The spike (S) protein of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) binds to a host cell receptor which facilitates viral entry. A polybasic motif detected at the cleavage site of ...the S protein has been shown to broaden the cell tropism and transmissibility of the virus. Here we examine the properties of SARS-CoV-2 variants with mutations at the S protein cleavage site that undergo inefficient proteolytic cleavage. Virus variants with S gene mutations generated smaller plaques and exhibited a more limited range of cell tropism compared to the wild-type strain. These alterations were shown to result from their inability to utilize the entry pathway involving direct fusion mediated by the host type II transmembrane serine protease, TMPRSS2. Notably, viruses with S gene mutations emerged rapidly and became the dominant SARS-CoV-2 variants in TMPRSS2-deficient cells including Vero cells. Our study demonstrated that the S protein polybasic cleavage motif is a critical factor underlying SARS-CoV-2 entry and cell tropism. As such, researchers should be alert to the possibility of de novo S gene mutations emerging in tissue-culture propagated virus strains.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Although the spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has resulted in a worldwide pandemic, there are currently no virus-specific drugs that are fully effective against ...SARS-CoV-2. Only a limited number of human-derived cells are capable of supporting SARS-CoV-2 replication and the infectivity of SARS-CoV-2 in these cells remains poor. In contrast, monkey-derived Vero cells are highly susceptibility to infection with SARS-CoV-2, although they are not suitable for the study of antiviral effects by small molecules due to their limited capacity to metabolize drugs compared to human-derived cells. In this study, our goal was to generate a virus-susceptible human cell line that would be useful for the identification and testing of candidate drugs. Towards this end, we stably transfected human lung-derived MRC5 cells with a lentiviral vector encoding angiotensin-converting enzyme 2 (ACE2), the cellular receptor for SARS-CoV-2. Our results revealed that SARS-CoV-2 replicates efficiently in MRC5/ACE2 cells. Furthermore, viral RNA replication and progeny virus production were significantly reduced in response to administration of the replication inhibitor, remdesivir, in MRC5/ACE2 cells compared with Vero cells. We conclude that the MRC5/ACE2 cells will be important in developing specific anti-viral therapeutics and will assist in vaccine development to combat SARS-CoV-2 infections.
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) utilizes host proteases, including a plasma membrane-associated transmembrane protease, serine 2 (TMPRSS2) to cleave and activate the ...virus spike protein to facilitate cellular entry. Although TMPRSS2 is a well-characterized type II transmembrane serine protease (TTSP), the role of other TTSPs on the replication of SARS-CoV-2 remains to be elucidated. Here, we have screened 12 TTSPs using human angiotensin-converting enzyme 2-expressing HEK293T (293T-ACE2) cells and Vero E6 cells and demonstrated that exogenous expression of TMPRSS11D and TMPRSS13 enhanced cellular uptake and subsequent replication of SARS-CoV-2. In addition, SARS-CoV-1 and SARS-CoV-2 share the same TTSPs in the viral entry process. Our study demonstrates the impact of host TTSPs on infection of SARS-CoV-2, which may have implications for cell and tissue tropism, for pathogenicity, and potentially for vaccine development.
We previously showed that 5-ethynyl-(1-β-D-ribofuranosyl)imidazole-4-carboxamide (1; EICAR) is a potent anti-dengue virus (DENV) compound but is cytotoxic to some cell lines, while its 4-thio ...derivative, 5-ethynyl-(4-thio-1-β-D-ribofuranosyl)imidazole-4-carboxamide (2; 4′-thioEICAR), has less cytotoxicity but also less anti-DENV activity. Based on the hypothesis that the lower anti-DENV activity of 2 is due to reduced susceptibility to phosphorylation by cellular kinase(s), we investigated whether a monophosphate prodrug of 2 can improve its activity. Here, we first prepared two types of prodrug of 1, which revealed that the S-acyl-2-thioethyl (SATE) prodrug had stronger anti-DENV activity than the aryloxyphosphoramidate (so-called ProTide) prodrug. Based on these findings, we next prepared the SATE prodrug of 4′-thioEICAR 18. As expected, the resulting 18 showed potent anti-DENV activity, which was comparable to that of 1; however, its cytotoxicity was also increased relative to 2. Our findings suggest that prodrugs of 4′-thioribonucleoside derivatives such as EICAR (1) represent an effective approach to developing potent biologically active compounds; however, the balance between antiviral activity and cytotoxicity remains to be addressed.
Abstract
Objectives
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiological agent of coronavirus disease 2019 (COVID-19) and a devastating worldwide health concern. ...Development of safe and effective treatments is not only important for interventions during the current pandemic, but also for providing general treatment options moving forward. We have developed ensitrelvir, an antiviral compound that targets the 3C-like protease of SARS-CoV-2. In this study, a delayed-treatment mouse model was used to clarify the potential in vivo efficacy of ensitrelvir.
