNAc-T6, can potentially stabilize the trimeric S protein structure. Our work provides deep molecular insights of how viral infection harnesses the host O-glycosyltransferases to dynamically regulate the O-glycosylation level of the viral envelope protein responsible for membrane fusion.

Molecular Changes in Dengue Envelope Protein Domain III upon Interaction with Glycosaminoglycans

Dengue fever is a rapidly emerging vector-borne viral disease with a growing global burden of approximately 390 million new infections per annum. The Dengue virus (DENV) is a flavivirus spread by female mosquitos of the aedes genus, but the mechanism of viral endocytosis is poorly understood at a molecular level, preventing the development of effective transmission blocking vaccines (TBVs). Recently, glycosaminoglycans (GAGs) have been identified as playing a role during initial viral attachment through interaction with the third domain of the viral envelope protein (EDIII). Here, we report a systematic study investigating the effect of a range of biologically relevant GAGs on the structure and oligomeric state of recombinantly generated EDIII. We provide novel in situ biophysical evidence that heparin and chondroitin sulphate C induce conformational changes in EDIII at the secondary structure level. Furthermore, we report the ability of chondroitin sulphate C to bind EDIII and induce higher-order dynamic molecular changes at the tertiary and quaternary structure levels which are dependent on pH, GAG species, and the GAG sulphation state. Lastly, we conducted ab initio modelling of Small Angle Neutron Scattering (SANS) data to visualise the induced oligomeric state of EDIII caused by interaction with chondroitin sulphate C, which may aid in TBV development.