Viral Membrane Proteins Research Articles

Differential Severe Acute Respiratory Syndrome Coronavirus 2-Specific Humoral Response in Inactivated Virus-Vaccinated, Convalescent, and Breakthrough-Infected Subjects.

We sought to identify potential antigens for discerning between humoral responses elicited after vaccination with CoronaVac (a severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] inactivated vaccine), natural infection, or breakthrough infection. Serum samples obtained from volunteers immunized with CoronaVac (2 and 3 doses), breakthrough case patients, and from convalescent individuals were analyzed to determine the immunoglobulin (Ig) G responses against 3 structural and 8 nonstructural SARS-CoV-2 antigens. Immunization with CoronaVac induced higher levels of antibodies against the viral membrane (M) protein compared with convalescent subjects both after primary vaccination and after a booster dose. Individuals receiving a booster dose displayed equivalent levels of IgG antibodies against the nucleocapsid (N) protein, similar to convalescent subjects. Breakthrough case patients produced the highest antibody levels against the N and M proteins. Antibodies against nonstructural viral proteins were present in >50% of the convalescent subjects. Vaccinated individuals elicited a different humoral response compared to convalescent subjects. The analysis of particular SARS-CoV-2 antigens could be used as biomarkers for determining infection in subjects previously vaccinated with CoronaVac.

Optimization of infectious bronchitis virus-like particle expression in Nicotiana benthamiana as potential poultry vaccines.

Infectious bronchitis (IB) is a highly contagious, acute respiratory disease in chickens, with a severe economic impact on poultry production globally. The rapid emergence of regional variants of this Gammacoronavirus warrants new vaccine approaches that are more humane and rapid to produce than the current embryonated chicken egg-based method used for IB variant vaccine propagation (chemically-inactivated whole viruses). The production of virus-like particles (VLPs) expressing the Spike (S) glycoprotein, the major antigen which induces neutralizing antibodies, has not been achieved in planta up until now. In this study, using the Agrobacterium-mediated Nicotiana benthamiana (tobacco plant) transient expression system, the highest levels of VLPs displaying a modified S protein of a QX-like IB variant were obtained when the native transmembrane (TM) domain and cytoplasmic tail were substituted with that of the Newcastle disease virus (NDV) fusion glycoprotein, co-infiltrated with the NDV Matrix protein. In comparison, the native IB modified S co-infiltrated with IB virus membrane, envelope and nucleocapsid proteins, or substituted with the TM and CT of an H6-subtype influenza A virus hemagglutinin glycoprotein yielded lower VLP expression levels. Strong immunogenicity was confirmed in specific pathogen free chickens immunized intramuscularly with VLPs adjuvanted with Emulsigen®-P, where birds that received doses of 5 μg or 20 μg (S protein content) seroconverted after two weeks with mean hemaggluttination inhibition titres of 9.1 and 10 log2, respectively. Plant-produced IB VLP variant vaccines are safer, more rapid and cost effective to produce than VLPs produced in insect cell expression systems or the traditional egg-produced inactivated whole virus oil emulsion vaccines currently in use, with great potential for improved IB disease control in future.

Vaccination against Epstein-Barr Latent Membrane Protein 1 Protects against an Epstein-Barr Virus-Associated B Cell Model of Lymphoma.

In this study, we demonstrate that expression of viral latent membrane protein 1 (LMP1) in a mouse B cell line renders the animals responsive to protection from a 38C13-LMP1 tumor challenge with a novel vaccine. The Epstein-Barr virus (EBV) preferentially infects circulating B lymphocytes, has oncogenic potential, and is associated with a wide variety of B cell lymphomas. EBV is ectotrophic to human cells, and currently there are no B cell animal models of EBV-associated lymphoma that can be used to investigate vaccine immunotherapy. Since most EBV-infected human tumor cells express latent membrane protein 1 (LMP1) on their surface, this viral antigen was tested as a potential target for an anticancer vaccine in a mouse model. Here, we describe a new mouse model of LMP1-expressing B cell lymphoma produced with plasmid transduction of 38C13 into mouse B cells. The expression of LMP-1 was confirmed with a western blot analysis and immunocytochemistry. We then designed a novel LMP1 vaccine, by fusing viral antigen LMP1 surface loop epitopes to the surface of a viral antigen carrier, the Tobacco Mosaic virus (TMV). Vaccinated mice produced high titer antibodies against the TMV-LMP1 vaccine; however, cellular responses were at the baseline, as measured with IFNγ ELISpot. Despite this, the vaccine showed significant protection from a 38C13-LMP1 tumor challenge. To provide additional immune targets, we compared TMV-LMP1 peptide immunization with DNA immunization with the full-length LMP1 gene. Anti-LMP1 antibodies were significantly higher in TMV-LMP1-vaccinated mice compared to the DNA-immunized mice, but, as predicted, DNA-vaccinated mice had improved cellular responses using IFNγ ELISpot. Surprisingly, the TMV-LMP1 vaccine provided protection from a 38C13-LMP1 tumor challenge, while the DNA vaccine did not. Thus, we demonstrated that LMP1 expression in a mouse B cell line is responsive to antibody immunotherapy that may be applied to EBV-associated disease.