Methods
Female BALB/cAJcl mice of different ages were infected with the SARS-CoV-2 gamma strain (hCoV-19/Japan/TY7-501/2021) or mouse-adapted SARS-CoV-2 MA-P10 and then 24 h post-infection orally administered various doses of ensitrelvir or vehicle. Viral titres and RNA levels in the lungs were quantified using VeroE6/TMPRSS2 cells and RT–qPCR, respectively. Body weight loss, survival, lung weight, cytokine/chemokine production, nucleocapsid protein expression and lung pathology were evaluated to investigate the in vivo efficacy of ensitrelvir.
Results
Based on infectious viral titres and viral RNA levels in the lungs of infected mice, ensitrelvir reduced viral loads in a dose-dependent manner. The antiviral efficacy correlated with increased survival, reduced body weight loss, reduced pulmonary lesions and suppression of inflammatory cytokine/chemokine levels.
Conclusions
This was the first evaluation of the in vivo anti-SARS-CoV-2 efficacy of ensitrelvir in a delayed-treatment mouse model. In this model, ensitrelvir demonstrated high antiviral potential and suppressed lung inflammation and lethality caused by SARS-CoV-2 infection. The findings support the continued clinical development of ensitrelvir as an antiviral agent to treat patients with COVID-19.
Tick-borne flaviviruses (TBFVs) infect mammalian hosts through tick bites and can cause various serious illnesses, such as encephalitis and hemorrhagic fevers, both in humans and animals. Despite ...their importance to public health, there is limited epidemiological information on TBFV infection in Africa. Herein, we report that a novel flavivirus, Mpulungu flavivirus (MPFV), was discovered in a Rhipicephalus muhsamae tick in Zambia. MPFV was found to be genetically related to Ngoye virus detected in ticks in Senegal, and these viruses formed a unique lineage in the genus Flavivirus. Analyses of dinucleotide contents of flaviviruses indicated that MPFV was similar to those of other TBFVs with a typical vertebrate genome signature, suggesting that MPFV may infect vertebrate hosts. Bioinformatic analyses of the secondary structures in the 3'-untranslated regions (UTRs) revealed that MPFV exhibited unique exoribonuclease-resistant RNA (xrRNA) structures. Utilizing biochemical approaches, we clarified that two xrRNA structures of MPFV in the 3'-UTR could prevent exoribonuclease activity. In summary, our findings provide new information regarding the geographical distribution of TBFV and xrRNA structures in the 3'-UTR of flaviviruses.
Scavenger receptor class B type 1 (SR-B1) and low-density lipoprotein receptor (LDLR) are known to be involved in entry of hepatitis C virus (HCV), but their precise roles and their interplay are not ...fully understood. In this study, deficiency of both SR-B1 and LDLR in Huh7 cells was shown to impair the entry of HCV more strongly than deficiency of either SR-B1 or LDLR alone. In addition, exogenous expression of not only SR-B1 and LDLR but also very low-density lipoprotein receptor (VLDLR) rescued HCV entry in the SR-B1 and LDLR double-knockout cells, suggesting that VLDLR has similar roles in HCV entry. VLDLR is a lipoprotein receptor, but the level of its hepatic expression was lower than those of SR-B1 and LDLR. Moreover, expression of mutant lipoprotein receptors incapable of binding to or uptake of lipid resulted in no or slight enhancement of HCV entry in the double-knockout cells, suggesting that binding and/or uptake activities of lipid by lipoprotein receptors are essential for HCV entry. In addition, rescue of infectivity in the double-knockout cells by the expression of the lipoprotein receptors was not observed following infection with pseudotype particles bearing HCV envelope proteins produced in non-hepatic cells, suggesting that lipoproteins associated with HCV particles participate in the entry through their interaction with lipoprotein receptors. Buoyant density gradient analysis revealed that HCV utilizes these lipoprotein receptors in a manner dependent on the lipoproteins associated with HCV particles. Collectively, these results suggest that lipoprotein receptors redundantly participate in the entry of HCV.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) possesses a discriminative polybasic cleavage motif in its spike protein that is recognized by the host furin protease. Proteolytic ...cleavage activates the spike protein, thereby affecting both the cellular entry pathway and cell tropism of SARS-CoV-2. Here, we investigated the impact of the furin cleavage site on viral growth and pathogenesis using a hamster animal model infected with SARS-CoV-2 variants bearing mutations at the furin cleavage site (S gene mutants). In the airway tissues of hamsters, the S gene mutants exhibited low growth properties. In contrast to parental pathogenic SARS-CoV-2, hamsters infected with the S gene mutants showed no body weight loss and only a mild inflammatory response, thereby indicating the attenuated variant nature of S gene mutants. This transient infection was sufficient for inducing protective neutralizing antibodies that cross-react with different SARS-CoV-2 lineages. Consequently, hamsters inoculated with S gene mutants showed resistance to subsequent infection with both the parental strain and the currently emerging SARS-CoV-2 variants belonging to lineages B.1.1.7 and P.1. Taken together, our findings revealed that the loss of the furin cleavage site causes attenuation in the airway tissues of hamsters and highlighted the potential benefits of S gene mutants as potential immunogens.
