Development and characterization of a packaging cell line for pseudo-infectious yellow fever virus particle generation

Pseudo-infectious yellow fever viral particles (YFV-PIVs) have been used to study vaccines and viral packaging. Here, we report the development of a packaging cell line, which expresses the YFV prM/E proteins.

METHODS:

HEK293 cells were transfected with YFV prM/E and C (84 nt) genes to generate HEK293-YFV-PrM/E-opt. The cells were evaluated for their ability to express the heterologous proteins and to package the replicon repYFV-17D-LucIRES, generating YFV-PIVs.

RESULTS:

The expression of prM/E proteins was confirmed, and the cell line trans-packaged the replicon for recovery of a reporter for the YFV-PIVs.

CONCLUSIONS:

HEK293-YFV-prM/E-opt trans-packaging capacity demonstrates its possible biotechnology application.

Keywords:
Yellow fever virus; Trans-packaging; Pseudo-infectious particles

Authorship

Sabrina Ribeiro de Almeida Queiroz

Departamento de Virologia e Terapia Experimental, Instituto Aggeu Magalhães, Fundação Oswaldo Cruz, Recife, PE, Brasil. Fundação Oswaldo CruzBrazilRecife, PE, BrazilDepartamento de Virologia e Terapia Experimental, Instituto Aggeu Magalhães, Fundação Oswaldo Cruz, Recife, PE, Brasil.

José Valter Joaquim Silva Júnior

Andréa Nazaré Monteiro Rangel da Silva

Laboratório de Virologia, Instituto de Ciências Biológicas, Universidade Federal do Pará-UFPA, Belém, Pará, Brasil. Universidade Federal do ParáBrazilBelém, Pará, BrazilLaboratório de Virologia, Instituto de Ciências Biológicas, Universidade Federal do Pará-UFPA, Belém, Pará, Brasil.

Amanda Gomes de Oliveira Carvalho

Jefferson José da Silva Santos

Department of Population Health, Poultry Diagnostic and Research Center, University of Georgia, Athens, USA. University of GeorgiaUSAAthens, USADepartment of Population Health, Poultry Diagnostic and Research Center, University of Georgia, Athens, USA.

Laura Helena Vega Gonzales Gil

Corresponding author: Dra. Laura Helena Vega Gonzales Gil. e-mail:laura@cpqam.fiocruz.br

Conflict of interest: The authors declare that there is no conflict of interest.

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Figures

Figures (3)

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FIGURE 1:
(A): Indirect immunofluorescence (IFA) for the analysis of prM/E protein expression by the HEK293-YFV-prM/E-opt cell line. After fixation, HEK293-YFV-prM/E-opt cells were incubated with mouse polyclonal antibodies specific for flaviviruses (group B) and then, with the anti-mouse IgG secondary antibody (FITC). Magnification, 630×. (B): Evaluation of E protein expression by flow cytometry. HEK293-YFV-prM/E-opt cells were permeabilized and incubated with an anti-YFV-17D E protein oligoclonal antibody. Cells were then washed and incubated with an anti-IgG secondary antibody. Next, cells were washed and resuspended in formaldehyde, and the read was performed. R3: negative cells; R2: 90% cells, positive for E protein expression. (C): Western blotting to confirm E protein expression in the HEK293-YFV-prM/E-opt cells. The SSP and the HEK293-YFV-prM/E-opt cell pellets were lysed and subjected to SDS-PAGE. Protein bands were transferred to nitrocellulose membranes that were incubated with a YFV anti-E antibody and then with a peroxidase-conjugated antibody. The E protein bands were visualized with the Immobilon Western Chemiluminescent HRP substrate. 1 - supernatant from HEK293 cells infected with YFV-17D; 2 - supernatant from non-infected HEK293 cells; 3 - supernatant from HEK293-YFV-prM/E-opt cells. Western blot of the HEK293-YFV-prM/E-opt cell extract showed the same band pattern (data not shown).

