Epstein-Barr Virus BALF3 Has Nuclease Activity and Mediates Mature Virion Production during the Lytic Cycle

Abstract

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. IMPORTANCEVirus 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.

Epstein-Barr virus BALF3 mediates genomic instability and progressive malignancy in nasopharyngeal carcinoma

“…The cells expressing GFP-BALF3 exhibited more H2AX phosphorylation than the mock and pEGFP-C1 transfections, detected by immunofluorescence staining with an antibody specific to γH2AX (Figure 1G), suggesting that EBV BALF3 could induce DSBs. Moreover, a recent study in our laboratory has shown that alanine substitution of the glycine residue at amino acid 624 of EBV BALF3 results in reduced endonucleolytic activity [58]. Therefore, the coding region of BALF3 in pEGFP-C1-BALF3 was mutated by replacement of glycine 624 with alanine, with the result that the occurrence of micronuclei and H2AX phosphorylation induced by the G624A mutant was reduced to a significant extent, compared to the wild-type transfection (Figure 1C, D and G), confirming that EBV BALF3 could generate chromosome breakage and loss and DSBs.…”

“…To investigate the effect of BALF3 on uninfected cells, BALF3 was expressed alone in NPC cells and these were examined for genetic alterations using several methods, showing that the BALF3 expression led to the induction of micronuclei, DNA strand breaks and genomic CNAs (Figure 1, 2 and 3). BALF3 has nuclease activity and the BALF3-mediated nucleolytic reaction occurs not only in the terminal repeats of the EBV genome, which contain cleavage/packaging signals, but also in vitro in plasmid DNA without the terminal repeat sequence [58]. Similarly, nonspecific cleavage also has been reported for the terminases of human cytomegalovirus (HCMV) and bacteriophage T4.…”

“…HCMV UL56 can cleave plasmid DNA in vitro in the absence of the a sequence and bacteriophage T4 gp17 carries out random cleavage of the Escherichia coli genomic DNA in vivo [69, 70]. In addition, Figure 1D, F and G reveal that a mutation within the putative ATP-binding motif of BALF3, which is responsible for nuclease activity [58], significantly reduced the numbers of micronuclei and phosphorylation of H2AX detected following wild-type BALF3 expression. On this basis, the genetic alterations of cells caused by EBV BALF3 may be attributed to its nuclease activity.…”

“…Furthermore, EBV DNase and BGLF4, lytic gene products, were shown to be able to elicit genomic instability [56, 57]. EBV BALF3 is expressed after EBV reactivation and is a terminase, which cleaves newly synthesized viral DNA and enables the translocation of unit length genomes into procapsids during the lytic cycle [58]. In addition, our preliminary screening suggests that BALF3 is involved in the induction of host genomic instability.…”

Intermolecular Complementation between Two Varicella-Zoster Virus pORF30 Terminase Domains Essential for DNA Encapsidation

“…The protein complex that docks or interacts with the capsid portal protein consists of the heterotrimeric viral terminase. Terminases of DNA bacteriophages have been well described (2,3), and those of several members of the Herpesviridae, including herpes simplex virus 1 (HSV-1) (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), human cytomegalovirus (HCMV) (15)(16)(17)(18)(19)(20)(21), Epstein-Barr virus (EBV) (22), and varicella-zoster virus (VZV) (23)(24)(25), are under investigation.…”