Marisa Granato

Characterization and intracellular localization of the Epstein-Barr virus protein BFLF2: Interactions with BFRF1 and with the nuclear lamina

ABSTRACT We have reported in the accompanying paper that the BFRF1 protein of Epstein-Barr virus (EBV) is important for efficient primary viral envelopment and egress (A. Farina, R. Feederle, S. Raffa, R. Gonnella, R. Santarelli, L. Frati, A. Angeloni, M. R. Torrisi, A. Faggioni, and H.-J. Delecluse, J. Virol. 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.

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

ABSTRACT 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. IMPORTANCE This 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.

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 (ΔBFLF2) 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, ΔBFLF2 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 ΔBFLF2 and ΔUL31 phenotypes therefore only partly overlap, from which we infer that BFLF2 and UL31 have substantially diverged during evolution to fulfil related but distinct functions.

HSP70 inhibition by 2-phenylethynesulfonamide induces lysosomal cathepsin D release and immunogenic cell death in primary effusion lymphoma

Heat-shock protein (HSP) 70 is aberrantly expressed in different malignancies and has a cancer-specific cell-protective effect. As such, it has emerged as a promising target for anticancer therapy. In this study, the effect of the HSP70-specific inhibitor (PES), also Pifitrin-μ, on primary effusion lymphoma (PEL) cell viability was analyzed. PES treatment induced a dose- and time-dependent cytotoxic effect in BC3 and BCBL1 PEL cells by inducing lysosome membrane permeabilization, relocation of cathepsin D in the cytosol, Bid cleavage, mitochondrial depolarization with release and nuclear translocation of apoptosis-activating factor. The PES-induced cell death in PEL cells was characterized by the appearance of Annexin-V/propidium iodide double-positive cells from the early times of treatment, indicating the occurrence of an additional type of cell death other than apoptosis, which, accordingly, was not efficiently prevented by the pan-caspase inhibitor Z-VAD-fmk. Conversely, PES-induced cell death was robustly reduced by pepstatin A, which inhibits Bid and caspase 8 processing. In addition, PES was responsible for a block of the autophagic process in PEL cells. Finally, we found that PES-induced cell death has immunogenic potential being able to induce dendritic cell activation.

Identification and Characterization of the Product Encoded by ORF69 of Kaposi's Sarcoma-Associated Herpesvirus

ABSTRACT We report the identification and characterization of p33, the product of Kaposis sarcoma-associated herpesvirus (KSHV) open reading frame 69 (ORF69), a positional homolog of the conserved herpesvirus protein UL31. p33 is expressed upon induction of viral lytic cycle with early kinetics. Immunofluorescence analysis revealed that in infected cell lines, the protein is localized in the nucleus, both in dotted spots and along the nuclear membrane. Nuclear fractionation experiments showed that p33 partitions with the nuclear matrix, and both immunoblotting of purified virions and immunoelectron microscopy indicated that the novel protein is not a component of the mature virus. Following ectopic expression in KSHV-negative cells, the protein was never associated with the nuclear membrane, suggesting that p33 needs to interact with additional viral proteins to reach the nuclear rim. In fact, after cotransfection with the ORF67 gene, the KSHV positional homolog of UL34, the p33 intranuclear signal changed and the two proteins colocalized on the nuclear membrane. A similar result was obtained when ORF69 was cotransfected with BFRF1, the Epstein-Barr virus (EBV) positional homolog of UL34 and ORF67. Finally, upon cotransfection, ORF69 significantly increased nuclear membrane reduplications induced by BFRF1. The above results indicate that KSHV p33 shares many similarities with its EBV homolog BFLF2 and suggest that functional cross-complementation is possible between members of the gammaherpesvirus subfamily.

STAT3 activation by KSHV correlates with IL-10, IL-6 and IL-23 release and an autophagic block in dendritic cells

Kaposiss 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.

JNK and Macroautophagy Activation by Bortezomib Has a Pro-Survival Effect in Primary Effusion Lymphoma Cells

Understanding the mechanisms of autophagy induction and its role during chemotherapeutic treatments is of fundamental importance in order to manipulate it to improve the outcome of chemotherapy. In particular whether the bortezomib-induced autophagy plays a pro-survival or pro-death role is still controversial. In this study we investigated if bortezomib induced endoplasmic reticulum (ER) stress and activated autophagy in Primary Effusion Lymphoma (PEL) cells and how they influenced cell survival. We found that bortezomib induced up-regulation of the pro-survival and pro-death ER stress molecules BIP and CHOP and activated c-Jun NH2-terminal kinase (JNK), resulting in Bcl-2 phosphorylation and induction of autophagy. JNK and autophagy activation played a pro-survival role in this setting, thus their inhibition increased the bortezomib cytotoxic effect and PARP cleavage in PEL cells. Based on our results we suggest that the combination of bortezomib with JNK or autophagy inhibitors could be exploited to improve the outcome of therapy of this aggressive B cell lymphoma.

