Amino acid T25 in the substrate-binding domain of SARS-CoV-2 nsp5 is involved in viral replication in the mouse lung DOI Creative Commons

Yoshiro Sugiura,

Kenta Shimizu,

Tatsuki Takahashi

et al.

PLoS ONE, Journal Year: 2024, Volume and Issue: 19(12), P. e0312800 - e0312800

Published: Dec. 6, 2024

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) non-structural protein 5 (nsp5) is a cysteine protease involved in viral replication and suppression of the host immune system. The substrate-binding domain nsp5 important for its activity. However, relationship between activity remains unclear. We confirmed importance amino acid T25 using split luciferase assay. By generating recombinant viruses bacterial artificial chromosomes, we found that proliferation with T25I mutation was cell-dependent culture. Furthermore, mice infected mutant virus mouse acclimation backbone showed weight loss increased lung load, similar to wild-type (WT) group, up 3 days after infection. day 4, load significantly reduced T25I-infected group compared WT-infected group. This suggests pathogenesis SARS-CoV-2.

Language: Английский

Immunogenicity and safety of a live-attenuated SARS-CoV-2 vaccine candidate based on multiple attenuation mechanisms DOI Creative Commons

Mie Suzuki-Okutani,

Shinya Okamura,

Tang Gis

et al.

eLife, Journal Year: 2025, Volume and Issue: 13

Published: Feb. 11, 2025

mRNA vaccines against SARS-CoV-2 were rapidly developed and effective during the pandemic. However, some limitations remain to be resolved, such as short-lived induced immune response certain adverse effects. Therefore, there is an urgent need develop new that address these issues. While live-attenuated are a highly modality, they pose risk of effects, including virulence reversion. In current study, we constructed vaccine candidate, BK2102, combining naturally occurring virulence-attenuating mutations in NSP14 , NSP1 spike, ORF7-8 coding regions. Intranasal inoculation with BK2102 humoral cellular responses Syrian hamsters without apparent tissue damage lungs, leading protection D614G Omicron BA.5 strains. The neutralizing antibodies by persisted for up 364 days, which indicated confer long-term infection. Furthermore, confirmed safety using transgenic (Tg) mice expressing human ACE2 (hACE2) susceptible SARS-CoV-2. did not kill Tg mice, even when virus was administered at dose 10 6 plaque-forming units (PFUs), while 2 PFU strain or attenuated lacking furin cleavage site spike sufficient mice. These results suggest promising live-vaccine candidate confers significant virulence.

Language: Английский

Citations

2
Immunogenicity and safety of a live-attenuated SARS-CoV-2 vaccine candidate based on multiple attenuation mechanisms DOI Open Access

Mie Suzuki-Okutani,

Shinya Okamura,

Tanggis

et al.

Published: Jan. 6, 2025

mRNA vaccines against SARS-CoV-2 were rapidly developed and effective during the pandemic. However, some limitations remain to be resolved, such as short-lived induced immune response certain adverse effects. Therefore, there is an urgent need develop new that address these issues. While live-attenuated are a highly modality, they pose risk of effects, including virulence reversion. In current study, we constructed vaccine candidate, BK2102, combining naturally occurring virulence-attenuating mutations in NSP14 , NSP1 spike ORF7-8 coding regions. Intranasal inoculation with BK2102 humoral cellular responses Syrian hamsters without apparent tissue damage lungs, leading protection D614G Omicron BA.5 strains. The neutralizing antibodies by persisted for up 364 days, which indicated confer long-term infection. Furthermore, confirmed safety using transgenic (Tg) mice expressing human ACE2 (hACE2), susceptible SARS-CoV-2. did not kill Tg mice, even when virus was administered at dose 10 6 plaque-forming units (PFU), while 2 PFU strain or attenuated lacking furin cleavage site (FCS) sufficient mice. These results suggest promising live-vaccine candidate confers significant virulence.

Language: Английский

Citations

0
Amino acid substitutions in NSP6 and NSP13 of SARS-CoV-2 contribute to superior virus growth at low temperatures DOI Creative Commons
Yuri Furusawa, Maki Kiso, Ryuta Uraki

et al.

Journal of Virology, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 12, 2025

ABSTRACT In general, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates well at 37°C, which is the temperature of human lower tract, but it poorly 30°C‒32°C, upper tract. The replication efficiency SARS-CoV-2 in tract may directly affect its transmissibility. this study, an XBB.1.5 isolate showed superior replicative ability 32°C and 30°C, whereas most other Omicron sub-variant isolates limited growth. Deep sequencing analysis demonstrated that frequencies viruses possessing NSP6-S163P NSP13-P238S substitutions increased to more than 97% during propagation did not reach 55% 37°C. Reverse genetics revealed these contributed virus growth vitro low temperatures by improving genome replication. Mutant both slightly higher titers hamsters compared parental virus; however, transmissibility between was similar for mutant viruses. Taken together, our findings indicate contribute hamsters. IMPORTANCE Severe efficiently However, airway 30°C–32°C. Therefore, could influence we assessed sub-variants found ability. By deep reverse genetics, amino acid changes NSP6 NSP13 low-temperature growth; improved RNA polymerase activity enhanced Although alone drastically transmissibility, combination with substitutions, they humans. Furthermore, since enhance cultured cells, be used improve production inactivated or live attenuated vaccine virus.