No Evidence for Myocarditis or Other Organ Affection by Induction of an Immune Response against Critical SARS-CoV-2 Protein Epitopes in a Mouse Model Susceptible for Autoimmunity.

After Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) developed into a global pandemic, not only the infection itself but also several immune-mediated side effects led to additional consequences. Immune reactions such as epitope spreading and cross-reactivity may also play a role in the development of long-COVID, although the exact pathomechanisms have not yet been elucidated. Infection with SARS-CoV-2 can not only cause direct damage to the lungs but can also lead to secondary indirect organ damage (e.g., myocardial involvement), which is often associated with high mortality. To investigate whether an immune reaction against the viral peptides can lead to organ affection, a mouse strain known to be susceptible to the development of autoimmune diseases, such as experimental autoimmune myocarditis (EAM), was used. First, the mice were immunized with single or pooled peptide sequences of the virus's spike (SP), membrane (MP), nucleocapsid (NP), and envelope protein (EP), then the heart and other organs such as the liver, kidney, lung, intestine, and muscle were examined for signs of inflammation or other damage. Our results showed no significant inflammation or signs of pathology in any of these organs as a result of the immunization with these different viral protein sequences. In summary, immunization with different SARS-CoV-2 spike-, membrane-, nucleocapsid-, and envelope-protein peptides does not significantly affect the heart or other organ systems adversely, even when using a highly susceptible mouse strain for experimental autoimmune diseases. This suggests that inducing an immune reaction against these peptides of the SARS-CoV-2 virus alone is not sufficient to cause inflammation and/or dysfunction of the myocardium or other studied organs.

Nanomaterials as Theragnostic Tools of Detection and Fighting off the Virus

Abstract: The primary cause of death worldwide is an infectious disease, and viruses in particular, have a major worldwide impact on health and economic growth. A critical public health problem is the quick emergence of medication resistance to currently accessible treatments as well as negative side effects from repeated use. Therefore, the creation of novel treatment plans is necessary. In contrast to conventional chemical-based antiviral drugs, the use of nanoparticles offers an alluring potential for the development of innovative antiviral therapies with minimal risk of acquiring drug resistance. Nanomaterials make it simple to change the properties of accessible resources. Nanomaterials can be readily applied to modify the available detection platforms to improve their sensitivity. Due to their high surface-to-volume ratio, NPs readily bind to either the viral membrane proteins or the viral reproduction system (DNA/RNA), thus hindering the virus' ability to infect cells. Viral proliferation has been inhibited by the use of metal nanoparticles (NPs) such as gold, silver, and copper. Copper has repeatedly been examined for its strong anti-microbial qualities due to its lower cost than silver and gold. The virus's inactivation on copper surfaces after 4 hours raises the possibility that copper could be used to destroy the infection. Also, it was discovered that 50% of virus-like particles (VLPs) could be rendered inactive by copper composite nanoparticles in just 10 minutes. The medical community could benefit greatly from sophisticated smartphone and/or LFA-based detection techniques that can be used right at the patient's bedside. This review discusses methods based on nanotechnology for diagnosing and treating viral diseases, especially HIV and influenza.

