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J. Virol. doi:10.1128/JVI.02436-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Deletion of Epstein-Barr virus BFLF2 leads to impaired viral DNA packaging and primary egress as well as to the production of defective viral particles

Instituto Pasteur Fondazione Cenci Bolognetti, Dipartimento di Medicina Sperimentale e Patologia, Università La Sapienza, Roma, Italy; German Cancer Research Center, Department of Virus Associated Tumors, Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany

^* To whom correspondence should be addressed. Email: h.delecluse{at}dkfz.de.

	Abstract

Previous genetic and biochemical studies performed on several members of the alphaherpesviruses subfamily have shown that the UL31 and UL34 proteins are essential components of the molecular machinery that mediates primary egress of newly assembled capsids across the nuclear membrane. Further, there is substantial evidence that BFLF2 and BFRF1, the respective positional homologs of UL31 and UL34 in the Epstein-Barr virus genome (EBV) are also their functional homologs, i.e. that the UL31/UL34 pathway is common to distant herpesviruses. However, the low degree of protein sequence identity between UL31 and BFLF2 would argue against such an hypothesis. To further clarify this issue, we have constructed a recombinant EBV devoid of BFLF2 (BFLF2) and show that BFLF2 is crucial for efficient virus production but not for lytic DNA replication or B cell transformation. This defective phenotype could be efficiently restored by trans-complementation with a BFLF2 expression plasmid. Detailed analysis of replicating cells using electron microscopy revealed that not only, as expected, BFLF2 viruses failed to egress from the nucleus but also showed defective DNA packaging. Non-functional primary egress did not, however, impair production and extracellular release of enveloped but empty viral particles that comprised L particles containing tegument-like structures and a few virus-like particles carrying empty capsids. BFLF2 and UL31 phenotypes therefore only partly overlap, from which we infer that BFLF2 and UL31 have substantially diverged during evolution to fulfil related but distinct functions.