Language: Английский

Citations

0
The NSP6-L260F substitution in SARS-CoV-2 BQ.1.1 and XBB.1.16 lineages compensates for the reduced viral polymerase activity caused by mutations in NSP13 and NSP14 DOI Creative Commons
Yuri Furusawa, Kiyoko Iwatsuki‐Horimoto, Seiya Yamayoshi

et al.

Journal of Virology, Journal Year: 2025, Volume and Issue: unknown

Published: May 13, 2025

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants emerged at the end of 2021, and their subvariants are still circulating worldwide. While changes in S protein these have been extensively studied, roles amino acid substitutions non-structural proteins not fully revealed. In this study, we found that SARS-CoV-2 bearing NSP6-L260F substitution repeatedly when generated several by reverse genetics or passaged isolated from clinical samples it was selected under cell culture conditions. Although has detected BQ.1.1 XBB.1.16 circulated nature, its effect on viral properties is unclear. Here, with without promotes virus replication vitro vivo increasing polymerase activity enhancing pathogenicity hamsters. We also identified disadvantageous substitutions, NSP13-M233I NSP14-D222Y, reduced replication, respectively. These adverse effects were compensated for NSP6-L260F. Our findings suggest importance reveal part evolutionary process variants. IMPORTANCE severe continue to change through acquisition various explored. enhances important pathogenicity. addition, lineage NSP14-D222Y reduce activity, substitution. results provide insight into SARS-CoV-2.

Language: Английский

Citations

0
Immunogenicity and safety of a live-attenuated SARS-CoV-2 vaccine candidate based on multiple attenuation mechanisms DOI Creative Commons

Mie Suzuki-Okutani,

Shinya Okamura,

Tang Gis

et al.

eLife, Journal Year: 2024, Volume and Issue: 13

Published: April 26, 2024

mRNA vaccines against SARS-CoV-2 were rapidly developed and effective during the pandemic. However, some limitations remain to be resolved, such as short-lived induced immune response certain adverse effects. Therefore, there is an urgent need develop new that address these issues. While live-attenuated are a highly modality, they pose risk of effects, including virulence reversion. In current study, we constructed vaccine candidate, BK2102, combining naturally occurring virulence-attenuating mutations in NSP14 , NSP1 spike, ORF7-8 coding regions. Intranasal inoculation with BK2102 humoral cellular responses Syrian hamsters without apparent tissue damage lungs, leading protection D614G Omicron BA.5 strains. The neutralizing antibodies by persisted for up 364 days, which indicated confer long-term infection. Furthermore, confirmed safety using transgenic (Tg) mice expressing human ACE2 (hACE2) susceptible SARS-CoV-2. did not kill Tg mice, even when virus was administered at dose 10 6 plaque-forming units (PFUs), while 2 PFU strain or attenuated lacking furin cleavage site spike sufficient mice. These results suggest promising live-vaccine candidate confers significant virulence.

Language: Английский

Citations

1
Immunogenicity and safety of a live-attenuated SARS-CoV-2 vaccine candidate based on multiple attenuation mechanisms DOI Creative Commons

Mie Suzuki-Okutani,

Shinya Okamura,

Tanggis

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: March 4, 2024

Abstract mRNA vaccines against SARS-CoV-2 were rapidly developed and effective during the pandemic. However, some limitations remain to be resolved, such as short-lived induced immune response certain adverse effects. Therefore, there is an urgent need develop new that address these issues. While live-attenuated are a highly modality, they pose risk of effects, including virulence reversion. In current study, we constructed vaccine candidate, BK2102, combining naturally occurring virulence-attenuating mutations in NSP14 , NSP1 spike ORF7-8 coding regions. Intranasal inoculation with BK2102 humoral cellular responses Syrian hamsters without apparent tissue damage lungs, leading protection D614G Omicron BA.5 strains. The neutralizing antibodies by persisted for up 364 days, which indicated confer long-term infection. Furthermore, confirmed safety using transgenic (Tg) mice expressing human ACE2 (hACE2), susceptible SARS-CoV-2. did not kill Tg mice, even when virus was administered at dose 10 6 plaque-forming units (PFU), while 2 PFU strain or attenuated lacking furin cleavage site (FCS) sufficient mice. These results suggest promising live-vaccine candidate confers significant virulence.

Language: Английский

Citations

0
Amino acid T25 in the substrate-binding domain of SARS-CoV-2 nsp5 is involved in viral replication in the mouse lung DOI Creative Commons

Yoshiro Sugiura,

Kenta Shimizu,

Tatsuki Takahashi

et al.

PLoS ONE, Journal Year: 2024, Volume and Issue: 19(12), P. e0312800 - e0312800

Published: Dec. 6, 2024

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) non-structural protein 5 (nsp5) is a cysteine protease involved in viral replication and suppression of the host immune system. The substrate-binding domain nsp5 important for its activity. However, relationship between activity remains unclear. We confirmed importance amino acid T25 using split luciferase assay. By generating recombinant viruses bacterial artificial chromosomes, we found that proliferation with T25I mutation was cell-dependent culture. Furthermore, mice infected mutant virus mouse acclimation backbone showed weight loss increased lung load, similar to wild-type (WT) group, up 3 days after infection. day 4, load significantly reduced T25I-infected group compared WT-infected group. This suggests pathogenesis SARS-CoV-2.

Language: Английский

Citations

0