SARS-CoV-2 uses its spike protein to enter target cells. The spike protein is cleaved by a host protease, and this event facilitates viral entry and broadens cell tropism. In this study, we employed SARS-CoV-2 mutants lacking the S protein cleavage site and characterized their growth and pathogenicity using hamsters, a laboratory animal model for SARS-CoV-2 infection. These mutants exerted low pathogenicity but induced sufficient levels of neutralizing antibodies in hamsters, which protected hamsters from rechallenge with pathogenic clinical SARS-CoV-2 strains. These virus mutants may be used as protective immunogens against SARS-CoV-2 infection.
Group A rotaviruses (RVAs) are representative enteric virus species and major causes of diarrhea in humans and animals. The RVA virion is a triple-layered particle, and the outermost layer consists ...of the glycoprotein VP7 and spike protein VP4. To increase the infectivity of RVA, VP4 is proteolytically cleaved into VP5* and VP8* subunits by trypsin; and these subunits form a rigid spike structure on the virion surface. In this study, we investigated the growth of RVAs in cells transduced with type II transmembrane serine proteases (TTSPs), which cleave fusion proteins and promote infection by respiratory viruses, such as influenza viruses, paramyxoviruses, and coronaviruses. We identified TMPRSS2 and TMPRSS11D as host TTSPs that mediate trypsin-independent and multi-cycle infection by human and animal RVA strains.
cleavage assays revealed that recombinant TMPRSS11D cleaved RVA VP4. We also found that TMPRSS2 and TMPRSS11D promote the infectious entry of immature RVA virions, but they could not activate nascent progeny virions in the late phase of infection. This observation differed from the TTSP-mediated activation process of paramyxoviruses, revealing the existence of virus species-specific activation processes in TTSPs. Our study provides new insights into the interaction between RVAs and host factors, and TTSP-transduced cells offer potential advantages for RVA research and development.
Proteolytic cleavage of the viral VP4 protein is essential for virion maturation and infectivity in group A rotaviruses (RVAs). In cell culture, RVAs are propagated in culture medium supplemented with the exogenous protease trypsin, which cleaves VP4 and induces the maturation of progeny RVA virions. In this study, we demonstrated that the host proteases TMPRSS2 and TMPRSS11D mediate the trypsin-independent infection and growth of RVA. Our data revealed that the proteolytic activation of RVAs by TMPRSS2 and TMPRSS11D occurs at the viral entry step. Because TMPRSS2 and TMPRSS11D gene expression induced similar or higher levels of RVA growth as trypsin-supplemented culture, this approach offers potential advantages for RVA research and development.
This paper presents results from geotechnical centrifuge tests performed at the Japanese Institute of Occupational Safety and Health (JNIOSH) in Tokyo, Japan. A range of shallow footing ...configurations with different bearing pressures founded on saturated sand in a laminar box on a shake table was studied using harmonic excitations. The free-field condition was also studied as a reference. The study aimed to address the influence of stand-alone and closely adjacent shallow foundations on the dynamic response of the soil-foundation-structure system. A partially drained response of the soil and a minor dilative response for large values of shear strain were observed for the free-field case. In the case with footings, the settlement was found to be largely due to the footing penetrating the supporting sand. A dilative response of the soil beneath the footings was observed for the stand-alone configurations at the beginning of the base motions, this was not observed in the case of adjacent footings. Adjacent footings also tilt inwards consistently while stand-alone footings did not present a clear tilt pattern. In terms of the acceleration recorded in the near-surface soil beneath the footings, a reduction was observed after a few cycles in the free-field case. This reduction was considerably smaller in the case with footings with no significant differences between the response of the stand-alone and that of the closely adjacent footings.
•Centrifuge tests of stand-alone and adjacent footings on a laminar box filled with saturated loose sand.•The presence of footings modifies the portion of the soil with higher excess pore-pressure ratio.•A partially drained response of the soil and a dilative response at large shear deformation are relevant for the settlement of the footings.•The shallowest soil presented a similar response for all the base motions while the rest of the deposit tend to densify with subsequent shakes.
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