MARCH8 Targets Cytoplasmic Lysine Residues of Various Viral Envelope Glycoproteins

A member of the MARCH E3 ubiquitin ligase family, MARCH8, downregulates many different kinds of host transmembrane proteins, resulting in the regulation of cellular homeostasis. On the other hands, MARCH8 acts as an antiviral factor when it binds to and downregulates HIV-1 envelope glycoprotein and vesicular stomatitis virus G-glycoprotein that are viral transmembrane proteins.

Molecular Aspects of Spike–ACE2 Interaction

A new betacoronavirus (CoV-2) is responsible for the pandemic of severe acute respiratory syndrome (SARS) that began in China at the end of 2019, today known as COronaVIrus Disease 2019 (COVID-19). Subsequent studies confirmed the human angiotensin-converting enzyme 2 (hACE2) as the main cell receptor of spike trimeric glycoprotein, located on the viral envelope, mediating the CoV-2 invasion into the host cells through the receptor-binding domain (RBD) of the spike. Computational analysis of the known experimental 3D structures of spike–ACE2 complexes evidenced distinguishing features in the molecular interactions at the RBD-cell receptor binding interface between CoV-2 and previous CoV-1. The spike represents a key target for drug design as well as an optimal antigen for RNA/viral vector vaccines and monoclonal antibodies in order to maximize prevention and therapy of COVID-19.

Lipid-mediated biosynthetic labeling strategy for in vivo dynamic tracing of avian influenza virus infection

Monitoring the infection behavior of avian influenza viruses is crucial for understanding viral pathogenesis and preventing its epidemics among people. A number of viral labeling methods have been utilized for tracking viral infection process, but most of them are laborious or decreasing viral activity. Herein we explored a lipid biosynthetic labeling strategy for dynamical tracking the infection of H5N1 pseudotype virus (H5N1p) in host. Biotinylated lipids (biotinyl Cap-PE) were successfully incorporated into viral envelope when it underwent budding process by taking advantage of host cell-derived lipid metabolism. Biotin-H5N1p virus was effectively in situ–labeled with streptavidin-modified near-infrared quantum dots (NIR SA-QDs) using streptavidin-biotin conjugation with well-preserved virus activities. Dual-labeled imaging obviously shows that H5N1p viruses are primarily taken up in host cells via clathrin-mediated endocytosis. In animal models, Virus-conjugated NIR QDs displayed extraordinary photoluminescence, superior stability, and tissue penetration in lung, allowing us to long-term monitor respiratory viral infection in a noninvasive manner. Importantly, the co-localization of viral hemagglutinin protein and QDs in infected lung further conformed the dynamic infection process of virus in vivo. Hence, this in situ QD-labeling strategy based on cell natural biosynthesis provides a brand-new and reliable tool for noninvasion visualizing viral infection in body in a real-time manner.

Click chemistry and molecular modeling methods in computer-aided design and identification of potential HIV-1 inhibitors

An integrated approach including the click chemistry methodology, molecular docking, quantum mechanics, and molecular dynamics was used to perform the computer-aided design of potential HIV-1 inhibitors able to block the membrane- proximal external region (MPER) of HIV-1 gp41 that plays an important role in the fusion of the viral and host cell membranes. Evaluation of the binding efficiency of the designed compounds to the HIV-1 MPER peptide was performed using the methods of molecular modeling, resulting in nine chemical compounds that exhibit the high-affinity binding to this functionally important site of the trimeric “spike” of the viral envelope. The data obtained indicate that the identified compounds are promising for the development of novel antiviral drugs, HIV fusion inhibitors blocking the early stages of HIV infection.

Functional Trimeric SARS-CoV-2 Envelope Protein Expressed in Stable CHO Cells

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a β-coronavirus, is the causative agent of the COVID-19 pandemic. One of the three membrane-bound envelope proteins is the spike protein (S), the one responsible for docking to the cellular surface protein ACE2 enabling infection with SARS-CoV-2. Although the structure of the S-protein has distinct similarities to other viral envelope proteins, robust and straightforward protocols for recombinant expression and purification are not described in the literature. Therefore, most studies are done with truncated versions of the protein, like the receptor-binding domain. To learn more about the interaction of the virus with the ACE2 and other cell surface proteins, it is mandatory to provide recombinant spike protein in high structural quality and adequate quantity. Additional mutant variants will give new insights on virus assembly, infection mechanism, and therapeutic drug development. Here, we describe the development of a recombinant CHO cell line stably expressing the extracellular domain of a trimeric variant of the SARS CoV-2 spike protein and discuss significant parameters to be considered during the expression and purification process.

Inhibition of Viral Membrane Fusion by Peptides and Approaches to Peptide Design

Fusion of lipid-enveloped viruses with the cellular plasma membrane or the endosome membrane is mediated by viral envelope proteins that undergo large conformational changes following binding to receptors. The HIV-1 fusion protein gp41 undergoes a transition into a “six-helix bundle” after binding of the surface protein gp120 to the CD4 receptor and a co-receptor. Synthetic peptides that mimic part of this structure interfere with the formation of the helix structure and inhibit membrane fusion. This approach also works with the S spike protein of SARS-CoV-2. Here we review the peptide inhibitors of membrane fusion involved in infection by influenza virus, HIV-1, MERS and SARS coronaviruses, hepatitis viruses, paramyxoviruses, flaviviruses, herpesviruses and filoviruses. We also describe recent computational methods used for the identification of peptide sequences that can interact strongly with protein interfaces, with special emphasis on SARS-CoV-2, using the PePI-Covid19 database.

