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Lytic cycle gene regulation of Epstein-Barr virus.

ABSTRACT: Episomal reporter plasmids containing the Epstein-Barr virus (EBV) oriP sequence stably transfected into Akata Burkitt's lymphoma cells were used to analyze EBV lytic cycle gene regulation. First, we found that the Zp promoter of EBV, but not the Rp promoter, can be activated in the absence of protein synthesis in these oriP plasmids, casting doubt on the immediate early status of Rp. An additional level of regulation of Zp was implied by analysis of a mutation of the ZV element. Second, our analysis of late lytic cycle promoters revealed that the correct relative timing, dependence on ori lyt in cis, and sensitivity to inhibitors of DNA replication were reconstituted on the oriP plasmids. Late promoter luciferase activity from oriP plasmids also incorporating replication-competent ori lyt was phosphonoacetic acid sensitive, a hallmark of EBV late genes. A minimal ori lyt, which only replicates weakly, was sufficient to confer late timing of expression specifically on late promoters. Finally, deletion analysis of EBV late promoter sequences upstream of the transcription start site confirmed that sequences between -49 and +30 are sufficient for late gene expression, which is dependent on ori lyt in cis. However, the TATT version of the TATA box found in many late genes was not essential for late expression.

SUBMITTER: Amon W

PROVIDER: S-EPMC533939 | biostudies-other | 2004 Dec

REPOSITORIES: biostudies-other

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Project description:Epstein-Barr virus (EBV) lytic replication involves complex processes, including DNA synthesis, DNA cleavage and packaging, and virion egress. These processes require many different lytic gene products, but the mechanisms of their actions remain unclear, especially for DNA cleavage and packaging. According to sequence homology analysis, EBV BALF3, encoded by the third leftward open reading frame of the BamHI-A fragment in the viral genome, is a homologue of herpes simplex virus type 1 UL28. This gene product is believed to possess the properties of a terminase, such as nucleolytic activity on newly synthesized viral DNA and translocation of unit length viral genomes into procapsids. In order to characterize EBV BALF3, the protein was produced by and purified from recombinant baculoviruses and examined in an enzymatic reaction in vitro, which determined that EBV BALF3 acts as an endonuclease and its activity is modulated by Mg(2+), Mn(2+), and ATP. Moreover, in EBV-positive epithelial cells, BALF3 was expressed and transported from the cytoplasm into the nucleus following induction of the lytic cycle, and gene silencing of BALF3 caused a reduction of DNA packaging and virion release. Interestingly, suppression of BALF3 expression also decreased the efficiency of DNA synthesis. On the basis of these results, we suggest that EBV BALF3 is involved simultaneously in DNA synthesis and packaging and is required for the production of mature virions.Virus lytic replication is essential to produce infectious virions, which is responsible for virus survival and spread. This work shows that an uncharacterized gene product of the human herpesvirus Epstein-Barr virus (EBV), BALF3, is expressed during the lytic cycle. In addition, BALF3 mediates an endonucleolytic reaction and is involved in viral DNA synthesis and packaging, leading to influence on the production of mature virions. According to sequence homology and physical properties, the lytic gene product BALF3 is considered a terminase in EBV. These findings identify a novel viral gene with an important role in contributing to a better understanding of the EBV life cycle.

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Lytic cycle gene regulation of Epstein-Barr virus.

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