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Exploitation of herpesviral transactivation allows quantitative reporter gene-based assessment of virus entry and neutralization.

Reinhard H, Le VT, Ohlin M, Hengel H, Trilling M - PLoS ONE (2011)

Bottom Line: Based on promoter transactivation of a reporter gene we established a novel method to quantify herpesvirus entry and neutralization by antibodies.The response was MOI-dependent and immunoblots confirmed a correlation between luciferase induction and pp72-IE1 expression.Monoclonal antibodies, immune sera and purified immunoglobulin preparations decreased virus-dependent luciferase induction dose-dependently, qualifying this approach as surrogate virus neutralization test.

View Article: PubMed Central - PubMed

Affiliation: Institute for Virology, Heinrich-Heine-University, Düsseldorf, Germany.

ABSTRACT
Herpesviral entry is a highly elaborated process requiring many proteins to act in precise conjunction. Neutralizing antibodies interfere with this process to abrogate viral infection. Based on promoter transactivation of a reporter gene we established a novel method to quantify herpesvirus entry and neutralization by antibodies. Following infection with mouse and human cytomegalovirus and Herpes simplex virus 1 we observed promoter transactivation resulting in substantial luciferase expression (>1000-fold). No induction was elicited by UV-inactivated viruses. The response was MOI-dependent and immunoblots confirmed a correlation between luciferase induction and pp72-IE1 expression. Monoclonal antibodies, immune sera and purified immunoglobulin preparations decreased virus-dependent luciferase induction dose-dependently, qualifying this approach as surrogate virus neutralization test. Besides the reduced hands-on time, this assay allows analysis of herpesvirus entry in semi-permissive and non-adherent cells, which were previously non-assessable but play significant roles in herpesvirus pathology.

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The episomal vector pB45Neo-promLuc confers herpesvirus responsiveness.(A) HeLa cells were transiently transfected with the pB45Neo-promLUC construct by nucleofection. Cells were infected with HSV-1 F strain (0.5 or 3 PFU/cell) or left uninfected. Cells were lysed and luciferase activity was determined. (B) G418-selected stable M2-10B4 cell clones (#1, #3 and #4) were infected with 5 PFU/cell HSV-1 F strain for 16 hours. Cells were lysed and luciferase activity was determined.
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pone-0014532-g002: The episomal vector pB45Neo-promLuc confers herpesvirus responsiveness.(A) HeLa cells were transiently transfected with the pB45Neo-promLUC construct by nucleofection. Cells were infected with HSV-1 F strain (0.5 or 3 PFU/cell) or left uninfected. Cells were lysed and luciferase activity was determined. (B) G418-selected stable M2-10B4 cell clones (#1, #3 and #4) were infected with 5 PFU/cell HSV-1 F strain for 16 hours. Cells were lysed and luciferase activity was determined.

Mentions: To allow establishment of stable cell lines harbouring the herpesvirus-responsive element, we sub-cloned the luciferase gene together with the corresponding promoter from the pTA-Control vector into a bovine papillomavirus-derived episomal vector (pB45Neo) [27]. Upon transient transfection of this vector (pB45Neo-promLUC) into HeLa cells, infection with HSV-1 induced luciferase expression (Fig. 2a). Clonal M2-10B4 cell lines harbouring pB45Neo-promLUC (selected by G418/geneticin) also responded to HSV-1 infection with robust luciferase induction (Fig. 2b).


Exploitation of herpesviral transactivation allows quantitative reporter gene-based assessment of virus entry and neutralization.

Reinhard H, Le VT, Ohlin M, Hengel H, Trilling M - PLoS ONE (2011)

The episomal vector pB45Neo-promLuc confers herpesvirus responsiveness.(A) HeLa cells were transiently transfected with the pB45Neo-promLUC construct by nucleofection. Cells were infected with HSV-1 F strain (0.5 or 3 PFU/cell) or left uninfected. Cells were lysed and luciferase activity was determined. (B) G418-selected stable M2-10B4 cell clones (#1, #3 and #4) were infected with 5 PFU/cell HSV-1 F strain for 16 hours. Cells were lysed and luciferase activity was determined.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3022015&req=5

pone-0014532-g002: The episomal vector pB45Neo-promLuc confers herpesvirus responsiveness.(A) HeLa cells were transiently transfected with the pB45Neo-promLUC construct by nucleofection. Cells were infected with HSV-1 F strain (0.5 or 3 PFU/cell) or left uninfected. Cells were lysed and luciferase activity was determined. (B) G418-selected stable M2-10B4 cell clones (#1, #3 and #4) were infected with 5 PFU/cell HSV-1 F strain for 16 hours. Cells were lysed and luciferase activity was determined.
Mentions: To allow establishment of stable cell lines harbouring the herpesvirus-responsive element, we sub-cloned the luciferase gene together with the corresponding promoter from the pTA-Control vector into a bovine papillomavirus-derived episomal vector (pB45Neo) [27]. Upon transient transfection of this vector (pB45Neo-promLUC) into HeLa cells, infection with HSV-1 induced luciferase expression (Fig. 2a). Clonal M2-10B4 cell lines harbouring pB45Neo-promLUC (selected by G418/geneticin) also responded to HSV-1 infection with robust luciferase induction (Fig. 2b).

Bottom Line: Based on promoter transactivation of a reporter gene we established a novel method to quantify herpesvirus entry and neutralization by antibodies.The response was MOI-dependent and immunoblots confirmed a correlation between luciferase induction and pp72-IE1 expression.Monoclonal antibodies, immune sera and purified immunoglobulin preparations decreased virus-dependent luciferase induction dose-dependently, qualifying this approach as surrogate virus neutralization test.

View Article: PubMed Central - PubMed

Affiliation: Institute for Virology, Heinrich-Heine-University, Düsseldorf, Germany.

ABSTRACT
Herpesviral entry is a highly elaborated process requiring many proteins to act in precise conjunction. Neutralizing antibodies interfere with this process to abrogate viral infection. Based on promoter transactivation of a reporter gene we established a novel method to quantify herpesvirus entry and neutralization by antibodies. Following infection with mouse and human cytomegalovirus and Herpes simplex virus 1 we observed promoter transactivation resulting in substantial luciferase expression (>1000-fold). No induction was elicited by UV-inactivated viruses. The response was MOI-dependent and immunoblots confirmed a correlation between luciferase induction and pp72-IE1 expression. Monoclonal antibodies, immune sera and purified immunoglobulin preparations decreased virus-dependent luciferase induction dose-dependently, qualifying this approach as surrogate virus neutralization test. Besides the reduced hands-on time, this assay allows analysis of herpesvirus entry in semi-permissive and non-adherent cells, which were previously non-assessable but play significant roles in herpesvirus pathology.

Show MeSH
Related in: MedlinePlus