A Luciferase Gene Driven by an Alphaherpesviral Promoter Also Responds to Immediate Early Antigens of the Betaherpesvirus HCMV, Allowing Comparative Analyses of Different Human Herpesviruses in One Reporter Cell Line
| Autor: | Anna Katharina Maier, Raimund Jung, Clarissa Villinger, Axel Schubert, Paul Walther, Christian Sinzger, Diana Lieber |
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| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
Physiology
viruses lcsh:Medicine Cytomegalovirus Gene Expression Immunostaining Alphaherpesvirinae Virus Replication Biochemistry Viral Packaging Genes Reporter Immune Physiology Medicine and Health Sciences Small interfering RNAs lcsh:Science Luciferases Promoter Regions Genetic Antigens Viral physiology [Cytomegalovirus] Staining Immune System Proteins virus diseases Enzymes Nucleic acids genetics [Alphaherpesvirinae] immunology [Immediate-Early Proteins] ultrastructure [Cytomegalovirus] Oxidoreductases Luciferase genetics [Luciferases] Research Article Immunology ultrastructure [Virion] Genome Viral Research and Analysis Methods Microbiology Antibodies Cell Line Immediate-Early Proteins Gene Types Virology Genetics Animals Humans ddc:610 Non-coding RNA lcsh:R metabolism [Immediate-Early Proteins] Virion Biology and Life Sciences Proteins immunology [Antigens Viral] metabolism [Antigens Viral] Viral Replication Gene regulation Viral Tropism Specimen Preparation and Treatment Enzymology RNA lcsh:Q Reporter Genes |
| Zdroj: | PLoS ONE PLOS ONE 12(1), e0169580 (2017). doi:10.1371/journal.pone.0169580 PLoS ONE, Vol 12, Iss 1, p e0169580 (2017) |
| ISSN: | 1932-6203 |
| DOI: | 10.1371/journal.pone.0169580 |
| Popis: | Widely used methods for quantification of human cytomegalovirus (HCMV) infection in cell culture such as immunoblotting or plaque reduction assays are generally restricted to low throughput and require time-consuming evaluation. Up to now, only few HCMV reporter cell lines have been generated to overcome these restrictions and they are afflicted with other limitations because permanently expandable cell lines are normally not fully permissive to HCMV. In this work, a previously existing epithelial cell line hosting a luciferase gene under control of a Varicella-zoster virus promoter was adopted to investigate HCMV infection. The cells were susceptible to different HCMV strains at infection efficiencies that corresponded to their respective degree of epithelial cell tropism. Expression of early and late viral antigens, formation of nuclear inclusions, release of infectious virus progeny, and focal growth indicated productive viral replication. However, viral release and spread occurred at lower levels than in primary cell lines which appears to be due to a malfunction of virion morphogenesis during the nuclear stage. Expression of the luciferase reporter gene was specifically induced in HCMV infected cultures as a function of the virus dose and dependent on viral immediate early gene expression. The level of reporter activity accurately reflected infection efficiencies as determined by viral antigen immunostaining, and hence could discriminate the cell tropism of the tested virus strains. As proof-of-principle, we demonstrate that this cell line is applicable to evaluate drug resistance of clinical HCMV isolates and the neutralization capacity of human sera, and that it allows comparative and simultaneous analysis of HCMV and human herpes simplex virus type 1. In summary, the permanent epithelial reporter cell line allows robust, rapid and objective quantitation of HCMV infection and it will be particularly useful in higher throughput analyses as well as in comparative analyses of different human herpesviruses. |
| Databáze: | OpenAIRE |
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