SARS-CoV-2 infection causes periodontal fibrotic pathogenesis through deregulating mitochondrial beta-oxidation

The global high prevalence of COVID-19 is a major challenge for health professionals and patients. SARS-CoV-2 virus has four structural protein components: the spike protein, envelope protein, membrane protein, and nucleocapsid protein. The SARS-CoV-2 virus mutates predominantly in the spike proteins, whilst the other key viral components usually remain stable. Essentially the pathological functions of the SARS-CoV-2 virus on different cell types are still largely unknown. Previous studies have shown that the human oral cavity can potentially act as reservoir of the SARS-CoV-2 virus. However, the consequence of SARS-CoV-2 viral infection on human oral health has not been systematically examined. COVID-19 can cause severe oral mucosa lesions and is likely to be connected with poor periodontal conditions. Fibroblasts are the major cell type inside periodontal ligament (PDL) and express the SARS-CoV-2 receptor: Angiotensin-converting enzyme 2 (ACE2), whose expression level can increase upon bacterial infection hence potentially provide a direct route of SARS-CoV-2 infection to PDL fibroblasts. In this research, we aimed to study the pathogenicity of SARS-CoV-2 viral components on human fibroblasts. We found that by exposing to SARS-CoV-2, especially to the viral envelope and membrane proteins, the human periodontal fibroblasts could develop fibrotic pathogenic phenotypes, including hyperproliferation that was simultaneously induced with increased apoptosis and senescence. The fibrotic degeneration was mediated by a down-regulation of mitochondrial β-oxidation in the fibroblasts. Fatty acid β-oxidation inhibitor, etomoxir treatment could mirror the same pathological consequence on the cells, similar to SARS-CoV-2 infection. Our results therefore provide novel mechanistic insights into how SARS-CoV-2 infection can affect human periodontal health at the cell and molecular level with potential new therapeutic targets for COVID-19 induced fibrosis.

Reticulons promote formation of ER-derived double-membrane vesicles that facilitate SARS-CoV-2 replication.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the etiologic agent for the global COVID-19 pandemic, triggers the formation of endoplasmic reticulum (ER)-derived replication organelles, including double-membrane vesicles (DMVs), in the host cell to support viral replication. Here, we clarify how SARS-CoV-2 hijacks host factors to construct the DMVs. We show that the ER morphogenic proteins reticulon-3 (RTN3) and RTN4 help drive DMV formation, enabling viral replication, which leads to productive infection. Different SARS-CoV-2 variants, including the delta variant, use the RTN-dependent pathway to promote infection. Mechanistically, our results reveal that the membrane-embedded reticulon homology domain (RHD) of the RTNs is sufficient to functionally support viral replication and physically engage NSP3 and NSP4, two viral non-structural membrane proteins known to induce DMV formation. Our findings thus identify the ER morphogenic RTN3 and RTN4 membrane proteins as host factors that help promote the biogenesis of SARS-CoV-2-induced DMVs, which can act as viral replication platforms.

MARCO Inhibits Porcine Reproductive and Respiratory Syndrome Virus Infection through Intensifying Viral GP5-Induced Apoptosis.

Studying viral glycoprotein-host membrane protein interactions contributes to the discovery of novel cell receptors or entry facilitators for viruses. Glycoprotein 5 (GP5), which is a major envelope protein of porcine reproductive and respiratory syndrome virus (PRRSV) virions, is a key target for the control of the virus. Here, the macrophage receptor with collagenous structure (MARCO), which is a member of the scavenger receptor family, was identified as one of the host interactors of GP5 through a DUALmembrane yeast two-hybrid screening. MARCO was specifically expressed on porcine alveolar macrophages (PAMs), and PRRSV infection downregulated MARCO expression both in vitro and in vivo. MARCO was not involved in viral adsorption and internalization processes, indicating that MARCO may not be a PRRSV-entry facilitator. Contrarily, MARCO served as a host restriction factor for PRRSV. The knockdown of MARCO in PAMs enhanced PRRSV proliferation, whereas overexpression suppressed viral proliferation. The N-terminal cytoplasmic region of MARCO was responsible for its inhibitory effect on PRRSV. Further, we found that MARCO was a proapoptotic factor in PRRSV-infected PAMs. MARCO knockdown weakened virus-induced apoptosis, whereas overexpression aggravated apoptosis. MARCO aggravated GP5-induced apoptosis, which may result in its proapoptotic function in PAMs. The interaction between MARCO and GP5 may contribute to the intensified apoptosis induced by GP5. Additionally, the inhibition of apoptosis during PRRSV infection weakened the antiviral function of MARCO, suggesting that MARCO inhibits PRRSV through the regulation of apoptosis. Taken together, the results of this study reveal a novel antiviral mechanism of MARCO and suggest a molecular basis for the potential development of therapeutics against PRRSV. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) has been one of the most serious threats to the global swine industry. Glycoprotein 5 (GP5) exposed on the surface of PRRSV virions is a major glycoprotein, and it is involved in viral entry into host cells. A macrophage receptor with collagenous structure (MARCO), which is a member of the scavenger receptor family, was identified to interact with PRRSV GP5 in a DUALmembrane yeast two-hybrid screening. Further investigation demonstrated that MARCO may not serve as a potential receptor to mediate PRRSV entry. Instead, MARCO was a host restriction factor for the virus, and the N-terminal cytoplasmic region of MARCO was responsible for its anti-PRRSV effect. Mechanistically, MARCO inhibited PRRSV infection through intensifying virus-induced apoptosis in PAMs. The interaction between MARCO and GP5 may contribute to GP5-induced apoptosis. Our work reveals a novel antiviral mechanism of MARCO and advances the development of control strategies for the virus.

