In a recent study posted to the bioRxiv* preprint server, researchers assessed the impact of targeting intracellular Neu1 on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.
There is an urgent need to develop coronavirus disease 2019 (COVID-19) treatments and SARS-CoV-2 antivirals. Additionally, studies show that the novel SARS-CoV-2 mutations are less resistant to the current vaccinations. Therefore, to create therapeutic drugs, it is vital to comprehend the SARS-CoV-2 pathogenicity mechanisms and the host response.
About the study
In the present study, researchers demonstrated that host Neu1 controlled the sialylation of the coronavirus nucleocapsid protein, thus regulating coronavirus replication.
The team employed mass spectrometry to study the N- and O-glycans on the nucleocapsid (N) protein from the human coronavirus OC43 (HCoV-OC43), a beta coronavirus that causes mild respiratory symptoms. A lectin blot was performed using samples immunoprecipitated from the serum of SARS-CoV-2-infected patients and healthy controls, as well as cell lysates infected with HCoV-OC43 and treated with anti-N protein antibodies.
The lectin blot ascertained whether sialylation occurred on N protein corresponding to coronavirus HCoV-OC43 and SARS-CoV-2. The immunoprecipitated samples were either subjected or not to sialidase treatment before undergoing sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separation.
The team investigated how the sialylation on the N protein might impact its capacity to bind ribonucleic acid (RNA). Nucleic acid-binding tests were performed with a 32-mer stem-loop II (32m) motif single-stranded RNA (ssRNA) and its 32-mer ss-deoxyribonucleic acid (DNA) mimic to evaluate the nucleic acid-binding affinity of N protein. The HEK293T cells were lysed 48 hours after the SARS-CoV-2 N protein expression vector was transfected.
Subsequently, cells were administered with or without sialidase for nucleic acid-binding experiments. Using THP-1 cell lines, the team assessed the role of endogenous sialidases in the sialylation of N protein.
The team assessed how sialylation might impact viral replication. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to measure the viral infection using primers targeting the viral N gene’s coding region. After the viral challenge, cell RNA was collected at the designated times, and viral transcripts were measured. Additionally, supernatants underwent processing for the 50% tissue culture infective dose (TCID50) assay, which was used to measure the virus titer.
Results
The study results showed that while N- and O-link glycosylation were observed on the N protein in vitro overexpress system, it is unclear if the N protein from the virion is glycosylated or not. The SARS-CoV-2 spike (S), envelope (E), and membrane (M) proteins are all glycosylated. N protein was significantly sialylated in cells from both COVID-19- and HCoV-OC43-infected patients. Most of the sialic acid on N protein was attached in alpha-2, 6 links, while sialidase treatment verified N protein sialylation. A SARS-CoV-2 N protein expressed in HEK293T cells, HCoV-OC43-N protein expressed in THP-1 cells, and HCoV-OC43 virion were all found to be sialylated.
In the presence of anti-N protein antibodies, a SARS-CoV-2 N protein formed a potent complex with 32-mer ssRNA and 32-mer ssDNA that was supershifted, demonstrating that this complex was exclusive to N protein. As anticipated, lysates of HEK293T cells transfected with an empty vector failed to bind 32-mer ssRNA and ssDNA. Furthermore, the amount of 32-mer ssDNA and ssRNA bound to N protein after sialidase treatment rose significantly. These results corroborated the crucial function of N protein sialylation in RNA binding by showing a considerable increase in N protein RNA binding activity following sialidase treatment.
After being infected with HCoV-OC43 for 72 hours, Neu1 expression was considerably elevated, but not Neu2, Neu3, or Neu4, according to real-time PCR and western blot analyses. Notably, patients with COVID-19 had upregulated Neu1 as well. Additionally, in cells infected with HCoV-OC43, N protein was associated with Neu1.
Two days after the viral challenge, the replication of HCoV-OC43 was more than 10-fold greater in cell culture supernatants of cells that overexpressed Neu1 as compared to cells that expressed empty vectors at the level of viral transcripts and viral titers. In contrast, HCoV-OC43 replication was over 100-fold lower in cells that overexpressed short hairpin RNA (shRNA) for Neu1 than those that expressed scrambled shRNA, both in terms of viral transcripts and viral titers.
In line with the knockdown effectiveness of shRNA, Neu1sh3 dramatically reduced HCoV-OC43 replication more than Neu1sh1 and Neu1sh2 did. N protein levels were also noticeably higher in Neu1 overexpressing cells than in Neu1 knockdown cells. These findings suggested that host Neu1 controlled HCoV-OC43 replication in THP-1 cells. The most effective protection was provided by Neu5Ac2en-OAcOMe, while viral neuraminidase inhibitors Zanamivir and oseltamivir displayed little inhibitory activity. These results supported earlier reports that Zanamivir and oseltamivir have weak anti-human sialidases activity.
The HCoV-OC43 challenge resulted in the death of all vehicle-treated mice but only 50% of animals treated with Neu5Ac2en-OAcOMe survived the entire observation period. Compared to mice treated with Neu5Ac2en-OAcOMe, vehicle-treated mice had considerably lower body weight. Mice treated with Neu5Ac2en-OAcOMe also reduced HCoV-OC43 viral replication in the blood, brain, and lungs.
The study findings showed that a newly developed sialidase inhibitor, Neu5Ac2en-OAcOMe, targeted the intracellular host sialidase Neu1 selectively and suppressed coronavirus replication, accounting for the roles of the host as well as the pathogen in disease manifestation.
*Important notice
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
