Metalloproteinase-dependent and TMPRSS2-independnt cell surface entry pathway of SARS-CoV-2 requires the furin-cleavage site and the S2 domain of spike protein

The ongoing global vaccination program to prevent SARS-CoV-2 infection, the causative agent of COVID-19, has had significant success. However, recently virus variants have emerged that can evade the immunity in a host achieved through vaccination. Consequently, new therapeutic agents that can efficiently prevent infection from these new variants, and hence COVID-19 spread are urgently required. To achieve this, extensive characterization of virus-host cell interactions to identify effective therapeutic targets is warranted. Here, we report a cell surface entry pathway of SARS-CoV-2 that exists in a cell type-dependent manner is TMPRSS2-independent but sensitive to various broad-spectrum metalloproteinase inhibitors such as marimastat and prinomastat. Experiments with selective metalloproteinase inhibitors and gene-specific siRNAs revealed that a disintegrin and metalloproteinase 10 (ADAM10) is partially involved in the metalloproteinase pathway. Consistent with our finding that the pathway is unique to SARS-CoV-2 among highly pathogenic human coronaviruses, both the furin cleavage motif in the S1/S2 boundary and the S2 domain of SARS-CoV-2 spike protein are essential for metalloproteinase-dependent entry. In contrast, the two elements of SARS-CoV-2 independently contributed to TMPRSS2-dependent S2 priming. The metalloproteinase pathway is involved in SARS-CoV-2-induced syncytia formation and cytopathicity, leading us to theorize that it is also involved in the rapid spread of SARS-CoV-2 and the pathogenesis of COVID-19. Thus, targeting the metalloproteinase pathway in addition to the TMPRSS2 and endosome pathways could be an effective strategy by which to cure COVID-19 in the future.

Virtual Screening and In Silico Interactions Studies for Potential Antivirals and Diagnostics against the Spike protein from the Novel Coronavirus SARS-Cov-2

COVID-19 is a newly-emerged respiratory disease that is caused by the SARS-CoV-2, the seventh known Coronaviruses strain that has struck a global pandemic. The sharp increase in the number of positive cases worldwide necessitates highly-sensitive diagnostics kits and effective antiviral drugs to be developed for the populations. One of the antigens that is targeted for antibody neutralisation is the coronavirus Spike protein that consists of the S1 and S2 subunits, which mediated the entry pathway into the host’s cell. Thus, the Spike protein has been suggested as a potential target for Covid-19 diagnostics and drug design. This study aims to evaluate the interactions between the SARS-CoV-2 Spike protein and the known monoclonal antibodies from Coronaviruses and to screen for potential Spike protein inhibitors. Virtual screening was conducted based on two compounds, N‐acetyl‐D‐glucosamine (NAG) and Hesperetin, which is a small molecule that binds to the SARS-CoV-2 Spike protein structure and a natural compound that has prophylactic agents against SARS-CoV-2 infection as it binds to Spike protein, respectively. Protein-protein interaction studies were conducted by using the STRING webserver, prior to performing rigid docking using SWISSDOCK and visualised using USCF Chimera. Meanwhile, ligand-based screening was conducted through Ultrafast Shape Recognition Virtual Screening Database (USR-VS), and structure-based screening was performed via AutoDock4 software. The toxicity of the compounds was predicted using ProTox-II database. Possible interactions have been observed between the known monoclonal antibodies with the SARS-CoV-2 Spike protein, where M396 monoclonal antibody has shown the strongest interaction with a binding energy of -8.50 kcal/mol. Meanwhile, virtual screening has yielded several compounds that indicate the possibility to inhibit the SARS-CoV-2 Spike protein, where Tamarixetin has shown the strongest binding energy of -7.93 kcal/mol. These findings have potentials to be further evaluated in the future for the development of improved diagnostic kits and potential therapeutic drugs that specifically target the Spike protein of SARS-CoV-2.