In silico discovery of potent inhibitors against monkeypox’s major structural proteins

Monkeypox virus (MPXV) outbreak in non-endemic countries is a worldwide public health emergency. An enveloped double-stranded DNA virus belongs to the genus Orth poxvirus. A viral zoonotic infection known as monkeypox has been a serious risk to public health, especially in Africa. However, it has recently spread to other continents, so it might soon become a worldwide problem. There is an increased risk of transmission of the virus because there is a lack of effective treatment that cures the disease. To stop the multi-country outbreak from spreading, it is important to discover effective medications urgently. The objective of the current study is to swiftly find new treatments for the monkeypox virus using advanced computational approaches. By investigating five potential MPXV targets (DNA ligase, Palmytilated Extracellular Enveloped Virus (EEV) membrane protein, Scaffold protein D13, Thymidylate Kinase, and Viral core cysteine proteinase), this research was carried out using cutting-edge computational techniques against human monkeypox virus infection. Here we present the accurate 3D structures and their binding cavities of the selected targets with higher confidence using AlphaFold 2 and SiteMap analysis. Molecular docking and MD simulation analysis revealed the top five potential lead compounds with higher binding affinity and stability toward selected targets. Binding free energy calculations and other essential dynamics analysis supports the finding. The selected lead compounds utilizing virtual screening and drug repurposing approach reported in this study are beneficial for medical scientists and experimental biologists in drug development for the treatment of human MPXV.Communicated by Ramaswamy H. Sarma

Displaying and delivering viral membrane antigens via WW domain-activated extracellular vesicles.

Membrane proteins expressed on the surface of enveloped viruses are conformational antigens readily recognized by B cells of the immune system. An effective vaccine would require the synthesis and delivery of these native conformational antigens in lipid membranes that preserve specific epitope structures. We have created an extracellular vesicle-based technology that allows viral membrane antigens to be selectively recruited onto the surface of WW domain-activated extracellular vesicles (WAEVs). Budding of WAEVs requires secretory carrier-associated membrane protein 3, which through its proline-proline-alanine-tyrosine motif interacts with WW domains to recruit fused viral membrane antigens onto WAEVs. Immunization with influenza and HIV viral membrane proteins displayed on WAEVs elicits production of virus-specific neutralizing antibodies and, in the case of influenza antigens, protects mice from the lethal viral infection. WAEVs thus represent a versatile platform for presenting and delivering membrane antigens as vaccines against influenza, HIV, and potentially many other viral pathogens.

