. 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...

Autophagy is a catabolic pathway that helps cells to survive under stressful conditions. Cells also use autophagy to clear microbiological infections, but microbes have learned how to manipulate the autophagic pathway for their own benefit. The experimental evidence obtained in this study suggests that the autophagic flux is blocked at the final steps during the reactivation of Epstein-Barr virus (EBV) from latency. This is indicated by the level of the lipidated form of LC3 that does not increase in the presence of bafilomycin and by the lack of colocalization of autophagosomes with lysosomes, which correlates with reduced Rab7 expression. Since the inhibition of the early phases of autophagy impaired EBV replication and viral particles were observed in autophagic vesicles in the cytoplasm of producing cells, we suggest that EBV exploits the autophagic machinery for its transportation in order to enhance viral production. The autophagic block was not mediated by ZEBRA, an immediate-early EBV lytic gene, whose transfection in Ramos, Akata, and 293 cells promoted a complete autophagic flux. The block occurred only when the complete set of EBV lytic genes was expressed. We suggest that the inhibition of the early autophagic steps or finding strategies to overcome the autophagic block, allowing viral degradation into the lysosomes, can be exploited to manipulate EBV replication. IMPORTANCEThis study shows, for the first time, that autophagy is blocked at the final degradative steps during EBV replication in several cell types. Through this block, EBV hijacks the autophagic vesicles for its intracellular transportation and enhances viral production. A better understanding of virus-host interactions could help in the design of new therapeutic approaches against EBV-associated malignancies.

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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...

Kaposis's sarcoma associated herpesvirus (KSHV) has been reported to infect, among others, monocytes and dendritic cells DCs impairing their function. However, the underlying mechanisms remain not completely elucidated yet. Here we show that DC exposure to active or UV-inactivated KSHV resulted in STAT3 phosphorylation. This effect, partially dependent on KSHV-engagement of DC-SIGN, induced a high release of IL-10, IL-6 and IL-23, cytokines that in turn might maintain STAT3 in a phosphorylated state. STAT3 activation also correlated with a block of autophagy in DCs, as indicated by LC3II reduction and p62 accumulation. The IL-10, IL-6 and IL-23 release and the autophagic block could be overcome by inhibiting STAT3 activation, highlighting the role of STAT3 in mediating such effects. In conclusion, here we show that STAT3 activation can be one of the molecular mechanisms leading to KSHV-mediated DC dysfunction, that might allow viral persistence and the onset of KSHV-associated malignancies.