Characterization and Intracellular Localization of the Epstein-Barr Virus Protein BFLF2: Interactions with BFRF1 and with the Nuclear Lamina

Abstract

. 79:3703-3712). Here we describe the characterization of the product of the EBV BFLF2 gene, which belongs to a family of conserved herpesviral genes which include the UL31 genes of herpes simplex virus and of pseudorabies virus and whose products are known to interact with UL34, the positional homolog of BFRF1. BFLF2 is an early transcript and is expressed in a variety of cell lines upon EBV lytic cycle activation. Western blotting of purified virion preparations showed that BFLF2 is a component of intracellular virions but is absent from mature extracellular virions. Coimmunoprecipitation experiments indicated that BFLF2 interacts with BFRF1, which was confirmed by immunofluorescence confocal microscopy showing that the two proteins colocalize on the nuclear membrane not only upon cotransfection in epithelial cells but also during viral replication. In cells carrying an EBV mutant with the BFRF1 gene deleted (293-BFRF1-KO cells) BFLF2 expression was low, and it was restored to wild-type levels upon treatment of the cells with the proteasome inhibitor MG132. Furthermore, recomplementing the 293-BFRF1-KO cells by BFRF1 transfection restored BFLF2 expression to the wild-type level. In addition, when expressed alone BFLF2 was localized diffusely inside the nucleus, whereas in the presence of BFRF1 the two proteins colocalized at the nuclear rim. Finally, 293 epithelial cells transfected with either protein or cotransfected were analyzed by electron microscopy to investigate potential alterations in the morphology of the nuclear membrane. The ultrastructural analysis revealed that (i) BFRF1 caused duplications of the nuclear membrane, similar to those reported to occur during the course of herpesviral replication, and (ii) while BFLF2 alone did not cause any apparent alteration, coexpression of the two proteins dramatically induced profound convolutions of the duplicated nuclear membrane. Both biochemical and morphological analysis showed association of the BFRF1-BFLF2 complex with a component of the nuclear lamina, lamin B. Taken together, these results and those of the accompanying paper (Farina et al., J. Virol. 79:3703-3712) indicate an important role of BFRF1 and BFLF2 in the early steps of EBV maturation at the nuclear membrane.Two conserved herpesvirus proteins, designated UL34 and UL31, of herpes simplex virus (HSV) and pseudorabies virus (PrV) are involved in the early steps of viral maturation at the nuclear envelope (reviewed in reference 26). With many similarities and a few differences, accumulating evidence indicates that these proteins and their homologs play similar roles in nuclear egress of both alpha-and betaherpesviruses (9,17,23,30,33,34,36,37,40,48,50). The physical interaction between the two proteins appears to be important to facilitate virion envelopment at the inner nuclear membrane, a process which probably also involves nuclear lamina disruption, to allow nucleocapsids to gain access at the interior face of the nuclear envelope (28, 39).We initially identified and characterized the prod...

Vesicle formation from the nuclear membrane is induced by coexpression of two conserved herpesvirus proteins

“…Alterations of the nuclear envelope have also been observed after coexpression of pUL31 and pUL34 homologs of EpsteinBarr virus (18). However, they did not include formation of perinuclear vesicles but rather presented as reduplications of the nuclear membrane.…”

“…Both proteins interact, and formation of the pUL34/pUL31 complex is required for proper positioning of both complex partners, local dissolution of the nuclear lamina, modification of the host cell chromatin, and efficient release of nucleocapsids from the nucleus (14-21). pUL31 and pUL34 bind nuclear lamins A/C and B (15,18,20) and are required for recruitment of cellular protein kinase C (PKC). Phosphorylation of lamins presumably leads to local dissolution of the lamina (12,21).…”

Identification of Synonymous Codon Usage Bias in the Pseudorabies Virus UL31 Gene

“…It is reported that synonymous codon usage bias may be connected with different biological factors (6,(10)(11)(12)(13). Further analysis found that synonymous codon usage pattern changed at distinct sites along a coding sequence (14), balances of strong versus weak base pair bonding (15,16), maintenance of DNA and RNA secondary structure (17), and translational efficiency and fidelity (6). Aujeszky's disease, which is provoked by the pathogenic factor of Pseudorabies virus (PRV) (also known as Suid herpesvirus 1,, is a regularly lethal disease with a global distribution that influences swine, mainly and other domestic and wild animals incidentally (18)(19)(20)(21)(22).…”

Role of tegument proteins in herpesvirus assembly and egress

“…In EBV, the pUL34 positional homolog BFRF1 and the pUL31 positional homolog BFLF2 also interact with each other. HSV-1 UL34 or EBV BFRF1 mutant viruses showed a phenotype of nuclear retention (Lake and HuttFletcher, 2004;Farina et al, 2005;Gonnella et al, 2005;Calderwood et al, 2007). EBV BFLF2 functions in both viral DNA packaging and primary envelopment, as deletion of BFLF2 led to sequestration of nucleocapsids in the nucleus and production of more empty capsids without viral genomes (Granato et al, 2008).…”