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FIGURE 2:
Schematic representation of the repYFV-17D-LucIRES and repYFV-17D-Luc replicons. The repYFV-17D-LucIRES (A): and repYFV-17D-Luc. (B): replicons contain all of the coding regions for the YFV non-structural proteins and are distinguished by the presence of the complete capsid region only in the repYFV-17D-LucIRES construct. YFV: yellow fever virus; Luc: luceferase gene; IRES: internal ribosome entry site; UTR: untranslated regions; NS: non-structural proteins.

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FIGURE 3:
Evaluation of the trans-packaging capacity of the HEK293-YFV-prM/E-opt cell line. After transfection of the HEK293-YFV-prM/E-opt cell line with the repYFV-17D-LucIRES or repYFV-17D-Luc replicon, the supernatants from both transfections were inoculated into another HEK293-YFV-prM/E-opt or HEK293 cell culture. Luciferase activity was detected only in the cells infected with the supernatant from the transfection with repYFV-17D-LucIRES. YFV: yellow fever virus; Luc: luceferase gene; IRGS: internal ribosome entry site.

FIGURE 1: (A): Indirect immunofluorescence (IFA) for the analysis of prM/E protein expression by the HEK293-YFV-prM/E-opt cell line. After fixation, HEK293-YFV-prM/E-opt cells were incubated with mouse polyclonal antibodies specific for flaviviruses (group B) and then, with the anti-mouse IgG secondary antibody (FITC). Magnification, 630×. (B): Evaluation of E protein expression by flow cytometry. HEK293-YFV-prM/E-opt cells were permeabilized and incubated with an anti-YFV-17D E protein oligoclonal antibody. Cells were then washed and incubated with an anti-IgG secondary antibody. Next, cells were washed and resuspended in formaldehyde, and the read was performed. R3: negative cells; R2: 90% cells, positive for E protein expression. (C): Western blotting to confirm E protein expression in the HEK293-YFV-prM/E-opt cells. The SSP and the HEK293-YFV-prM/E-opt cell pellets were lysed and subjected to SDS-PAGE. Protein bands were transferred to nitrocellulose membranes that were incubated with a YFV anti-E antibody and then with a peroxidase-conjugated antibody. The E protein bands were visualized with the Immobilon Western Chemiluminescent HRP substrate. 1 - supernatant from HEK293 cells infected with YFV-17D; 2 - supernatant from non-infected HEK293 cells; 3 - supernatant from HEK293-YFV-prM/E-opt cells. Western blot of the HEK293-YFV-prM/E-opt cell extract showed the same band pattern (data not shown).

FIGURE 2: Schematic representation of the repYFV-17D-LucIRES and repYFV-17D-Luc replicons. The repYFV-17D-LucIRES (A): and repYFV-17D-Luc. (B): replicons contain all of the coding regions for the YFV non-structural proteins and are distinguished by the presence of the complete capsid region only in the repYFV-17D-LucIRES construct. YFV: yellow fever virus; Luc: luceferase gene; IRES: internal ribosome entry site; UTR: untranslated regions; NS: non-structural proteins.

FIGURE 3: Evaluation of the trans-packaging capacity of the HEK293-YFV-prM/E-opt cell line. After transfection of the HEK293-YFV-prM/E-opt cell line with the repYFV-17D-LucIRES or repYFV-17D-Luc replicon, the supernatants from both transfections were inoculated into another HEK293-YFV-prM/E-opt or HEK293 cell culture. Luciferase activity was detected only in the cells infected with the supernatant from the transfection with repYFV-17D-LucIRES. YFV: yellow fever virus; Luc: luceferase gene; IRGS: internal ribosome entry site.

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Sociedade Brasileira de Medicina Tropical - SBMT Sociedade Brasileira de Medicina Tropical - SBMT, Núcleo de Medicina Tropical UnB, Sala 43C 70904-970, E-mails: rsbmt@uftm.edu.br | artes.rsbmt@gmail.com | sbmt@sbmt.org.br , WhatsApp: SBMT (61) 9.9192-6496, WhatsApp: RSBMT (34) 9.9996-5807 - Brasília - DF - Brazil
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Development and characterization of a packaging cell line for pseudo-infectious yellow fever virus particle generation

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[1] Corresponding author: Dra. Laura Helena Vega Gonzales Gil. e-mail:laura@cpqam.fiocruz.br