Transactive response DNA-binding Protein (TDP-43) regulates early HIV-1 entry and infection.

The transactive response DNA-binding protein (TDP-43) is an important regulator of mRNA, being reported to stabilize the anti-HIV factor, histone deacetylase 6 (HDAC6). However, little is known about the role of TDP-43 in HIV infection. In this work, we seek for the TDP-43 function on regulating CD4+ T cell permissibility to HIV infection. We observed that over-expression of wt-TDP-43 in CD4+ T cells stabilized HDAC6, increasing mRNA and the protein levels of this antiviral enzyme. Under this experimental condition, HIV-1 infection was impaired, independently of the viral envelope glycoprotein (Env) complex tropism. The results obtained by using an HIV-1 Env-mediated cell-to-cell fusion model, under the same experimental conditions, suggest that the increase in TDP-43 levels negatively affects the viral Env fusion capacity. Moreover, the specific siRNA silencing of endogenous TDP-43 in target cells lead to a significant decrease in the levels of HDAC6 which consistently induces an increase in the fusogenic and infection activities of the HIV-1 Env. These observations were confirmed by using primary viral Envs from HIV+ individuals with different clinical phenotypes. An increase in the level of expression of wt-TDP-43 strongly reduced the Envs infection activity of viremic non-progressors (VNP) and rapid progressors (RP) HIV+ individuals down to the levels of the inefficient HIV-1 Envs from long-term non-progressor elite controllers (LTNP-EC) individuals. On the contrary, low levels of endogenous TDP-43, obtained after specific siRNA-TDP-43 knocking-down, significantly favors the infection activity of primary HIV-1 Envs of VNP and RP individuals, leading to an increase in the infection ability of the primary HIV-1/LTNP-EC Envs. Based on this evidence, we interpret that TDP-43 conditions cell permissibility to HIV infection by affecting viral Env fusion and infection capacities, at least by altering the cellular levels of the antiviral enzyme HDAC6.

Durable nanocomposite face masks with high particulate filtration and rapid inactivation of coronaviruses

The COVID-19 pandemic presents a unique challenge to the healthcare community due to the high infectivity rate and need for effective personal protective equipment. Zinc oxide nanoparticles have shown promising antimicrobial properties and are recognized as a safe additive in many food and cosmetic products. This work presents a novel nanocomposite synthesis approach, which allows zinc oxide nanoparticles to be grown within textile and face mask materials, including melt-blown polypropylene and nylon-cotton. The resulting nanocomposite achieves greater than 3 log10 reduction (≥ 99.9%) in coronavirus titer within a contact time of 10 min, by disintegrating the viral envelope. The new nanocomposite textile retains activity even after 100 laundry cycles and has been dermatologist tested as non-irritant and hypoallergenic. Various face mask designs were tested to improve filtration efficiency and breathability while offering antiviral protection, with Claros’ design reporting higher filtration efficiency than surgical masks (> 50%) for particles ranged 200 nm to 5 µm in size.

Analysis of Hendra Virus Fusion Protein N-Terminal Transmembrane Residues

Hendra virus (HeV) is a zoonotic enveloped member of the family Paramyoxviridae. To successfully infect a host cell, HeV utilizes two surface glycoproteins: the attachment (G) protein to bind, and the trimeric fusion (F) protein to merge the viral envelope with the membrane of the host cell. The transmembrane (TM) region of HeV F has been shown to have roles in F protein stability and the overall trimeric association of F. Previously, alanine scanning mutagenesis has been performed on the C-terminal end of the protein, revealing the importance of β-branched residues in this region. Additionally, residues S490 and Y498 have been demonstrated to be important for F protein endocytosis, needed for the proteolytic processing of F required for fusion. To complete the analysis of the HeV F TM, we performed alanine scanning mutagenesis to explore the residues in the N-terminus of this region (residues 487–506). In addition to confirming the critical roles for S490 and Y498, we demonstrate that mutations at residues M491 and L492 alter F protein function, suggesting a role for these residues in the fusion process.

Influenza A virus hemagglutinin prevents extensive membrane damage upon dehydration

While the molecular mechanisms of virus infectivity are rather well known, the detailed consequences of environmental factors on virus biophysical properties are poorly understood. 20 Seasonal influenza outbreaks are usually connected to the low winter temperature, but also to the low relative air humidity. Indeed, transmission rates increase in cold regions during winter. While low temperature must slow degradation processes, the role of low humidity is not clear. We studied the effect of relative humidity on a model of influenza A H1N1 virus envelope, a supported lipid bilayer containing the surface glycoprotein hemagglutinin (HA), which is 25 present in the viral envelope in very high density. For complete cycles of hydration, dehydration and rehydration, we evaluate the membrane properties in terms of structure and dynamics, which we assess by combining confocal fluorescence microscopy, raster image correlation spectroscopy, line-scan fluorescence correlation spectroscopy and atomic force microscopy. Our findings indicate that the presence of HA prevents macroscopic membrane 30 damage after dehydration. Without HA, fast membrane disruption is followed by irreversible loss of lipid and protein mobility. Although our model is principally limited by the membrane composition, the macroscopic effects of HA under dehydration stress reveal new insights on the stability of the virus at low relative humidity.