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

Le Coupanec A, Desforges M, Meessen-Pinard M, Dubé M, Day R, Seidah NG, Talbot PJ - PLoS Pathog. (2015)

Bottom Line: Human coronaviruses (HCoV) are respiratory pathogens that may be associated with the development of neurological diseases, in view of their neuroinvasive and neurotropic properties.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Neuroimmunovirology, INRS-Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada.

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↓ G758 to RRSR↓R758), 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.

No MeSH data available.


Related in: MedlinePlus

Cleavage of S glycoprotein is also observed in human LA-N-5 cells for mutant virus.The differentiated human neuroblastoma cell line (LA-N-5) was infected with rOC/ATCC or rOC/SG758R at MOI 0.1. Proteins in association with cell or in supernatant were extracted at 24 and 48 hpi, and kinetics of viral replication was evaluated over a period of 48 hpi for (A) cell-associated virus or (B) free virus (supernatant). Titers of cell-associated and free virus were significantly higher for rOC/SG758R compared to rOC/ATCC (* P≤0.05 and ** P≤0.01). Western blot analysis of whole cell lysates (C) or cell culture supernatant (D) (10 μg of proteins) revealed the presence of the uncleaved form of the S glycoprotein (180 kDa), and of a cleaved form at around 100 kDa (S1/S2). Results shown are the mean values (with standard deviations) of three independent experiments.
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ppat.1005261.g007: Cleavage of S glycoprotein is also observed in human LA-N-5 cells for mutant virus.The differentiated human neuroblastoma cell line (LA-N-5) was infected with rOC/ATCC or rOC/SG758R at MOI 0.1. Proteins in association with cell or in supernatant were extracted at 24 and 48 hpi, and kinetics of viral replication was evaluated over a period of 48 hpi for (A) cell-associated virus or (B) free virus (supernatant). Titers of cell-associated and free virus were significantly higher for rOC/SG758R compared to rOC/ATCC (* P≤0.05 and ** P≤0.01). Western blot analysis of whole cell lysates (C) or cell culture supernatant (D) (10 μg of proteins) revealed the presence of the uncleaved form of the S glycoprotein (180 kDa), and of a cleaved form at around 100 kDa (S1/S2). Results shown are the mean values (with standard deviations) of three independent experiments.

Mentions: In order to further study the role of the G758R mutation on the biology of both HCoV-OC43 variants, we first evaluated the kinetics of viral replication and spread within mixed primary CNS cultures from BALB/c mice over a period of 72 h post-infection (hpi). Using immunofluorescence, we observed no change in cell tropism, with neurons remaining the main target of infection by both virus variants (Fig 5), even though astrocytes could also be infected later in the infection (S3 Fig) as we previously reported [10]. Interestingly, we did observe a delay in viral spread in neurons for the mutant virus at 8 and 24 hpi compared to the reference strain (Fig 5). Interestingly, even though the infection was shown to be productive for both variants in primary CNS cultures from BALB/c mice, there was a significant increase in the total amount of infectious virus in the cell culture supernatant (free virus) between 48 and 72 hpi for the mutant virus compared to the reference virus rOC/ATCC (Fig 6B). As the G758R mutation creates a putative furin-like cleavage site [16] in the S glycoprotein previously reported to influence viral infectivity [20–22, 24], we wished to evaluate whether cleavage was indeed associated with the delayed spreading in neuronal cells, the increased release of infectious virus and eventually with neurovirulence. As seen in Fig 6, our data correlated with a much stronger cleavage of the S protein of the rOC/SG758R mutant into S1/S2 fragments, compared to reference virus rOC/ATCC at 24 and 48 hpi (Fig 6C; whole cell lysate), which was even more obvious at 48 hpi in the cell supernatant (Fig 6D). In order to evaluate whether this cleavage of the viral S protein also took place in human cells, we made use of the differentiated LA-N-5 neuronal cell line described in the Materials and Methods section [27] and showed, first, that the kinetics of viral replication was similar to that observed between both viruses in murine primary cells (Fig 7A and 7B), as there was a significant increase of virus release for the rOC/SG758R mutant and, second, that the cleavage of the S protein into S1/S2 fragments was again predominantly detected in the cell culture supernatant (Fig 7D) compared to the protein associated with cells (Fig 7C). Again, this cleavage was more evident for mutant than for reference virus. Similar results were obtained with HRT-18 cells. Even though the S protein of HCoV-OC43 reference virus was present mostly in the uncleaved form, our results also show that there are intermediate size bands between the uncleaved and furin-like cleaved forms of the protein. These secondary bands may be unspecific degradation products, but we suggest that among these intermediate size fragments seen on SDS-PAGE, there could be a fragment corresponding to the S protein cleaved at a potential alternative site (S2’ in Figs 6, 7, 8 and S4, the latter showing corresponding overexpositions).


