Trypsin enhances SARS-CoV-2 infection by facilitating viral entry

Kim, Yeeun; Jang, Guehwan; Lee, Duri; Kim, Nara; Seon, Jeong Won; Kim, Young-Hoan; Lee, Changhee

doi:10.1007/s00705-021-05343-0

Cited by 35 publications

(31 citation statements)

References 40 publications

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“…Assuming that this could be a factor explaining non-productive SARS-CoV-2 infection in these cell types, we analyzed virus replication in the presence of exogenous serine protease. SARS-CoV-2 infection of SC pre-incubated with 5 μg/mL trypsin, a serine protease, which at this concentration does not interfere with cell attachment and has been used by others to enhance SARS-CoV-2 entry in other cell types (42), also did not result in increased intracellular virus RNA (data not shown) or infectious virions in the supernatant (Fig. 1D).…”

mentioning

confidence: 68%

In vitro evidence against productive SARS-CoV-2 infection of human testicular cells: Bystander effects of infection mediate testicular injury

Giannakopoulos

Strange

Jiyarom

et al. 2022

Preprint

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The hallmark of severe COVID-19 involves systemic cytokine storm and multi-organ failure including testicular injury and germ cell depletion. The ACE2 receptor is also expressed in the resident testicular cells however, SARS-CoV-2 infection and mechanisms of testicular injury are not fully understood. The testicular injury can likely result either from direct virus infection of resident cells or by exposure to systemic inflammatory mediators or virus antigens. We here characterized SARS-CoV-2 infection in different human testicular 2D and 3D models including primary Sertoli cells, Leydig cells, mixed seminiferous tubule cells (STC), and 3D human testicular organoids (HTO). Data shows that SARS-CoV-2 does not establish a productive infection in any testicular cell types. However, exposure of STC and HTO to inflammatory supernatant from infected airway epithelial cells and COVID-19 plasma depicted a significant decrease in cell viability and death of undifferentiated spermatogonia. Further, exposure to only SARS-CoV-2 envelope protein, but not Spike or nucleocapsid proteins led to cytopathic effects on testicular cells that was dependent on the TLR2 receptor. A similar trend was observed in the K18h-ACE2 mouse model which revealed gross pathology in the absence of virus replication in the testis. Collectively, data strongly indicates that the testicular injury is not due to direct infection of SARS-CoV-2 but more likely an indirect effect of exposure to systemic inflammation or SARS-CoV-2 antigens. Data also provide novel insights into the mechanism of testicular injury and could explain the clinical manifestation of testicular symptoms associated with severe COVID-19.

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mentioning

confidence: 68%

In vitro evidence against productive SARS-CoV-2 infection of human testicular cells: Bystander effects of infection mediate testicular injury

Giannakopoulos

Strange

Jiyarom

et al. 2022

Preprint

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“…Rationale for the fusion assays pathways (Simmons et al, 2004;Matsuyama et al, 2005;Du et al, 2007;Bertram et al, 2011). Trypsin has also been found to activate SARS-CoV and SARS-CoV-2 infection and fusion (Letko et al, 2020;Xia et al, 2020;Kim et al, 2022). Trypsin cleaves at both the S1/S2 R667 and the S2′ R797 of the SARS-1-S (Belouzard et al, 2009) and at multiple sites within the S1/S2 of the SARS-2-S (Jaimes et al, 2020;Mustafa et al, 2021).…”

Section: Resultsmentioning

confidence: 99%

Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation

Chan

2022

Front. Pharmacol.

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Virus fusion process is evolutionarily conserved and provides a promising pan-viral target. Cell-cell fusion leads to syncytial formation and has implications in pathogenesis, virus spread and immune evasion. Drugs that target these processes can be developed into anti-virals. Here, we have developed sensitive, rapid, adaptable fusion reporter gene assays as models for plasma membrane and alternative fusion pathways as well as syncytial fusion in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and have confirmed their specificity using neutralizing antibodies and specific protease inhibitors. The fusion report gene assays are more sensitive and unbiased than morphological fusion assay. The fusion assays can differentiate between transmembrane serine protease 2 (TMPRSS2)-dependency in TMPRSS2(+) cells and trypsin-dependency in angiotensin-converting enzyme 2 (ACE2)(+)TMPRSS2(-) cells. Moreover, we have identified putative novel fusion processes that are triggered by an acidic pH with and without trypsin. Coupled with morphological fusion criteria, we have found that syncytia formation is enhanced by TMPRSS2 or trypsin. By testing against our top drug hits previously shown to inhibit SARS-CoV-2 pseudovirus infection, we have identified several fusion inhibitors including structurally related lopsided kite-shaped molecules. Our results have important implications in the development of universal blockers and synergistic therapeutics and the small molecule inhibitors can provide important tools in elucidating the fusion process.

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“…This variant has been demonstrated to rely primarily on endocytosis and not on membrane fusion for entry in vitro and in vivo [17]. Both virus isolates were pre-treated with trypsin prior to infection to proteolytically cleave the S protein and bypass the need of cellular proteases [18]. Under these conditions, infection of NTg Calu-3 cells with SARS-CoV-2 Omicron resulted in ~15-fold higher infection (as measured by % of N-positive cells at 24 hpt) than with the WA1 strain ( Figure 5C ).…”

Section: Resultsmentioning

confidence: 99%

ARF6 is a host factor for SARS-CoV-2 infectionin vitro

Mirabelli

Sherman

Wotring

et al. 2022

Preprint

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SARS-CoV-2 is a newly emerged beta-coronavirus that enter cells via two routes, direct fusion at the plasma membrane or endocytosis followed by fusion with the late endosome/lysosome. While the viral receptor, ACE2, multiple entry factors, and the mechanism of fusion of the virus at the plasma membrane have been extensively investigated, viral entry via the endocytic pathway is less understood. By using a human hepatocarcinoma cell line, Huh-7, which is resistant to the antiviral action of the TMPRSS2 inhibitor camostat, we discovered that SARS-CoV-2 entry is not dependent on dynasore but dependent on cholesterol. ADP-ribosylation factor 6 (ARF6) has been described as a host factor for SARS-CoV2 replication and it is involved in the entry and infection of several pathogenic viruses. By CRISPR-Cas9 genetic deletion, we found that ARF6 is important for SARS-CoV-2 uptake and infection in Huh-7. In addition, the ARF6 inhibitor NAV-2729, and the ARF6 agonist AA147, showed a dose-responsive inhibition or enhancement of viral infection, respectively. Importantly, ARF6 inhibition reduced SARS-CoV-2 viral loads also in more physiologic models of infection: Calu-3 and kidney organoids, suggesting a role also in post-entry steps. Together, these experiments points to a ARF6 as a putative target to develop antiviral strategies against SARS-CoV-2.

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Trypsin enhances SARS-CoV-2 infection by facilitating viral entry

Cited by 35 publications

References 40 publications

In vitro evidence against productive SARS-CoV-2 infection of human testicular cells: Bystander effects of infection mediate testicular injury

In vitro evidence against productive SARS-CoV-2 infection of human testicular cells: Bystander effects of infection mediate testicular injury

Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation

ARF6 is a host factor for SARS-CoV-2 infectionin vitro

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