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Epstein-Barr virus mRNA export factor EB2 is essential for production of infectious virus
Henri Gruffat1, Julien Batisse, Dagmar Pich
1Laboratoire de Virologie Humaine, INSERM U412, ENS-Lyon, F-69364 Lyon Cedex 07, France. hgruffat@ens-lyons.fr
Abstract:
The splicing machinery which positions a protein export complex near the exon-exon junction mediates nuclear export of mRNAs generated from intron-containing genes. Many Epstein-Barr virus (EBV) early and late genes are intronless, and an alternative pathway, independent of splicing, must export the corresponding mRNAs. Since the EBV EB2 protein induces the cytoplasmic accumulation of intronless mRNA, it is tempting to speculate that EB2 is a viral adapter involved in the export of intronless viral mRNA. If this is true, then the EB2 protein is essential for the production of EBV infectious virions. To test this hypothesis, we generated an EBV mutant in which the BMLF1 gene, encoding the EB2 protein, has been deleted (EBV(BMLF1-KO)). Our studies show that EB2 is necessary for the production of infectious EBV and that its function cannot be transcomplemented by a cellular factor. In the EBV(BMLF1-KO) 293 cells, oriLyt-dependent DNA replication was greatly enhanced by EB2. Accordingly, EB2 induced the cytoplasmic accumulation of a subset of EBV early mRNAs coding for essential proteins implicated in EBV DNA replication during the productive cycle. Two herpesvirus homologs of the EB2 protein, the herpes simplex virus type 1 protein ICP27 and, the human cytomegalovirus protein UL69, only partly rescued the phenotype of the EBV(BMLF1-KO) mutant, indicating that some EB2 functions in virus production cannot be transcomplemented by ICP27 and UL69.
Insights
The Epstein-Barr virus (EBV) EB2 protein is crucial for producing infectious EBV virions. This viral protein facilitates the export of intronless viral mRNAs, essential for DNA replication and virus production.
Area of Science:
- Virology
- Molecular Biology
- Cellular Biology
Background:
- Nuclear export of messenger RNAs (mRNAs) typically relies on splicing machinery for intron-containing genes.
- Epstein-Barr virus (EBV) utilizes intronless genes, necessitating alternative mRNA export pathways.
- The EBV EB2 protein's role in accumulating intronless mRNA suggests involvement in viral mRNA export.
Purpose of the Study:
- To investigate the essentiality of the EBV EB2 protein in the production of infectious EBV virions.
- To determine if cellular factors can compensate for the loss of EB2 function.
- To elucidate EB2's specific roles in viral DNA replication and mRNA export.
Main Methods:
- Generation of an EBV mutant lacking the BMLF1 gene (encoding EB2) – EBV(BMLF1-KO).
- Analysis of infectious virion production in EBV(BMLF1-KO) cells.
- Assessment of oriLyt-dependent DNA replication and cytoplasmic accumulation of viral mRNAs.
- Functional complementation studies using EB2 homologs (ICP27, UL69).
Main Results:
- EB2 is essential for infectious EBV production; its function cannot be replaced by cellular factors.
- EB2 significantly enhances oriLyt-dependent DNA replication in EBV(BMLF1-KO) cells.
- EB2 induces cytoplasmic accumulation of a subset of EBV early mRNAs critical for DNA replication.
- Herpes simplex virus type 1 ICP27 and human cytomegalovirus UL69 partially rescue EB2's function, but not fully.
Conclusions:
- The EBV EB2 protein is indispensable for infectious EBV production.
- EB2 plays a critical role in enhancing viral DNA replication and regulating the export of specific viral mRNAs.
- EB2's function in EBV production is not fully redundant with related proteins from other herpesviruses.