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A Herpesvirus Specific Motif of Epstein-Barr Virus DNA Polymerase Is Required for the Efficient Lytic Genome Synthesis.

ABSTRACT: Epstein-Barr virus (EBV) is associated with several malignancies, including Burkitt lymphoma and nasopharyngeal carcinoma. To overcome such disorders, understanding the molecular mechanisms of the EBV replication is important. The EBV DNA polymerase (Pol) is one of the essential factors for viral lytic DNA replication. Although it is well known that its C-terminal half, possessing DNA polymerase and 3'-5' exonuclease activity, is highly conserved among Family B Pols, the NH2-terminal half has yet to be characterized in detail. In this study, we show that a stretch of hydrophobic amino acids within the pre-NH2-terminal domain of EBV Pol plays important role. In addition, we could identify the most essential residue for replication in the motif. These findings will shed light on molecular mechanisms of viral DNA synthesis and will help to develop new herpesviruses treatments.

SUBMITTER: Narita Y

PROVIDER: S-EPMC4485236 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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A Herpesvirus Specific Motif of Epstein-Barr Virus DNA Polymerase Is Required for the Efficient Lytic Genome Synthesis.

Narita Yohei Y Sugimoto Atsuko A Kawashima Daisuke D Watanabe Takahiro T Kanda Teru T Kimura Hiroshi H Tsurumi Tatsuya T Murata Takayuki T

Scientific reports 20150630

Epstein-Barr virus (EBV) is associated with several malignancies, including Burkitt lymphoma and nasopharyngeal carcinoma. To overcome such disorders, understanding the molecular mechanisms of the EBV replication is important. The EBV DNA polymerase (Pol) is one of the essential factors for viral lytic DNA replication. Although it is well known that its C-terminal half, possessing DNA polymerase and 3'-5' exonuclease activity, is highly conserved among Family B Pols, the NH2-terminal half has ye ...[more]

PMID: 26123572

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Project description:Epstein-Barr virus (EBV)-associated diseases of epithelial cells, including tumors that have latent infection, such as nasopharyngeal carcinoma (NPC), and oral hairy leukoplakia (OHL) lesions that have lytic infection, frequently express the viral latent membrane protein 1 (LMP1). In lytically infected cells, LMP1 expression is activated by the BRLF1 (R) immediate early (IE) protein. However, the mechanisms by which LMP1 expression is normally regulated in epithelial cells remain poorly understood, and its potential roles in regulating lytic reactivation in epithelial cells are as yet unexplored. We previously showed that the differentiation-dependent cellular transcription factors KLF4 and BLIMP1 induce lytic EBV reactivation in epithelial cells by synergistically activating the two EBV immediate early promoters (Zp and Rp). Here we show that epithelial cell differentiation also induces LMP1 expression. We demonstrate that KLF4 and BLIMP1 cooperatively induce the expression of LMP1, even in the absence of the EBV IE proteins BZLF1 (Z) and R, via activation of the two LMP1 promoters. Furthermore, we found that differentiation of NOKs-Akata cells by either methylcellulose suspension or organotypic culture induces LMP1 expression prior to Z and R expression. We show that LMP1 enhances the lytic infection-inducing effects of epithelial cell differentiation, as well as 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate treatment, in EBV-infected epithelial cells by increasing expression of the Z and R proteins. Our results suggest that differentiation of epithelial cells activates a feed-forward loop in which KLF4 and BLIMP1 first activate LMP1 expression and then cooperate with LMP1 to activate Z and R expression.IMPORTANCE The EBV protein LMP1 is expressed in EBV-associated epithelial cell diseases, regardless of whether these diseases are due to lytic infection (such as oral hairy leukoplakia) or latent infection (such as nasopharyngeal carcinoma). However, surprisingly little is known about how LMP1 expression is regulated in epithelial cells, and there are conflicting reports about whether it plays any role in regulating viral lytic reactivation. In this study, we show that epithelial cell differentiation induces LMP1 expression by increasing expression of two cellular transcription factors (KLF4 and BLIMP1) which cooperatively activate the two LMP1 promoters. We also demonstrate that LMP1 promotes efficient lytic reactivation in EBV-infected epithelial cells by enhancing expression of the Z and R proteins. Thus, in EBV-infected epithelial cells, LMP1 expression is promoted by differentiation and positively regulates lytic viral reactivation.

Project description:Epstein-Barr virus (EBV) is linked to the development of both lymphoid and epithelial malignancies worldwide. The M81 strain of EBV, isolated from a Chinese patient with nasopharyngeal carcinoma (NPC), demonstrates spontaneous lytic replication and high-titer virus production in comparison to the prototype B95-8 EBV strain. Genetic comparisons of M81 and B95-8 EBVs were previously been performed in order to determine if the hyperlytic property of M81 is associated with sequence differences in essential lytic genes. EBV SM is an RNA-binding protein expressed during early lytic replication that is essential for virus production. We compared the functions of M81 SM and B95-8 SM and demonstrate that polymorphisms in SM do not contribute to the lytic phenotype of M81 EBV. However, the expression level of the EBV DNA polymerase protein was much higher in M81- than in B95-8-infected cells. The relative deficiency in the expression of B95-8 DNA polymerase was related to the B95-8 genome deletion, which truncates the BALF5 3' untranslated region (UTR). Similarly, the insertion of bacmid DNA into the widely used recombinant B95-8 bacmid creates an inefficient BALF5 3' UTR. We further showed that the while SM is required for and facilitates the efficient expression of both M81 and B95-8 mRNAs regardless of the 3' UTR, the BALF5 3' UTR sequence is important for BALF5 protein translation. These data indicate that the enhanced lytic replication and virus production of M81 compared to those of B95-8 are partly due to the robust translation of EBV DNA polymerase required for viral DNA replication due to a more efficient BALF5 3' UTR in M81.IMPORTANCE Epstein-Barr virus (EBV) infects more than 90% of the human population, but the incidence of EBV-associated tumors varies greatly in different parts of the world. Thus, understanding the connection between genetic polymorphisms from patient isolates of EBV, gene expression phenotypes, and disease is important and may help in developing antiviral therapy. This study examines potential causes of the enhanced lytic replicative properties of M81 EBV isolated from a nasopharyngeal carcinoma (NPC) patient and provides new evidence for the role of the BALF5 gene 3' UTR sequence in DNA polymerase protein expression during lytic replication. Variation in the gene structure of the DNA polymerase gene may therefore contribute to lytic virus reactivation and pathogenesis.

Dataset Information

A Herpesvirus Specific Motif of Epstein-Barr Virus DNA Polymerase Is Required for the Efficient Lytic Genome Synthesis.

Publications

A Herpesvirus Specific Motif of Epstein-Barr Virus DNA Polymerase Is Required for the Efficient Lytic Genome Synthesis.

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