Abstract
Primary varicella-zoster virus (VZV) infection leads to varicella and the establishment of lifelong latency in sensory ganglion neurons. Reactivation of latent VZV causes herpes zoster, which is frequently associated with chronic pain. Latent viral gene expression is restricted to the VZV latency-associated transcript (VLT) and VLT-ORF63 (VLT63) fusion transcripts. Since VLT and VLT63 encode proteins that are expressed during lytic infection, we investigated whether pVLT and pVLT-ORF63 are essential for VZV replication by performing VZV genome mutagenesis using CRISPR/Cas9 and BAC technologies. We first established that CRISPR/Cas9 can efficiently mutate VZV genomes in lytically VZV-infected cells through targeting non-essential genes ORF8 and ORF11 and subsequently show recovery of viable mutant viruses. By contrast, the VLT region was markedly resistant to CRISPR/Cas9 editing. Whereas most mutants expressed wild-type or N-terminally altered versions of pVLT and pVLT-ORF63, only a minority of the resulting mutant viruses lacked pVLT and pVLT-ORF63 coding potential. Growth curve analysis showed that pVLT/pVLT-ORF63 negative viruses were viable, but impaired in growth in epithelial cells. We confirmed this phenotype independently using BAC-derived pVLT/pVLT-ORF63 negative and repaired viruses. Collectively, these data demonstrate that pVLT and/or pVLT-ORF63 are dispensable for lytic VZV replication but promote efficient VZV infection in epithelial cells.
| Original language | English |
|---|---|
| Article number | 2289 |
| Journal | Viruses |
| Volume | 13 |
| Issue number | 11 |
| DOIs | |
| Publication status | Published - Nov 2021 |
Bibliographical note
Funding Information:S.E.B. was in part supported by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) project 022.005.032. G.M.G.M.V. and W.J.D.O. are supported by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health under Award Number R01AI151290; The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. D.P.D. is supported by a DZIF (Deutsches Zentrum f?r Infektionsforschung) Professorship. T.S. received funding from the Tokyo Biochemical Research Foundation, the Takeda Science Foundation, and the Japan Society for the Promotion of Science (JSPS KAKENHI JP24590551) and the Ministry of Education, Culture, Sports, Science and Technology (MEXT KAKENHI JP21H02741). We thank Stipan Jonjic for providing the mouse monoclonal anti-VZV ORF8 an-tibody, Yasuko Mori (Kobe University) for the pOka-BAC genome and Gregory Smith (Northwestern University) for GS1783 E. coli.
Funding Information:
Funding: S.E.B. was in part supported by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) project 022.005.032. G.M.G.M.V. and W.J.D.O. are supported by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health under Award Number R01AI151290; The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. D.P.D. is supported by a DZIF (Deutsches Zentrum für Infektionsforschung) Professorship. T.S. received funding from the Tokyo Biochemical Research Foundation, the Takeda Science Foundation, and the Japan Society for the Promotion of Science (JSPS KAKENHI JP24590551) and the Ministry of Education, Culture, Sports, Science and Technology (MEXT KAKENHI JP21H02741).
Publisher Copyright:
© 2021 by the authors. Licensee MDPI, Basel, Switzerland.