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

Le Coupanec A, Desforges M, Meessen-Pinard M, Dubé M, Day R, Seidah NG, Talbot PJ - PLoS Pathog. (2015)

Cleavage of S glycoprotein is also observed in human LA-N-5 cells for mutant virus.The differentiated human neuroblastoma cell line (LA-N-5) was infected with rOC/ATCC or rOC/SG758R at MOI 0.1. Proteins in association with cell or in supernatant were extracted at 24 and 48 hpi, and kinetics of viral replication was evaluated over a period of 48 hpi for (A) cell-associated virus or (B) free virus (supernatant). Titers of cell-associated and free virus were significantly higher for rOC/SG758R compared to rOC/ATCC (* P≤0.05 and ** P≤0.01). Western blot analysis of whole cell lysates (C) or cell culture supernatant (D) (10 μg of proteins) revealed the presence of the uncleaved form of the S glycoprotein (180 kDa), and of a cleaved form at around 100 kDa (S1/S2). Results shown are the mean values (with standard deviations) of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4636366&req=5

ppat.1005261.g007: Cleavage of S glycoprotein is also observed in human LA-N-5 cells for mutant virus.The differentiated human neuroblastoma cell line (LA-N-5) was infected with rOC/ATCC or rOC/SG758R at MOI 0.1. Proteins in association with cell or in supernatant were extracted at 24 and 48 hpi, and kinetics of viral replication was evaluated over a period of 48 hpi for (A) cell-associated virus or (B) free virus (supernatant). Titers of cell-associated and free virus were significantly higher for rOC/SG758R compared to rOC/ATCC (* P≤0.05 and ** P≤0.01). Western blot analysis of whole cell lysates (C) or cell culture supernatant (D) (10 μg of proteins) revealed the presence of the uncleaved form of the S glycoprotein (180 kDa), and of a cleaved form at around 100 kDa (S1/S2). Results shown are the mean values (with standard deviations) of three independent experiments.
Mentions: In order to further study the role of the G758R mutation on the biology of both HCoV-OC43 variants, we first evaluated the kinetics of viral replication and spread within mixed primary CNS cultures from BALB/c mice over a period of 72 h post-infection (hpi). Using immunofluorescence, we observed no change in cell tropism, with neurons remaining the main target of infection by both virus variants (Fig 5), even though astrocytes could also be infected later in the infection (S3 Fig) as we previously reported [10]. Interestingly, we did observe a delay in viral spread in neurons for the mutant virus at 8 and 24 hpi compared to the reference strain (Fig 5). Interestingly, even though the infection was shown to be productive for both variants in primary CNS cultures from BALB/c mice, there was a significant increase in the total amount of infectious virus in the cell culture supernatant (free virus) between 48 and 72 hpi for the mutant virus compared to the reference virus rOC/ATCC (Fig 6B). As the G758R mutation creates a putative furin-like cleavage site [16] in the S glycoprotein previously reported to influence viral infectivity [20–22, 24], we wished to evaluate whether cleavage was indeed associated with the delayed spreading in neuronal cells, the increased release of infectious virus and eventually with neurovirulence. As seen in Fig 6, our data correlated with a much stronger cleavage of the S protein of the rOC/SG758R mutant into S1/S2 fragments, compared to reference virus rOC/ATCC at 24 and 48 hpi (Fig 6C; whole cell lysate), which was even more obvious at 48 hpi in the cell supernatant (Fig 6D). In order to evaluate whether this cleavage of the viral S protein also took place in human cells, we made use of the differentiated LA-N-5 neuronal cell line described in the Materials and Methods section [27] and showed, first, that the kinetics of viral replication was similar to that observed between both viruses in murine primary cells (Fig 7A and 7B), as there was a significant increase of virus release for the rOC/SG758R mutant and, second, that the cleavage of the S protein into S1/S2 fragments was again predominantly detected in the cell culture supernatant (Fig 7D) compared to the protein associated with cells (Fig 7C). Again, this cleavage was more evident for mutant than for reference virus. Similar results were obtained with HRT-18 cells. Even though the S protein of HCoV-OC43 reference virus was present mostly in the uncleaved form, our results also show that there are intermediate size bands between the uncleaved and furin-like cleaved forms of the protein. These secondary bands may be unspecific degradation products, but we suggest that among these intermediate size fragments seen on SDS-PAGE, there could be a fragment corresponding to the S protein cleaved at a potential alternative site (S2’ in Figs 6, 7, 8 and S4, the latter showing corresponding overexpositions).

Bottom Line: Human coronaviruses (HCoV) are respiratory pathogens that may be associated with the development of neurological diseases, in view of their neuroinvasive and neurotropic properties.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Neuroimmunovirology, INRS-Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada.

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↓ G758 to RRSR↓R758), 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.

No MeSH data available.


Related in: MedlinePlus