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BackgroundKaposi’s Sarcoma Herpesvirus (KSHV) is a gammaherpesvirus strongly linked to human cancer. The virus is also able to induce immune suppression, effect that contributes to onset/progression of the viral-associated malignancies. As KSHV may infect macrophages and these cells abundantly infiltrate Kaposi’s sarcoma lesions, in this study we investigated whether KSHV-infection could affect macrophage polarisation to promote tumorigenesis.MethodsFACS analysis was used to detect macrophage markers and PD-L1 expression. KSHV infection and the molecular pathways activated were investigated by western blot analysis and by qRT-PCR while cytokine release was assessed by Multi-analyte Kit.ResultsWe found that KSHV infection reduced macrophage survival and skewed their polarisation towards M2 like/TAM cells, based on the expression of CD163, on the activation of STAT3 and STAT6 pathways and the release of pro-tumorigenic cytokines such as IL-10, VEGF, IL-6 and IL-8. We also found that KSHV triggered Ire1 α-XBP1 axis activation in infected macrophages to increase the release of pro-tumorigenic cytokines and to up-regulate PD-L1 surface expression.ConclusionsThe findings that KSHV infection of macrophages skews their polarisation towards M2/TAM and that activate Ire1 α-XBP1 to increase the release of pro-tumorigenic cytokines and the expression of PD-L1, suggest that manipulation of UPR could be exploited to prevent or improve the treatment of KSHV-associated malignancies.

The Tyr705 STAT3 constitutive activation, besides promoting PEL cell survival, contributes to the maintenance of viral latency. We found indeed that its de-phosphorylation by AG490 induced KSHV lytic cycle. Moreover, Tyr705 STAT3 de-phosphorylation, mediated by the activation of tyrosine phosphatases, together with the increase of Ser727 STAT3 phosphorylation contributed to KSHV lytic cycle induction by TPA. We then observed that p53-p21 axis, essential for the induction of KSHV replication, was activated by the inhibition of Tyr705 and by the increase of Ser727 STAT3 phosphorylation. As a possible link between STAT3, p53-p21 and KSHV lytic cycle, we found that TPA and AG490 reduced the expression of KAP-1, promoting p53 stability, p21 transcription and KSHV lytic cycle activation in PEL cells.

Viral egress and autophagy are two mechanisms that seem to be strictly connected in Herpesviruses's biology. Several data suggest that the autophagic machinery facilitates the egress of viral capsids and thus the production of new infectious particles. In the Herpesvirus family, viral nuclear egress is controlled and organized by a well conserved group of proteins named Nuclear Egress Complex (NEC). In the case of EBV, NEC is composed by BFRF1 and BFLF2 proteins, although the alterations of the nuclear host cell architecture are mainly driven by BFRF1, a multifunctional viral protein anchored to the inner nuclear membrane of the host cell. BFRF1 shares a peculiar distribution with several nuclear components and with them it strictly interacts. In this study, we investigated the possible role of BFRF1 in manipulating autophagy, pathway that possibly originates from nucleus, regulating the interplay between autophagy and viral egress.

Human Herpes Virus-6 (HHV-6), Epstein-Barr Virus (EBV) and Kaposi Sarcoma Herpes Virus (KSHV) are viruses that share with other member of the Herpesvirus family the capacity to interfere with the autophagic process. In this paper, mainly based on the findings of our laboratory, we describe how, through different mechanisms, these viruses converge in reducing autophagy to impair DC immune function and how, by infecting and dysregulating autophagy in different cell types, they promote the pathologies associated with their infection, from the neurodegenerative diseases such Alzheimer’s disease to cancer.

The oncogenic gammaherpesvirus Epstein&ndash, Barr virus (EBV) immortalizes in vitro B lymphocytes into lymphoblastoid cell lines (LCLs), a model that gives the opportunity to explore the molecular mechanisms driving viral tumorigenesis. In this study, we addressed the potential of quercetin, a widely distributed flavonoid displaying antioxidant, anti-inflammatory, and anti-cancer properties, in preventing EBV-driven B cell immortalization. The results obtained indicated that quercetin inhibited thectivation of signal transducer and activator of transcription 3 (STAT3) induced by EBV infection and reduced molecules such as interleukin-6 (IL-6) and reactive oxidative species (ROS) known to be essential for the immortalization process. Moreover, we found that quercetin promoted autophagy and counteracted the accumulation of sequestosome1/p62 (SQSTM1/p62), ultimately leading to the prevention of B cell immortalization. These findings suggest that quercetin may have the potential to be used to counteract EBV-driven lymphomagenesis, especially if its stability is improved.

