Generation and characterization of an EICP0 null mutant of equine herpesvirus 1.
Abstract: The EICP0 gene (gene 63) of equine herpesvirus 1 (EHV-1) encodes an early regulatory protein that is a promiscuous trans-activator of all classes of viral genes. Bacterial artificial chromosome (BAC) technology and RecE/T cloning were employed to delete the EICP0 gene from EHV-1 strain KyA. Polymerase chain reaction, Southern blot analysis, and DNA sequencing confirmed the deletion of the EICP0 gene and its replacement with a kanamycin resistance gene in mutant KyA. Transfection of rabbit kidney cells with the EICP0 mutant genome produced infectious virus, indicating that the EICP0 gene is not essential for KyA replication in cell culture. Experiments to assess the effect of the EICP0 deletion on EHV-1 gene programming revealed that mRNA expression of the immediate-early gene and representative early and late genes as well as the synthesis of these viral proteins were reduced as compared to the kinetics of viral mRNA and protein synthesis observed for the wild type virus. However, the transition from early to late viral gene expression was not prevented or delayed, suggesting that the absence of the EICP0 gene did not disrupt the temporal aspects of EHV-1 gene regulation. The extracellular virus titer and plaque areas of the EICP0 mutant virus KyADeltaEICP0, in which the gp2-encoding gene 71 gene that is absent in the KyA BAC was restored, were reduced by 10-fold and 19%, respectively, when compared to parental KyA virus; while the titer and plaque areas of mutant KyADeltaEICP0Deltagp2 that lacks both the EICP0 gene and gene 71 were reduced more than 50-fold and 67%, respectively. The above results show that the EICP0 gene is dispensable for EHV-1 replication in cell culture, and that the switch from early to late viral gene expression for the representative genes examined does not require the EICP0 protein, but that the EICP0 protein may be structurally required for virus egress and cell-to-cell spread.
Publication Date: 2003-12-09 PubMed ID: 14659563DOI: 10.1016/j.virusres.2003.09.007Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- P.H.S.
Summary
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The research paper explores the role of the EICP0 gene in the replication of equine herpesvirus 1 (EHV-1) in cells. The EICP0 gene was deleted from the virus, and it was observed that the lack of the EICP0 gene affects the virus’s gene programming and cell-to-cell spread but does not stop its replication.
Generation of EICP0 Null Mutant
- The EICP0 gene, which usually plays a significant role in activating all classes of viral genes, was removed from the EHV-1 strain KyA using bacterial artificial chromosome (BAC) technology and RecE/T cloning.
- Methods such as Polymerase chain reaction, Southern blot analysis, and DNA sequencing confirmed that the EICP0 gene was successfully removed and replaced with a kanamycin resistance gene.
Impact on KyA Replication and Viral Gene Programming
- The researchers found out that the replication of the virus is still possible even without the EICP0 gene. This was seen when rabbit kidney cells that were transfected with the EICP0 mutant genome produced the virus.
- Furthermore, it was observed that the absence of the EICP0 gene reduces mRNA expression of the immediate-early gene and other representative early and late genes, which are crucial in virus proliferation and development. It also significantly affects the synthesis of these viral proteins resulting in slower kinetics.
- Despite this, the transition from early to late viral gene expression, an important cycle for viral survival, was not prevented or delayed, although the EICP0 gene may play a significant role in the timely aspect of this transition process.
Effect on Virus Egress and Cell-to-Cell Spread
- The EICP0 gene might have a structural role in virus egress and cell-to-cell spread, as the extracellular virus titer and plaque areas were reduced up to 19% and 67% respectively when the EICP0 gene and gene 71 were removed.
- Therefore, the analysis indicates that while the EICP0 gene is not integral for EHV-1 replication in cell culture or for the switch from early to late viral gene expression, it appears to be essential for virus egress and spread between cells.
Cite This Article
APA
Yao H, Osterrieder N, O'Callaghan DJ.
(2003).
Generation and characterization of an EICP0 null mutant of equine herpesvirus 1.
Virus Res, 98(2), 163-172.
https://doi.org/10.1016/j.virusres.2003.09.007 Publication
Researcher Affiliations
- Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, 1501 Kings Highway, P.O. Box 33932, Shreveport, LA 71130-3932, USA.
MeSH Terms
- Animals
- Cells, Cultured
- Gene Expression Regulation, Viral
- Herpesvirus 1, Equid / genetics
- Herpesvirus 1, Equid / metabolism
- Viral Regulatory and Accessory Proteins / genetics
- Viral Regulatory and Accessory Proteins / metabolism
Grant Funding
- AI-22001 / NIAID NIH HHS
Citations
This article has been cited 8 times.- Roy P, Noad R. Use of bacterial artificial chromosomes in baculovirus research and recombinant protein expression: current trends and future perspectives. ISRN Microbiol 2012;2012:628797.
- Warden C, Tang Q, Zhu H. Herpesvirus BACs: past, present, and future. J Biomed Biotechnol 2011;2011:124595.
- Ahn BC, Zhang Y, O'Callaghan DJ. The equine herpesvirus-1 (EHV-1) IR3 transcript downregulates expression of the IE gene and the absence of IR3 gene expression alters EHV-1 biological properties and virulence. Virology 2010 Jul 5;402(2):327-37.
- Everett RD, Boutell C, McNair C, Grant L, Orr A. Comparison of the biological and biochemical activities of several members of the alphaherpesvirus ICP0 family of proteins. J Virol 2010 Apr;84(7):3476-87.
- Breitenbach JE, Ebner PD, O'Callaghan DJ. The IR4 auxiliary regulatory protein expands the in vitro host range of equine herpesvirus 1 and is essential for pathogenesis in the murine model. Virology 2009 Jan 20;383(2):188-94.
- Ahn BC, Breitenbach JE, Kim SK, O'Callaghan DJ. The equine herpesvirus-1 IR3 gene that lies antisense to the sole immediate-early (IE) gene is trans-activated by the IE protein, and is poorly expressed to a protein. Virology 2007 Jun 20;363(1):15-25.
- Buczynski KA, Kim SK, O'Callaghan DJ. Initial characterization of 17 viruses harboring mutant forms of the immediate-early gene of equine herpesvirus 1. Virus Genes 2005 Oct;31(2):229-39.
- Kim SK, Albrecht RA, O'Callaghan DJ. A negative regulatory element (base pairs -204 to -177) of the EICP0 promoter of equine herpesvirus 1 abolishes the EICP0 protein's trans-activation of its own promoter. J Virol 2004 Nov;78(21):11696-706.
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