RESUMEN
The protein activator of protein kinase R (PKR) (PACT) has been shown to play a crucial role in stimulating the host antiviral response through the activation of PKR, retinoic acid-inducible gene I, and melanoma differentiation-associated protein 5. Whether PACT can inhibit viral replication independent of known mechanisms is still unrevealed. In this study, we show that, like many viruses, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks GSK-3ß to facilitate its replication. GSK-3ß-induced phosphorylation on N protein increased the interaction between N protein and nsp3. Thus, GSK-3ß-N-nsp3 cascade promotes viral replication. Although SARS-CoV-2 can sabotage the activation of AKT, the upstream proteins suppressing the activation of GSK-3ß, we found that the host can use PACT, another protein kinase, instead of AKT to decrease the activity of GSK-3ß and the interaction between PACT and GSK-3ß is enhanced upon viral infection. Moreover, PACT inhibited the activity of GSK-3ß independent of its well-studied double-stranded RNA binding and PKR activating ability. In summary, this study identified an unknown function of PACT in inhibiting SARS-CoV-2 replication through the blockage of GSK-3ß-N-nsp3 cascade.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , SARS-CoV-2/metabolismo , Línea Celular , Proteínas Proto-Oncogénicas c-akt/metabolismo , FosforilaciónRESUMEN
SARS-CoV Spike (S) protein shares considerable homology with SARS-CoV-2 S, especially in the conserved S2 subunit (S2). S protein mediates coronavirus receptor binding and membrane fusion, and the latter activity can greatly influence coronavirus infection. We observed that SARS-CoV S is less effective in inducing membrane fusion compared with SARS-CoV-2 S. We identify that S813T mutation is sufficient in S2 interfering with the cleavage of SARS-CoV-2 S by TMPRSS2, reducing spike fusogenicity and pseudoparticle entry. Conversely, the mutation of T813S in SARS-CoV S increased fusion ability and viral replication. Our data suggested that residue 813 in the S was critical for the proteolytic activation, and the change from threonine to serine at 813 position might be an evolutionary feature adopted by SARS-2-related viruses. This finding deepened the understanding of Spike fusogenicity and could provide a new perspective for exploring Sarbecovirus' evolution.
Asunto(s)
COVID-19 , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , Humanos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Proteolisis , Replicación Viral , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del Virus , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismoRESUMEN
Understanding the process of replication and transcription of SARS-CoV-2 is essential for antiviral strategy development. The replicase polyprotein is indispensable for viral replication. However, whether all nsps derived from the replicase polyprotein of SARS-CoV-2 are indispensable is not fully understood. In this study, we utilized the SARS-CoV-2 replicon as the system to investigate the role of each nsp in viral replication. We found that except for nsp16, all the nsp deletions drastically impair the replication of the replicon, and nsp14 could recover the replication deficiency caused by its deletion in the viral replicon. Due to the unsuccessful expressions of nsp1, nsp3, and nsp16, we could not draw a conclusion about their in trans-rescue functions. Our study provided a new angle to understand the role of each nsp in viral replication and transcription, helping the evaluation of nsps as the target for antiviral drug development.