KSHV and EBV are gammaherpesviruses strictly linked to human cancers. Even if the majority of cancer cells harbor a latent infection, the few cells that undergo viral replication may contribute to the pathogenesis and maintenance of the virus-associated malignancies. Cytotoxic drugs used for the therapies of cancers harboring virus-infection often have, as side effect, the activation of viral lytic cycle. Therefore it is important to investigate whether they affect viral reactivation and understand the underlying mechanisms involved. In this study, we found that proteasome inhibitor bortezomib, a cytotoxic drug that efficiently target gammaherpesvirus-associated B cell lymphomas, triggered KSHV or EBV viral lytic cycle by activating JNK, in the course of ER stress, and inducing autophagy. These results suggest that the manipulation of these pathways could limit viral spread and improve the outcome of bortezomib treatment in patients affected by gammaherpesvirus-associated lymphomas.

KSHV is a gammaherpesvirus strongly associated to human cancers such as Primary Effusion Lymphoma (PEL) and Kaposi's Sarcoma. The naturally virus-infected tumor cells usually display latent infection since a minority of cells undergoes spontaneous viral replication. The lytic cycle can be induced in vitro upon appropriate stimuli such as TPA (T), alone or in combination with butyrate (B), (T/B). In previous studies, Protein Kinase C (PKC) δ, Extracellular Signal-regulated Kinase1/2 (ERK1/2) and p53-p21 axis have been separately reported to play a role in KSHV reactivation from latency. Here, we found that these pathways were interconnected to induce KSHV lytic cycle in PEL cells treated with T/B. T/B also increased H2O2 that played an important role in the activation of these pathways. Oxidant specie production correlated with PKC δ activation, as the PKC δ inhibitor rottlerin reduced both H2O2 and KSHV lytic antigen expression. H2O2 contributed to T/B-mediated ERK1/2 activation that mediated p53 phosphorylation at serine 15 (Ser15) and increased p21 expression. Oxidant specie inhibition by quercetin indeed strongly reduced the activation of these pathways, lytic antigen expression and interestingly it also increased T/B-induced cell death. The use of ERK inhibitor PD98059 or p53 silencing demonstrated the importance of p53Ser15 phosphorylation and of p53-p21 axis in KSHV lytic cycle activation. Understanding the role of oxidant species and the molecular mechanisms involved in KSHV lytic cycle induction is particularly important since oxidant species represent the most physiological stimulus for viral reactivation in vivo and it is known that viral production contributes to the maintenance/progression of KSHV associated malignancies.

We have previously shown that Kaposi sarcoma-associated herpesvirus (KSHV) impairs monocyte differentiation into dendritic cells (DCs). Macroautophagy/autophagy has been reported to be essential in such a differentiating process. Here we extended these studies and found that the impairment of DC formation by KSHV occurs through autophagy inhibition. KSHV indeed reduces CAST (calpastatin) and consequently decreases ATG5 expression in both THP-1 monocytoid cells and primary monocytes. We unveiled a new mechanism put in place by KSHV to escape from immune control. The discovery of viral immune suppressive strategies that contribute to the onset and progression of viral-associated malignancies is of fundamental importance for finding new therapeutic approaches against them.

This study confirms that autophagy is activated concomitantly with KSHV lytic cycle induction, and that autophagy inhibition by BECN1 knockdown reduces viral lytic gene expression. In addition, we extend previous observations and show that autophagy is blocked at late steps, during viral replication. This is indicated by the lack of colocalization of autophagosomes and lysosomes and by the LC3-II level that does not increase in the presence of bafilomycin A1 in primary effusion lymphoma (PEL) cells induced to enter the lytic cycle, either by TPA/sodium butyrate (BC3 and BCBL1) or by doxycycline (TRExBCBL1-Rta). The autophagic block correlates with the downregulation of RAB7, whose silencing with specific siRNA results in an autophagic block in the same cells. Finally, by electron microscopy analysis, we observed viral particles inside autophagic vesicles in the cytoplasm of PEL cells undergoing viral replication, suggesting that they may be involved in viral transport.

Reverse-transcriptase polymerase chain reaction has been used to analyze the expression of 2 latent genes (EBNA-1 and LMP-1) and one replicative gene (BZLF-1) of Epstein-Barr virus in mononuclear cells from bone marrow and peripheral blood of healthy donors. EBV-gene transcription was detected in 8 out of 15 bone-marrow samples. Among these, 5 allowed the detection of latency-associated transcripts in the absence of BZLF-1 expression. Only one sample showed positivity for expression of both latent and lytic genes. In 2 cases, BZLF-1 was the only transcript detected. In peripheral blood, 4 out of 7 samples showed evidence of EBNA-1 transcription; LMP-1 was expressed in 5 samples, and in 2 cases concomitant expression of EBNA-1 and BZLF-1 was detected. These results provide a direct demonstration by RT-PCR of EBV-gene transcription in bone-marrow-resident viral infected cells and suggest, in contrast to previous studies on peripheral blood, that LMP-1 and BZLF-1 are frequently transcribed also in absence of EBV-related disease. The heterogeneous viral gene expression found makes it difficult to define a pattern of viral latency in vivo which coincides with that described for lymphoblastoid or Burkitt's-lymphoma cell lines at different stages of differentiation. Int. J. Cancer 70:524–529. © 1997 Wiley-Liss Inc.