Valproate-coenzyme A conjugate blocks opening of receptor binding domains in the spike trimer of SARS-CoV-2 through an allosteric mechanism

The receptor-binding domains (RBDs) of the SARS-CoV-2 spike trimer exhibit "up" and "down" conformations often targeted by neutralizing antibodies. Only in the “up” configuration can RBDs bind to the ACE2 receptor of the host cell and initiate the process of viral multiplication. Here, we identify a lead compound (3-oxo-valproate-coenzyme A conjugate or Val-CoA) that stabilizes the spike trimer with RBDs in the down conformation. Val-CoA interacts with three R408 residues, one from each RBD, which significantly reduces the inter-subunit R408-R408 distance by ∼ 13 Å and closes the central pore formed by the three RBDs. Experimental evidence is presented that R408 is part of a triggering mechanism that controls the prefusion to postfusion state transition of the spike trimer. By stabilizing the RBDs in the down configuration, this and other related compounds can likely attenuate viral transmission. The reported findings for binding of Val-CoA to the spike trimer suggest a new approach for the design of allosteric antiviral drugs that do not have to compete for specific virus-receptor interactions but instead hinder the conformational motion of viral membrane proteins essential for interaction with the host cell. Here, we introduce an approach to target the spike protein by identifying lead compounds that stabilize the RBDs in the trimeric “down” configuration. When these compounds trimerize monomeric RBD immunogens as co-immunogens, they could also induce new types of non-ACE2 blocking antibodies that prevent local cell-to-cell transmission of the virus, providing a novel approach for inhibition of SARS-CoV-2.

FEATURES OF THE COURSE OF COVID-19 IN WOMEN WITH HYPERPROLACTINEMIA

The 2019 coronavirus pandemic is a major stressful event. In turn, stressful situations are triggers for the exacerbation of existing and activation of new diseases caused by a violation of the immune response in the human body and the development of inflammatory diseases. A stressful state causes the release of stress-mediated hormones, which in turn contributes to an increase in the level of PRL. Obviously, it is a closed circle. Therefore, the questions become relevant: 1. Can stress against the background of Covid-19 really cause an increase in the level of PRL? 2. Can Sovid-19 worsen the course of an already existing HP? Purpose: to conduct an analysis of available medical research on the peculiarities of the course of Covid-19 in women with HP, highlighting research on this topic in order to systematize new views on the diagnosis and treatment of Covid-19 against the background of already existing HP. Materials and methods. 10 articles from 2018 to 2022 with the keywords prolactin, hyperprolactinemia and Covid-19 were analyzed. Research results and their discussion. A review and analysis of a closed cohort study in which 30 men and 15 women participated and passed the Trier social stress test showed that PRL levels were significantly increased in response to a stressor [10], which could be the Covid-19 pandemic. In SARS-CoV-2, the defined receptor binding domain of the viral membrane protein uses angiotensin-converting enzyme 2 (ACE2) as a receptor for virus-host membrane fusion [2]. ACE2 regulates the activity of dopamine decarboxylase (DDC), which is responsible for the synthesis of dopamine and serotonin. Thus, SARS-CoV-2 disrupts the biosynthesis of dopamine, which leads to an increase in the level of PRL. With Covid-19, the expression of TLR4 and NF-κB is activated and increases the induction release of pro-inflammatory cytokines with the development of ALI, ARDS and cytokines with MOF. Conclusions Based on the studies described above, it can be concluded that Covid-19 inhibits the synthesis of dopamine and thus increases the level of prolactin. Also, prolactin can exhibit both
 immunostimulating and immunosuppressive, as well as pro-inflammatory and anti-inflammatory effects. Treatment with agonists of dopamine receptors can lead to deepening of depression in Covid-19. Treatment of hyperprolactinemia should be carried out with caution, because in critically ill patients who require personal support, the use of amine derivatives with DRA can cause additional vasospasm and, as a result, a rapid increase in blood pressure.

Immunoinformatics-Aided Design of a Peptide Based Multiepitope Vaccine Targeting Glycoproteins and Membrane Proteins against Monkeypox Virus.

Monkeypox is a self-limiting zoonotic viral disease and causes smallpox-like symptoms. The disease has a case fatality ratio of 3-6% and, recently, a multi-country outbreak of the disease has occurred. The currently available vaccines that have provided immunization against monkeypox are classified as live attenuated vaccinia virus-based vaccines, which pose challenges of safety and efficacy in chronic infections. In this study, we have used an immunoinformatics-aided design of a multi-epitope vaccine (MEV) candidate by targeting monkeypox virus (MPXV) glycoproteins and membrane proteins. From these proteins, seven epitopes (two T-helper cell epitopes, four T-cytotoxic cell epitopes and one linear B cell epitopes) were finally selected and predicted as antigenic, non-allergic, interferon-γ activating and non-toxic. These epitopes were linked to adjuvants to design a non-allergic and antigenic candidate MPXV-MEV. Further, molecular docking and molecular dynamics simulations predicted stable interactions between predicted MEV and human receptor TLR5. Finally, the immune-simulation analysis showed that the candidate MPXV-MEV could elicit a human immune response. The results obtained from these in silico experiments are promising but require further validation through additional in vivo experiments.