Integrating Desire Thinking into the I-PACE Model: a Special Focus on Internet-Use Disorders

Purpose of Review This manuscript aims to propose an integration of desire thinking into the Interaction of Person-Affect-Cognition-Execution (I-PACE) model based on theoretical considerations within the Elaborated Intrusion Theory of Desire and Self-Regulatory Execution Function model and empirical evidence from the field of internet-use disorders. Recent Findings Theory and research on desire thinking in the context of internet-use disorders suggest considerable relations to craving, metacognitive beliefs, and emphasizes its nature when initiated as a reaction towards unpleasant triggers. Accordingly, we postulate that desire thinking may be located at the position for affective and cognitive reactions towards triggers within the I-PACE model. Summary The suggested integration of desire thinking into the I-PACE model specifically implies the assumption of a relief-oriented and pleasure-oriented entry pathway into desire thinking and a feedback loop between desire thinking and the experience of gratification and compensation. The model pathways proposed here may serve as a theoretical basis for future research and need further empirical verification.

Biological Pathway Introduction of Quarantine Animal Disease through International Waste at Soekarno Hatta International Airport, Indonesia

International flights coming to Indonesia, in addition to bringing in passengers, also brings in passenger’s food waste and garbage from the activities in the airplane. Soekarno Hatta International Airport (SHIA), as one of the busiest airports in Indonesia has great potential as a waste producer. International waste has a higher risk in terms of spreading disease to the environment around the airport, which will ultimately impact human and animal health in general. Waste risk assessment carried by international aircraft as a risk carrier of quarantine animal disease entering Indonesia has never been done. The aim of this study was to identify the biological pathway from the entry of pathogens through international waste and how to prevent it. The results, there were three pathway of international waste management at SHIA. The first pathway, the waste was not unloaded from international aircraft and returned to the country of origin. The second pathway, international waste was unloaded from international aircraft, then destroyed in incinerators inside the airport area. The third pathway, the waste was managed by the inflight catering company and taken out of the SHIA. The third pathway has a highest risk as an entry pathway of quarantine animal disease. There was possibility that food waste was reused as animal feed. Regulations regarding airport waste management is exist, but the implementation is not optimal. The authority and agencies related to international waste at SHIA need to enforce the existing rules about international waste management, to prevent the spread of diseases due to waste.

Recent Advances in Our Understanding of the Infectious Entry Pathway of Human Papillomavirus Type 16

Papillomaviruses are a diverse viral species, but several types such as HPV16 are given special attention due to their contribution towards the pathogenesis of several major cancers. In this review, we will summarize how the knowledge of HPV16 entry has expanded since the last comprehensive HPV16 entry review our lab published in 2017.

Challenges on the development of a pseudotyping assay for Zika glycoproteins

Introduction. Zika virus (ZIKV) emerged as a public health concern on the American continent during late 2015. As the number of infected grew so did the concerns about its capability to cause long-term damage especially with the appearance of the congenital Zika syndrome (CZS). Proteins from the TAM family of receptor tyrosine kinases (RTKs) were proposed as the cellular receptors, however, due to the ability of the virus to infect a variety of cell lines different strategies to elucidate the tropism of the virus should be investigated. Hypothesis. Pseudotyping is a powerful tool to interrogate the ability of the glycoprotein (GP) to permit entry of viruses. Aim. We aimed to establish a highly tractable pseudotype model using lenti- and retro-viral backbones to investigate the entry pathway of ZIKV. Methodology. We used different glycoprotein constructs and different lenti- or retro-viral backbones, in a matrix of ratios to investigate production of proteins and functional pseudotypes. Results. Varying the ratio of backbone and glycoprotein plasmids did not yield infectious pseudotypes. Moreover, the supplementation of the ZIKV protease or the substitution of the backbone had no positive impact on the infectivity. We showed production of the proteins in producer cells implying the lack of infectious pseudotypes is due to a lack of successful glycoprotein incorporation, rather than lack of protein production. Conclusion. In line with other reports, we were unable to successfully produce infectious pseudotypes using the variety of methods described. Other strategies may be more suitable in the development of an efficient pseudotype model for ZIKV and other flaviviruses.

