AIM: To determine if calnexin(CANX), RAB1 and alphatubulin were involved in the production of hepatitis C virus(HCV) particles by baby hamster kidney-West Nile virus(BHK-WNV) cells. METHODS: Using a si RNA-based appro...AIM: To determine if calnexin(CANX), RAB1 and alphatubulin were involved in the production of hepatitis C virus(HCV) particles by baby hamster kidney-West Nile virus(BHK-WNV) cells. METHODS: Using a si RNA-based approach complemented with immuno-fluorescence confocal microscope and Western blot studies, we examined the roles of CANX, RAB1 and alpha-tubulin in the production of HCV particles by permissive BHK-WNV cells expressing HCV structural proteins or the full-length genome of HCV genotype 1a. Immuno-fluorescence studies in producer cells were performed with monoclonal antibodies against HCV structural proteins, as well as immunoglobulin from the serum of a patient recently cured from an HCV infection of same genotype. The cellular compartment stained by the serum immunoglobulin was also observedin thin section transmission electron microscopy. These findings were compared with the JFH-1 strain/Huh-7.5 cell model.RESULTS: We found that CANX was necessary for the production of HCV particles by BHK-WNV cells. This process involved the recruitment of a subset of HCV proteins, detected by immunoglobulin of an HCV-cured patient, in a compartment of rearranged membranes bypassing the endoplasmic reticulum-Golgi intermediary compartment and surrounded by mitochondria. It also involved the maturation of N-linked glycans on HCV envelope proteins, which was required for assembly and/or secretion of HCV particles. The formation of this specialized compartment required RAB1; upon expression of HCV structural genes, this compartment developed large vesicles with viral particles. RAB1 and alpha-tubulin were required for the release of HCV particles. These cellular factors were also involved in the production of HCVcc in the JFH-1 strain/Huh-7.5 cell system, which involves HCV RNA replication. The secretion of HCV particles by BHK-WNV cells presents similarities with a pathway involving caspase-1; a caspase-1 inhibitor was found to suppress the production of HCV particles from a full-length genome.CONCLUSION: Prior activity of the WNV subgenomic replicon in BHK-21 cells promoted re-wiring of host factors for the assembly and release of infectious HCV in a caspase-1-dependent mechanism.展开更多
Viral protein R(Vpr) plays an important role in the replication and pathogenesis of Human immunodeficiency virus type 1(HIV-1). Some of the various functions attributed to Vpr, including the induction of G2/M cell cyc...Viral protein R(Vpr) plays an important role in the replication and pathogenesis of Human immunodeficiency virus type 1(HIV-1). Some of the various functions attributed to Vpr, including the induction of G2/M cell cycle arrest, activating the NF-κB pathway, and promoting viral reverse transcription, might be interrelated. To test this hypothesis, a panel of Vpr mutants were investigated for their ability to induce G2/M arrest and to activate the NF-κB pathway. The results showed that the Vpr mutants that failed to activate NF-κB also lost the activity to induce G2/M arrest, which suggests that inducing G2/M arrest via Vpr depends at least partially on the activation of NF-κB. This latter possibility is supported by data showing that knocking down the key factors in the NF-κB pathway – p65, Rel B, IKKα, or IKKβ– partially rescued the G2/M arrest induced by Vpr.Our results suggest that the NF-κB pathway is probably involved in Vpr-induced G2/M cell cycle arrest.展开更多
The generation of stable clones for biomolecule production is a common but lengthy and labor-intensive process.For complex molecules,such as viruses or virus-like particles(VLPs),the timeline becomes even more cumbers...The generation of stable clones for biomolecule production is a common but lengthy and labor-intensive process.For complex molecules,such as viruses or virus-like particles(VLPs),the timeline becomes even more cumbersome.Thus,in the early stages of development,transient production methods serve as a reasonable alternative to stable clone construction.In this work,an investigation of a polyethylenimine-(PEI-)based transfection method for the transient production of Chikungunya(Chik)VLPs,a vaccine candidate molecule,was undertaken.This effort focuses on tracking cell population responses during transfection,understanding how process changes affect these responses,and monitoring patterns in cell performance over the culture duration.Plasmid labeling and VLP staining were employed to comprehensively track cells via flow cytometry and to draw correlations between plasmid DNA(pDNA)uptake and the resulting VLP expression.The method detected high transfection efficiency(≥97%)in all samples tested and demonstrated the capability to track kinetics of plasmid-cell binding.With varied transfection cell concentrations,the pDNA binding kinetics are altered and saturation binding is observed in the lowest cell concentration sample tested in less than 3 hours of incubation.Interestingly,in all samples,the flow cytometry analysis of relative pDNA amount versus VLP expression staining showed that cells which contained fewer pDNA complexes resulted in the highest levels of VLP stain.Finally,to determine the potential breadth of our observations,we compared daily expression patterns of ChikVLP with a reporter,monomeric GFP molecule.The similarities detected suggest the interpretations presented here to likely be more broadly informative and applicable to PEI-based transient production of additional biological products as well.展开更多