An improved Fuzzy based GWO algorithm for predicting the potential host receptor of COVID-19 infection

Coronavirus disease (COVID-19) is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and has infected millions worldwide. SARS-CoV-2 spike protein uses Angiotensin-converting enzyme 2 (ACE2) and Transmembrane serine protease 2 (TMPRSS2) for entering and fusing the host cell membrane. However, interaction with spike protein receptors and protease processing are not the only factors determining coronaviruses’ entry. Several proteases mediate the entry of SARS-CoV-2 virus into the host cell. Identifying receptor factors helps understand tropism, transmission, and pathogenesis of COVID-19 infection in humans. The paper aims to identify novel viral receptor or membrane proteins that are transcriptionally and biologically similar to ACE2 and TMPRSS2 through a fuzzy clustering technique that employs the Grey wolf optimizer (GWO) algorithm for finding the optimal cluster center. The exploratory and exploitation capability of GWO algorithm is improved by hybridizing mutation and crossover operators of the evolutionary algorithm. Also, the genetic diversity of the grey wolf population is enhanced by eliminating weak individuals from the population. The proposed clustering algorithm’s effectiveness is shown by detecting novel viral receptors and membrane proteins associated with the pathogenesis of SARS-CoV-2 infection. The expression profiles of ACE2 protein and its co-receptor factor are analyzed and compared with single-cell transcriptomics profiling using the Seurat R toolkit, mass spectrometry (MS), and immunohistochemistry (IHC). Our advanced clustering method infers that cell that expresses high ACE2 level are more affected by SARS-CoV-infection. So, SARS-CoV-2 virus affects lung, intestine, testis, heart, kidney, and liver more severely than brain, bone marrow, skin, spleen, etc.We have identified 58 novel viral receptors and 816 membrane proteins, and their role in the pathogenicity mechanism of SARS-CoV-2 infection has been studied. Besides, our study confirmed that Neuropilins (NRP1), G protein-coupled receptor 78 (GPR78), C-type lectin domain family 4 member M (CLEC4M), Kringle containing transmembrane protein 1 (KREMEN1), Asialoglycoprotein receptor 1 (ASGR1), A Disintegrin and metalloprotease 17 (ADAM17), Furin, Neuregulin-1,(NRG1), Basigin or CD147 and Poliovirus receptor (PVR) are the potential co-receptors of SARS-CoV-2 virus. A significant finding is that heparin derivative glycosaminoglycans could block the replication of SARS-CoV-2 virus inside the host cytoplasm. The membrane protein N-Deacetylase/N-Sulfotransferase-2 (NDST2), Extostosin protein (EXT1, EXT2, and EXT3), Glucuronic acid epimerase (GLCE), and Xylosyltransferase I, II (XYLT1, XYLT2) could act as the therapeutic target for inhibiting the spread of SARS-CoV-2 infection. Drugs such as carboplatin and gemcitabine are effective in such situations.

Vaccinia virus H7-protein is required for the organization of the viral scaffold protein into hexamers

Viruses of the giant virus family are characterized by a structurally conserved scaffold-capsid protein that shapes the icosahedral virion. The vaccinia virus (VACV) scaffold protein D13, however, transiently shapes the newly assembled viral membrane in to a sphere and is absent from the mature brick-shaped virion. In infected cells D13, a 62 kDa polypeptide, forms trimers that arrange in hexamers and a honey-comb like lattice. Membrane association of the D13-lattice may be mediated by A17, an abundant 21 kDa viral membrane protein. Whether membrane binding mediates the formation of the honey-comb lattice or if other factors are involved, remains elusive. Here we show that H7, a 17 kDa protein conserved among poxviruses, mediates proper formation of D13-hexamers, and hence the honey comb lattice and spherical immature virus. Without H7 synthesis D13 trimers assemble into a large 3D network rather than the typical well organized scaffold layer observed in wild-type infection, composed of short D13 tubes of discrete length that are tightly associated with the endoplasmic reticulum (ER). The data show an unexpected role for H7 in D13 organization and imply that formation of the honey-comb, hexagonal, lattice is essential for VACV membrane assembly and production of infectious progeny. The data are discussed with respect to scaffold proteins of other giant viruses.

