Human cytomegaloviruses expressing yellow fluorescent fusion proteins--characterization and use in antiviral screening.
Recombinant viruses labelled with fluorescent proteins are useful tools in molecular virology with multiple applications (e.g., studies on intracellular trafficking, protein localization, or gene activity). We generated by homologous recombination three recombinant cytomegaloviruses carrying the enhanced yellow fluorescent protein (EYFP) fused with the viral proteins IE-2, ppUL32 (pp150), and ppUL83 (pp65). In growth kinetics, the three viruses behaved all like wild type, even at low multiplicity of infection (MOI). The expression of all three fusion proteins was detected, and their respective localizations were the same as for the unmodified proteins in wild-type virus-infected cells. We established the in vivo measurement of fluorescence intensity and used the recombinant viruses to measure inhibition of viral replication by neutralizing antibodies or antiviral substances. The use of these viruses in a pilot screen based on fluorescence intensity and high-content analysis identified cellular kinase inhibitors that block viral replication. In summary, these viruses with individually EYFP-tagged proteins will be useful to study antiviral substances and the dynamics of viral infection in cell culture.
Straschewski
Sarah
S
Institute of Virology, Ulm University Hospital, Ulm, Germany.
Warmer
Martin
M
Frascaroli
Giada
G
Hohenberg
Heinrich
H
Mertens
Thomas
T
Winkler
Michael
M
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000998:Antiviral Agents / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000821:virology; D015774:Ganciclovir / Q000494:pharmacology; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008856:Microscopy, Fluorescence; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000187:drug effects
D000998:Antiviral Agents; C069186:IE2 protein, Cytomegalovirus; D017874:Immediate-Early Proteins; D008164:Luminescent Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014763:Viral Matrix Proteins; D014764:Viral Proteins; C065418:cytomegalovirus matrix protein 65kDa; C056367:pp150 protein, Cytomegalovirus; D015774:Ganciclovir