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Infection of Chinese hamster ovary cells by pseudorabies virus
R Nixdorf1, J Schmidt, A Karger
1Institute of Molecular Biology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, D-17498 Insel Riems, Germany.
Abstract:
Chinese hamster ovary (CHO) cells have recently been used for identification of receptors for several alphaherpesviruses, including pseudorabies virus (PrV) (R. J. Geraghty, C. Krummenacher, G. H. Cohen, R. J. Eisenberg, and P. G. Spear, Science 280:1618-1620, 1998). The experiments were based on the fact that CHO cells are inefficient target cells for PrV. However, a detailed analysis of the interaction between PrV and CHO wild-type and recombinant PrV-receptor bearing cells has not been performed. We show here that PrV has a growth defect on CHO cells which leads to a ca. 100-fold reduction in plating efficiency, strongly delayed penetration kinetics, and a 10(4)-fold reduction in one-step growth. Entry of PrV into CHO cells is significantly delayed but is not affected by inhibitors of endocytosis, suggesting that the mechanism of penetration resembles that on permissive cells. The defects in plating efficiency and penetration could be corrected by expression of herpesvirus entry mediators B (HveB), HveC, or HveD, with HveC being the most effective. However, the defects in one-step growth and plaque formation were not corrected by expression of PrV receptors, indicating an additional restriction in viral replication after entry. Surprisingly, PrV infection of CHO cells was sensitive to neutralization by a gB-specific monoclonal antibody, which does not inhibit PrV infection of other host cells. Moreover, the same monoclonal antibody neutralized PrV infectivity on cells displaying the interference phenomenon by overexpression of gD and subsequent intracellular sequestration of gD receptors. Thus, absence of gD receptors on two different host cells leads to an increased sensitivity of PrV toward gB neutralization. We hypothesize that this is due to the increased requirement for interaction of gB with a cellular surface protein in the absence of the gD-gD receptor interaction. As expected, CHO cells are as susceptible as other host cells to infection by PrV gD(-) Pass, an infectious gD-negative PrV mutant. However, PrV gD(-) Pass was also not able to form plaques on CHO cells.
Insights
Pseudorabies virus (PrV) shows growth defects in Chinese hamster ovary (CHO) cells, with entry mediated by specific receptors but replication restricted post-entry. This suggests a dual mechanism for PrV host cell interaction and infectivity.
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- Chinese hamster ovary (CHO) cells are known to be inefficient targets for pseudorabies virus (PrV).
- Previous studies identified receptors for alphaherpesviruses in CHO cells, but a detailed analysis of PrV interaction with these cells was lacking.
Purpose of the Study:
- To comprehensively analyze the interaction between PrV and both wild-type and recombinant PrV-receptor bearing CHO cells.
- To investigate the mechanisms underlying PrV's growth defect in CHO cells and identify factors influencing viral entry and replication.
Main Methods:
- Infection of CHO wild-type and recombinant cells with PrV.
- Analysis of plating efficiency, penetration kinetics, and one-step growth.
- Assessment of viral entry using endocytosis inhibitors.
- Correction of defects by expressing herpesvirus entry mediators (HveB, HveC, HveD).
- Neutralization assays using a gB-specific monoclonal antibody.
- Infection studies with a gD-negative PrV mutant (PrV gD(-) Pass).
Main Results:
- PrV exhibited a significant growth defect in CHO cells, with reduced plating efficiency and delayed penetration.
- Expression of HveB, HveC, or HveD partially corrected entry defects, with HveC being most effective.
- Receptor expression did not correct defects in viral replication or plaque formation, indicating post-entry restrictions.
- PrV infection of CHO cells was sensitive to gB-specific antibody neutralization, unlike in other host cells.
- Absence of gD receptors increased PrV sensitivity to gB neutralization, suggesting a greater reliance on gB-mediated interactions.
Conclusions:
- PrV entry into CHO cells is mediated by specific receptors (HveB, HveC, HveD), but viral replication is restricted post-entry.
- The absence of gD receptors on CHO cells enhances PrV sensitivity to gB-mediated neutralization.
- This suggests a complex interplay between viral glycoproteins and host cell factors governing PrV infectivity and host range.