基金Supported by Intramural Program of the National Institutes of Health,National Institute of Allergy and Infectious Diseases(Project No.1 ZIA AI000733-15:Enveloped Virus Glycoprotein/Receptor Interactions)to Edward A Berger,PhD(MSS,LVD,DIR,NIAID)ORISE Senior Fellow award(Award No.1238-1238-03:Department of Energy/Oak Ridge Institute for Science and Education)to Bertrand Saunier,MD,PhD
文摘AIM: To determine if calnexin(CANX), RAB1 and alphatubulin were involved in the production of hepatitis C virus(HCV) particles by baby hamster kidney-West Nile virus(BHK-WNV) cells. METHODS: Using a si RNA-based approach complemented with immuno-fluorescence confocal microscope and Western blot studies, we examined the roles of CANX, RAB1 and alpha-tubulin in the production of HCV particles by permissive BHK-WNV cells expressing HCV structural proteins or the full-length genome of HCV genotype 1a. Immuno-fluorescence studies in producer cells were performed with monoclonal antibodies against HCV structural proteins, as well as immunoglobulin from the serum of a patient recently cured from an HCV infection of same genotype. The cellular compartment stained by the serum immunoglobulin was also observedin thin section transmission electron microscopy. These findings were compared with the JFH-1 strain/Huh-7.5 cell model.RESULTS: We found that CANX was necessary for the production of HCV particles by BHK-WNV cells. This process involved the recruitment of a subset of HCV proteins, detected by immunoglobulin of an HCV-cured patient, in a compartment of rearranged membranes bypassing the endoplasmic reticulum-Golgi intermediary compartment and surrounded by mitochondria. It also involved the maturation of N-linked glycans on HCV envelope proteins, which was required for assembly and/or secretion of HCV particles. The formation of this specialized compartment required RAB1; upon expression of HCV structural genes, this compartment developed large vesicles with viral particles. RAB1 and alpha-tubulin were required for the release of HCV particles. These cellular factors were also involved in the production of HCVcc in the JFH-1 strain/Huh-7.5 cell system, which involves HCV RNA replication. The secretion of HCV particles by BHK-WNV cells presents similarities with a pathway involving caspase-1; a caspase-1 inhibitor was found to suppress the production of HCV particles from a full-length genome.CONCLUSION: Prior activity of the WNV subgenomic replicon in BHK-21 cells promoted re-wiring of host factors for the assembly and release of infectious HCV in a caspase-1-dependent mechanism.
基金supported by grants from the Chinese Ministry of Health (2012ZX10001006)the National Natural Science Foundation of China (81271812 and 31370182)+1 种基金111 Project (B08011)the Postgraduate Scholarship Program of the China Scholarship Council
文摘Viral protein R(Vpr) plays an important role in the replication and pathogenesis of Human immunodeficiency virus type 1(HIV-1). Some of the various functions attributed to Vpr, including the induction of G2/M cell cycle arrest, activating the NF-κB pathway, and promoting viral reverse transcription, might be interrelated. To test this hypothesis, a panel of Vpr mutants were investigated for their ability to induce G2/M arrest and to activate the NF-κB pathway. The results showed that the Vpr mutants that failed to activate NF-κB also lost the activity to induce G2/M arrest, which suggests that inducing G2/M arrest via Vpr depends at least partially on the activation of NF-κB. This latter possibility is supported by data showing that knocking down the key factors in the NF-κB pathway – p65, Rel B, IKKα, or IKKβ– partially rescued the G2/M arrest induced by Vpr.Our results suggest that the NF-κB pathway is probably involved in Vpr-induced G2/M cell cycle arrest.
基金This work was supported by the intramural research program of the Vaccine Research Center(VRC)National Institute of Allergy and Infectious Diseases(NIAID)National Insti-tutes of Health(NIH).
文摘The generation of stable clones for biomolecule production is a common but lengthy and labor-intensive process.For complex molecules,such as viruses or virus-like particles(VLPs),the timeline becomes even more cumbersome.Thus,in the early stages of development,transient production methods serve as a reasonable alternative to stable clone construction.In this work,an investigation of a polyethylenimine-(PEI-)based transfection method for the transient production of Chikungunya(Chik)VLPs,a vaccine candidate molecule,was undertaken.This effort focuses on tracking cell population responses during transfection,understanding how process changes affect these responses,and monitoring patterns in cell performance over the culture duration.Plasmid labeling and VLP staining were employed to comprehensively track cells via flow cytometry and to draw correlations between plasmid DNA(pDNA)uptake and the resulting VLP expression.The method detected high transfection efficiency(≥97%)in all samples tested and demonstrated the capability to track kinetics of plasmid-cell binding.With varied transfection cell concentrations,the pDNA binding kinetics are altered and saturation binding is observed in the lowest cell concentration sample tested in less than 3 hours of incubation.Interestingly,in all samples,the flow cytometry analysis of relative pDNA amount versus VLP expression staining showed that cells which contained fewer pDNA complexes resulted in the highest levels of VLP stain.Finally,to determine the potential breadth of our observations,we compared daily expression patterns of ChikVLP with a reporter,monomeric GFP molecule.The similarities detected suggest the interpretations presented here to likely be more broadly informative and applicable to PEI-based transient production of additional biological products as well.
