Cleavage of a Neuroinvasive Human Respiratory Virus Spike Glycoprotein by Proprotein Convertases Modulates Neurovirulence and Virus Spread within the Central Nervous System

Abstract

Human coronaviruses (HCoV) are respiratory pathogens that may be associated with the development of neurological diseases, in view of their neuroinvasive and neurotropic properties. The viral spike (S) glycoprotein is a major virulence factor for several coronavirus species, including the OC43 strain of HCoV (HCoV-OC43). In an attempt to study the role of this protein in virus spread within the central nervous system (CNS) and neurovirulence, as well as to identify amino acid residues important for such functions, we compared the sequence of the S gene found in the laboratory reference strain HCoV-OC43 ATCC VR-759 to S sequences of viruses detected in clinical isolates from the human respiratory tract. We identified one predominant mutation at amino acid 758 (from RRSR↓ G 758 to RRSR↓R 758), which introduces a putative furin-like cleavage (↓) site. Using a molecular cDNA infectious clone to generate a corresponding recombinant virus, we show for the first time that such point mutation in the HCoV-OC43 S glycoprotein creates a functional cleavage site between the S1 and S2 portions of the S protein. While the corresponding recombinant virus retained its neuroinvasive properties, this mutation led to decreased neurovirulence while potentially modifying the mode of virus spread, likely leading to a limited dissemination within the CNS. Taken together, these results are consistent with the adaptation of HCoV-OC43 to the CNS environment, resulting from the selection of quasi-species harboring mutations that lead to amino acid changes in viral genes, like the S gene in HCoV-OC43, which may contribute to a more efficient establishment of a less pathogenic but persistent CNS infection. This adaptative mechanism could potentially be associated with human encephalitis or other neurological degenerative pathologies.

The OC43 human coronavirus envelope protein is critical for infectious virus production and propagation in neuronal cells and is a determinant of neurovirulence and CNS pathology

“…Functional viroporins and viral PBM are associated with viral pathogenesis for a growing number of viruses and their studies to better understand virus-host interaction represent an emerging field (Javier and Rice, 2011;Nieva et al, 2012;Scott and Griffin, 2015). Considering that the HCoV-OC43 E protein seems to possess both functions and that they seem to be important for the induction of disease, future studies regarding their functionality and underlying mechanisms resulting in HCoV-OC43 neuropathogenesis previously described (Brison et al, 2011;Jacomy et al, 2006;Le Coupanec et al, 2015) are warranted and necessary as they will help to identify virus-host interfaces which could represent therapeutic target.…”

“…As HCoV-OC43 is naturally neuroinvasive and neurovirulent in mice (Brison et al, 2011;Desforges et al, 2014;Le Coupanec et al, 2015) and that the E protein is important for efficient propagation in neuronal cells (Figs. 3 and 4), we wished to investigate the importance of the two functional domains of E in the process of neuroinvasion.…”

“…Mixed primary cultures of mouse CNS cells were prepared as previously described (Le Coupanec et al, 2015). Briefly, embryos at 15 days of gestation were removed from pregnant anesthetized CD1 mice and their cortex and hippocampus were harvested and placed in Hanks balanced salt solution (HBSS) medium, without Ca 2+ and Mg 2+ , supplemented with 1 mM sodium pyruvate and 10 mM HEPES buffer.…”

“…Infection of 22-day-old female or 7-day-old male and female C57Bl/ 6 mice (Charles River) were performed as previously described (Le Coupanec et al, 2015). Briefly, mice were inoculated respectively by the IC route with 10 1.5 or the intranasal route with 10 3 of 50% tissue culture infective doses (TCID 50 ) recombinant virus.…”

The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade

“…They have the potential to cleave specifically viral envelope glycoproteins, thereby enhancing viral fusion with host cell membranes (Izaguirre, 2019;Moulard and Decroly, 2000). In the case of human-infecting coronaviruses such as HCoV-OC43 (Le Coupanec et al, 2015), MERS-CoV (Millet and Whittaker, 2014), and HKU1 (Chan et al, 2008) the spike protein has been demonstrated to be cleaved at an S1/S2 cleavage site (Fig. 2) generating the S1 and S2 subunits.…”