Zinc supplementation is required for the cytotoxic and immunogenic effects of chemotherapy in chemoresistant p53-functionally deficient cells

Optimal tumor eradication often results from the death of malignant cells, as induced by chemotherapeutic agents, coupled to the induction of antitumor immune responses. However, cancer cells frequently become resistant to the cytotoxic activity of chemotherapy. The aim of the present study was to evaluate whether zinc dichloride (ZnCl2), which was known to re-establish the chemosensitivity of cancer cells by reactivating p53, promotes immunogenic instances of cell death. We found that ZnCl2, in combination with chemotherapeutic agents such as cisplatin and adriamycin (ADR), favors the apoptotic demise of chemoresistant cells, while cisplatin and ADR alone fail to do so. The co-culture of immature dendritic cells (DCs) with cancer cells succumbing to the co-administration of chemotherapy and ZnCl2 led to DC activation, as indicated by the upregulation of the activation markers CD83 and CD86. In part, such process depended on cell death, as it was limited (but not abrogated) by the pan-caspase inhibitor Z-VAD-fmk. Moreover, DC activation relied on the ZnCl2-induced exposure of calreticulin (CRT) on the surface of cancer cells, correlating with the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), a marker of endoplasmic reticulum stress. The siRNA-mediated knockdown of CRT as well as the inhibition of CRT exposure with brefeldin A strongly impaired DC maturation, indicating CRT translocation as induced by that ZnCl2 is a key event in this setting. Altogether, these results suggest that ZnCl2, has the potential to enhance the therapeutic effects of antineoplastic agents not only by improving their cytotoxic activity but also by promoting CRT exposure.

Kaposi sarcoma associated herpesvirus (KSHV) induces AKT hyperphosphorylation, bortezomib-resistance and GLUT-1 plasma membrane exposure in THP-1 monocytic cell line

BackgroundPhosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway regulates multiple cellular processes such as cell proliferation, evasion from apoptosis, migration, glucose metabolism, protein synthesis and proper differentiation in immune cells. Kaposi sarcoma-associated herpesvirus (KSHV), an oncogenic virus associated with several human malignancies, expresses a variety of latent and lytic proteins able to activate PI3K/AKT pathway, promoting the growth of infected cells and a successful viral infection.ResultsWe found that KSHV latent infection of THP-1 cells, a human monocytic cell line derived from an acute monocytic leukemia patient, resulted in an increase of AKT phoshorylation, not susceptible to bortezomib-induced dephosphorylation, compared to the mock-infected THP-1. Accordingly, THP-1-infected cells displayed increased resistance to the bortezomib cytotoxic effect in comparison to the uninfected cells, which was counteracted by pre-treatment with AKT-specific inhibitors. Finally, AKT hyperactivation by KSHV infection correlated with plasma membrane exposure of glucose transporter GLUT1, particularly evident during bortezomib treatment. GLUT1 membrane trafficking is a characteristic of malignant cells and underlies a change of glucose metabolism that ensures the survival to highly proliferating cells and render these cells highly dependent on glycolysis. GLUT1 membrane trafficking in KSHV-infected THP-1 cells indeed led to increased sensitivity to cell death induced by the glycolysis inhibitor 2-Deoxy-D-glucose (2DG), further potentiated by its combination with bortezomib.ConclusionsKSHV confers to the THP-1 infected cells an oncogenic potential by altering the phosphorylation, expression and localization of key molecules that control cell survival and metabolism such as AKT and GLUT1. Such modifications in one hand lead to resistance to cell death induced by some chemotherapeutic drugs such as bortezomib, but on the other hand, offer an Achilles heel, rendering the infected cells more sensitive to other treatments such as AKT or glycolysis inhibitors. These therapeutic strategies can be exploited in the anticancer therapy of KSHV-associated malignancies.

Histone deacetylase inhibitors VPA and TSA induce apoptosis and autophagy in pancreatic cancer cells

PurposeHistone deacetylase inhibitors (HDACi) are anti-neoplastic agents that are known to affect the growth of different cancer types, but their underlying mechanisms are still incompletely understood. Here, we compared the effects of two HDACi, i.e., Trichostatin A (TSA) and Valproic Acid (VPA), on the induction of cell death and autophagy in pancreatic cancer-derived cells that exhibit a high metastatic capacity and carry KRAS/p53 double mutations.MethodsCell viability and proliferation tests were carried out using Trypan blue dye exclusion, MTT and BrdU assays. FACS analyses were carried out to assess cell cycle progression, apoptosis, reactive oxygen species (ROS) production and mitochondrial depolarization, while Western blot and immunoprecipitation analyses were employed to detect proteins involved in apoptosis and autophagy.ResultsWe found that both VPA and TSA can induce apoptosis in Panc1 and PaCa44 pancreatic cancer-derived cells by triggering mitochondrial membrane depolarization, Cytochrome c release and Caspase 3 activation, although VPA was more effective than TSA, especially in Panc1 cells. As underlying molecular events, we found that ERK1/2 was de-phosphorylated and that the c-Myc and mutant p53 protein levels were reduced after VPA and, to a lesser extent, after TSA treatment. Up-regulation of p21 and Puma was also observed, concomitantly with mutant p53 degradation. In addition, we found that in both cell lines VPA increased the pro-apoptotic Bim level, reduced the anti-apoptotic Mcl-1 level and increased ROS production and autophagy, while TSA was able to induce these effects only in PaCA44 cells.ConclusionsFrom our results we conclude that both VPA and TSA can induce pancreatic cancer cell apoptosis and autophagy. VPA appears have a stronger and broader cytotoxic effect than TSA and, thus, may represent a better choice for anti-pancreatic cancer therapy.