The Structure and Mechanism of Spike Protein, as Well as its Role in Numerous Viral Diseases

A spike protein is a protein that builds a huge spike that ejects from an enveloped virus’s membrane. The spike protein is the only virus membrane protein that enables the virus to penetrate through the cell. Spike Protein has three potential methods of action. Most common viral illnesses have relatively similar virus structures, which are predominantly made up of dimers or trimers of the spike glycoprotein, as well as analogous mechanisms of host cell invasion. The purpose of this paper is to explore the structure of the spike protein and its cell invasion method. The prevalence of spike protein in distinct viruses, as well as their similar invasion mechanisms, are also highlighted in the paper. We observed that many infectious viruses have very identical structures, predominantly constituted of spike glycoprotein, as well as similar processes of invasion into host cells. There are diverse sorts of pathogenesis that have been identified, especially those relating to host cell contamination and the means wherein the infection spreads and produces disease. The Spike protein must be operational for the virus to penetrate the host organism, and variations in the protein’s activation techniques are thought to have an influence in viral pathogenesis. Vaccines struggle to prevent the transmission of all virus variants due to variances in the spike protein in different viral versions, as well as modifications in them. More research into the structure of spike glycoproteins, as well as the creation of more effective vaccines to inhibit spike protein invasion and infection, are required.

Cleavage of the selective autophagy receptor SQSTM1/p62 by the SARS-CoV-2 main protease NSP5 prevents the autophagic degradation of viral membrane proteins

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the coronavirus disease 2019 (COVID-19) global pandemic. Omicron, a new variant of SARS-CoV-2, has the characteristics of strong transmission and pathogenicity, short incubation period, and rapid onset progression, and has spread rapidly around the world. The high replication rate and intracellular accumulation of SARS-CoV-2 are remarkable, but the underlying molecular mechanisms remain unclear. Autophagy acts as a conservative cellular defence mechanism against invading pathogens. Here, we provide evidence that the main protease of SARS-CoV-2, NSP5, effectively cleaves the selective autophagy receptor p62. NSP5 targets p62 for cleavage at glutamic acid 354 and thus abolishes the capacity of p62 to mediate selective autophagy. It was further shown that p62 specifically interacted with ubiquitinated SARS-CoV-2 M, the viral membrane protein, to promote its autophagic degradation. In the presence of NSP5, p62-mediated autophagic degradation of the M protein was inhibited. The cleaved products of p62 also cannot facilitate the degradation of the M protein. Collectively, our findings reveal that p62 is a novel host target of SARS-CoV-2 NSP5 and suggest that selective autophagy targets viruses and potential strategies by which the virus evades autophagic clearance. Our results may provide new ideas for the development of anti-COVID-19 drugs based on autophagy and NSP5.

The E3-ligase TRIM7 acts as an antiviral factor by ubiquitinating the SARS-CoV-2 membrane protein to limit apoptosis and virus replication

Abstract Cytokines, such as type-I Interferons (IFN-I), are essential to mount an effective antiviral response. The production of IFN-I is regulated by multiple mechanisms, including ubiquitination of host signaling factors. However, viruses can hijack the ubiquitin (Ub) system to promote viral replication. For example, we recently reported that the E3-Ub ligase TRIM7 ubiquitinates the envelope protein of Zika virus, which increases virus binding to its receptor favoring virus entry. Previous studies suggest that TRIM7 may play dual roles during infection by promoting or inhibiting IFN-I induction depending on the conditions. Here, we used our recently generated Trim7−/− mice to study its physiological role during SARS-CoV-2 (SCoV2) infection. We found that Trim7−/− mice have increased weight loss and lung virus titers compared to WT controls upon infection with SCoV2. We also found a dysregulated innate immune response, which included increased IFN-I induction and reduced neutrophil recruitment and production of inflammatory cytokines such as IL-6 in Trim7−/− mice. We observed a higher frequency of apoptotic cells in Trim7−/− compared to WT mice, specifically in CD45− lung epithelial cells. Mechanistically, using co-immunoprecipitation studies, we confirmed previous reports that TRIM7 interacts with the viral membrane protein (M), which can cause apoptosis. Using recombinant mutants that lack ubiquitination sites on M, we found that TRIM7-mediated ubiquitination of M protects cells against death. This suggests that TRIM7 restricts SCoV2 infection via a novel non-degradative mechanism involving ubiquitination of M. Understanding the role of TRIM7 in inhibiting SCoV2 replication could help develop treatments for COVID-19. Supported by grants from NIH R01 AI166668

