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

Abstract

Previous genetic and biochemical studies performed with several members of the Alphaherpesvirus subfamily have shown that the UL31 and UL34 proteins are essential components of the molecular machinery that mediates the 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 (EBV) genome, 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 a hypothesis. To further clarify this issue, we have constructed a recombinant EBV strain 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 by electron microscopy revealed that, as expected, ⌬BFLF2 viruses not only failed to egress from the nucleus but also showed defective DNA packaging. Nonfunctional primary egress did not, however, impair the 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. The ⌬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.The Epstein-Barr virus (EBV), a member of the gammaherpesvirus family, establishes lifelong clinical latency (or passive latency) in its hosts. In the absence of an effective cytotoxic T-cell response, EBV-infected B cells undergo permanent proliferation upon the synthesis of the EBV nuclear antigen (EBNA) and LMP latent protein family members (also called active latency) (28). The detection of latent gene products in EBV-associated human cancers established a link between neoplastic transformation and active latency (28). Consequently, latent genes have been and continue to be studied in great detail. In contrast, knowledge of the molecular mechanisms that underlie EBV lytic replication and maturation is not as deep as it is for alphaherpesviruses such as herpes simplex virus type 1 (HSV-1) or pseudorabies (PrV). It is, however, unlikely that information gathered from the study of alphaherpesviruses can immediately be translated to EBV, even if the two virus types show a substantial degree of homology. This might be relevant for therapeutic strategies that aim at targeting viral genes and proteins involved in virus replication and infection with, e.g., inhibitory peptides or small interfering RNA.Electron microscopy studies have led to the identification of several substructures within mature herpesvirus virions. The icosahedral viral capsid surrounds the inner nucleoprotein core, which contains the electron-dense...

Epstein-Barr Virus Blocks the Autophagic Flux and Appropriates the Autophagic Machinery To Enhance Viral Replication

“…A total of 10 4 Raji cells were infected into 96-wells plates with a serial dilution of virus stocked 100ϫ and scored for GFP expression after 3 days by UV microscopy. The number of green Raji units per milliliter was calculated as a measurement of the concentration of infectious particles in virus stocks (33). Three different infection experiments were performed.…”

Selection of HSV capsids for envelopment involves interaction between capsid surface components pU L 31, pU L 17, and pU L 25

“…Primary envelopment requires the products of genes U L 31 and U L 34 in the HSV system (3)(4)(5). Orthologs of U L 31 and U L 34 are present in all known herpesviruses, and for those systems in which it has been studied, the requirement for these proteins in primary envelopment is conserved (6)(7)(8)(9). U L 31 encodes a nucleoplasmic phosphoprotein, pU L 31 (10), that is maintained in close approximation to the INM by association with pU L 34, a type II integral membrane protein (5,(11)(12)(13).…”

The Prediction and Analysis of Inter- and Intra-Species Protein-Protein Interaction

“…BFLF2 interacts with BFRF1 and changes cellular localization (Gonnella et al, 2005). Deletion of BFLF2 also impairs viral DNA packaging (Granato et al, 2008). The most targeted human proteins are HOMER3 and GRN.…”