Isoform-Specific Properties of Orai Homologues in Activation, Downstream Signaling, Physiology and Pathophysiology

Ca2+ ion channels are critical in a variety of physiological events, including cell growth, differentiation, gene transcription and apoptosis. One such essential entry pathway for calcium into the cell is the Ca2+ release-activated Ca2+ (CRAC) channel. It consists of the Ca2+ sensing protein, stromal interaction molecule 1 (STIM1) located in the endoplasmic reticulum (ER) and a Ca2+ ion channel Orai in the plasma membrane. The Orai channel family includes three homologues Orai1, Orai2 and Orai3. While Orai1 is the “classical” Ca2+ ion channel within the CRAC channel complex and plays a universal role in the human body, there is increasing evidence that Orai2 and Orai3 are important in specific physiological and pathophysiological processes. This makes them an attractive target in drug discovery, but requires a detailed understanding of the three Orai channels and, in particular, their differences. Orai channel activation is initiated via Ca2+ store depletion, which is sensed by STIM1 proteins, and induces their conformational change and oligomerization. Upon STIM1 coupling, Orai channels activate to allow Ca2+ permeation into the cell. While this activation mechanism is comparable among the isoforms, they differ by a number of functional and structural properties due to non-conserved regions in their sequences. In this review, we summarize the knowledge as well as open questions in our current understanding of the three isoforms in terms of their structure/function relationship, downstream signaling and physiology as well as pathophysiology.

TRPM7-Mediated Calcium Transport in HAT-7 Ameloblasts

TRPM7 plays an important role in cellular Ca2+, Zn2+ and Mg2+ homeostasis. TRPM7 channels are abundantly expressed in ameloblasts and, in the absence of TRPM7, dental enamel is hypomineralized. The potential role of TRPM7 channels in Ca2+ transport during amelogenesis was investigated in the HAT-7 rat ameloblast cell line. The cells showed strong TRPM7 mRNA and protein expression. Characteristic TRPM7 transmembrane currents were observed, which increased in the absence of intracellular Mg2+ ([Mg2+]i), were reduced by elevated [Mg2+]i, and were inhibited by the TRPM7 inhibitors NS8593 and FTY720. Mibefradil evoked similar currents, which were suppressed by elevated [Mg2+]i, reducing extracellular pH stimulated transmembrane currents, which were inhibited by FTY720. Naltriben and mibefradil both evoked Ca2+ influx, which was further enhanced by the acidic intracellular conditions. The SOCE inhibitor BTP2 blocked Ca2+ entry induced by naltriben but not by mibefradil. Thus, in HAT-7 cells, TRPM7 may serves both as a potential modulator of Orai-dependent Ca2+ uptake and as an independent Ca2+ entry pathway sensitive to pH. Therefore, TRPM7 may contribute directly to transepithelial Ca2+ transport in amelogenesis.

Viral Envelope Membrane: A Special Entry Pathway and a Promising Drug Target

Enveloped viruses belong to a large class of pathogens responsible for multiple serious diseases. Their spread into new territories has been the cause of major epidemics throughout human history, including the Spanish flu in 1918 and the latest COVID-19 pandemic. Thanks to their outer membrane, consisting essentially of host lipids, enveloped viruses are more resistant to enzymes, and are also less susceptible to host immune defenses than their naked counterparts. Therefore, the development of effective approaches to combat enveloped virus infections represents a major challenge for antiviral therapy in the current century. This review focuses on the characteristics of enveloped viruses, their importance in the entry phase, drugs targeting envelope membrane-mediated entry, and those specifically designed to target the envelope. The broad-spectrum antiviral activity of these compounds can be attributed to their ability to affect the envelope, an essential structural feature common to several viruses. This makes this class of compounds agents of great interest when no specific drugs or vaccines are available to block viral infections.