Protein engineering and biochemical/biophysical approaches for structural studies of small membrane proteins and their complexes: Application to viroporins

We study the structure and function of small viral membrane proteins, which are expressed in the host and function in cellular membranes. Typically, they are multi‐domain multi‐functional proteins aiding virus adaptation and proliferation. Detailed characterization of the structure‐function relationship of these proteins will help to better understand virus‐host interactions and possibly develop protein inhibitors. However, such characterization is challenging due to the proteins’ small size and dynamic nature. Our focus is on viroporins, namely the Vpu, p7 and E proteins from HIV‐1, HCV, and CoV, respectively. Viroporins form ion‐conducting oligomers in cellular membranes leading to increased membrane permeability affecting ion homeostasis in infected cells. In addition, these proteins have soluble domains involved in interactions with host proteins. Currently, the structures of full‐length viroporins are largely uncharacterized and the information about the mechanism of oligomer formation and inter‐domain communication is very limited. Here, we report our effort to produce and solve the structure of stable viroporin oligomers in lipid environment. We use protein engineering to generate a range of fusion constructs aiming to increase the protein/oligomer size and reduce the conformational heterogeneity, thus making the protein amenable for structural determination by electron microscopy (EM) techniques. We produced, expressed in E. coli and purified, several soluble constructs of Vpu, p7 and E proteins fused at their N‐terminals to maltose binding protein (MBP). Thereafter, we interacted these proteins with lyso lipid mixtures of PC and PG, and their oligomerization was observed by negative staining EM and pulse electron paramagnetic resonance (EPR). We further use size‐exclusion chromatography (SEC) to assess the heterogeneity of viroporin oligomers in lyso PC/PG. Indeed, the protein elution exhibits a relatively broad chromatographic profile, indicating the presence of more than one oligomeric form. We further use SEC to test different protein‐to‐lipid molar ratios and the effect of viroporin inhibitor binding, aiming to shift the equilibrium toward stable functional viroporin oligomers. We will also discuss results from EM studies on these stable oligomers and EPR studies on spin‐labeled viroporin assembly. The progress we made in generating protein constructs and assessing protein oligomerization and stability in lipid provides a great promise for the development of successful methodology to determine the structure of full‐length viroporin. This methodology could also be applicable to other small oligomeric membrane proteins.

Generation of Mature DENVs via Genetic Modification and Directed Evolution.

ABSTRACTMaturation of dengue viruses (DENVs) alters the structure, immunity, and infectivity of the virion and highly mature particles represent the dominant form in vivo. The production of highly mature virions principally relies on the structure and function of the viral premature membrane protein (prM) and its cleavage by the host protease furin. We redeveloped a reliable clonal cell line (VF1) which produces single-round mature DENVs without the need for DENV reverse genetics. More importantly, using protein engineering and directed evolution of the prM cleavage site, we engineered genetically stable mature DENVs in all serotypes independent of cell or host, usually with minimal impact on viral yield. Using these complementary strategies to regulate maturation, we demonstrate that the resulting mature DENVs are antigenically distinct from their isogenic partially mature forms. Given the clinical importance of mature DENVs in immunity, our study provides reliable strategies and reagents for the production of stable, high-titer mature DENVs for DENV antibody neutralization and vaccination immunity studies. Biologically, our data from directed evolution across host species reveals distinct maturation-dependent selective pressures between mammalian and insect cells, verifying the substrate preference between mammalian and insect furin, while hinting at an evolutionary equilibrium of DENV prM cleavage site between its host and vector in nature.