p29, a newly identified Kaposi's sarcoma-associated herpesvirus (KSHV) protein, is the product of ORF67, the positional homolog of the conserved herpesvirus protein UL34. Like its homologues in other herpesviruses, p29 is expressed early during viral lytic cycle, and is localized on the nuclear rim. Upon chemical induction of viral replication in primary effusion lymphoma cells, p29 interacts with p33, encoded by ORF69, the positional homolog of the conserved herpesvirus protein UL31, and both proteins colocalize on the nuclear membrane. IFA and biochemical analysis of infected or transfected cells showed that p29 expression resulted in delocalization and hyperphosphorylation of emerin, whereas other nuclear lamin associated proteins, such as LUMA, LB1 and LBR were not affected. Mislocalization of emerin was robustly increased upon combined expression of p29 and p33, suggesting that emerin destabilization might represent the first step in nuclear lamina disassembling, a process necessary for nucleocapsid maturation.

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.

We report the identification and characterization of p33, the product of Kaposi's 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.

The switch from latency to lytic phase of Epstein–Barr virus (EBV) is coordinated by the expression of two viral transactivators known as ZEBRA and RTA. The BFRF1 gene has been shown to be transcribed during the early phases of EBV lytic cycle. Here, we characterized the BFRF1 promoter showing that ZEBRA transfection stimulated BFRF1 expression, whereas RTA induced BFRF1 only after the transfection of an amount of plasmid largely in excess than that sufficient to stimulate the expression of other RTA-responsive genes. However, a co-operative effect between ZEBRA and RTA in the expression of BFRF1 is evident since the transfection of RTA can rescue the transactivating capacity of a mutant of the ZEBRA protein, known as Z(S186A), that has a substitution affecting the DNA binding region. Moreover, we identified one ZEBRA-responsive element (ZRE) and one RTA-responsive element (RRE) within the BFRF1 promoter region.

The molecular mechanisms that underlie maturation and egress of Epstein-Barr virus (EBV) virions are only partially characterized. We have recently shown that the BFRF1 gene, the EBV positional homolog of herpes simplex virus type 1 and pseudorabies virus UL34, is expressed early during EBV lytic replication and that it is found predominantly on the nuclear membrane (A. Farina, R. Santarelli, R. Gonnella, R. Bei, R. Muraro, G. Cardinali, S. Uccini, G. Ragona, L. Frati, A. Faggioni, and A. Angeloni, J. Virol. 74:3235-3244, 2000). These data suggest that the BFRF1 protein might be involved in viral primary envelopment. To precisely determine the function of this protein, we have constructed an EBV mutant devoid of the BFRF1 gene (BFRF1-KO). 293 cells carrying BFRF1-KO showed no differences in comparison with wild-type EBV in terms of DNA lytic replication or expression of late viral proteins upon induction of the lytic cycle. However, binding assays and infection experiments using cell lines or human cord blood lymphocytes showed a clear reduction in the viral mutant titers. Complementation experiments with BFRF1-KO and a BFRF1 expression vector restored viral titers to levels similar to those for the wild-type control, showing that the modifications that we introduced were limited to the BFRF1 gene. Electron microscopic observations showed that the reduction in viral titers was due to sequestration of EBV nucleocapsids in the nuclei of lytically induced cells. This suggests that BFRF1 is involved in transport of the maturing virion across the nuclear membrane. This hypothesis was further supported by the observation that BFRF1 is present in maturing intracellular virions but not in their extracellular counterparts. This implies that BFRF1 is a key protein for EBV maturation.

A novel protein encoded by the BFRF1 gene of the Epstein-Barr virus was identified recently [Farina et al. (2000) J Virol 74:3235-3244], which is antigenic "in vivo" and expressed early in the viral replicative cycle. In the present study, its subcellular localization was examined in greater detail comparing Epstein-Barr virus (EBV) induced producing and nonproducing cell lines by immunofluorescence: in 12-0-tetradecanoyl phorbol-13-acetate (TPA)-induced Raji and B95-8 cells, as well as in anti-IgG-stimulated Akata cells, the protein appeared to be localized over the cell nuclear membrane. A similar nuclear membrane localization was observed in epithelial cells of oral hairy leukoplakia, a pathological manifestation of permissive EBV infection. In contrast, upon transfection of BFRF1 in the EBV-negative Burkitt's lymphoma cell line DG75, the protein was localized predominantly over the plasma membrane. The membrane localization was abolished when DG75 cells were transfected with a C-terminal deletion mutant of BFRF1 lacking the transmembrane domain. Because induced Raji cells do not produce virus, the above observations indicate that the nuclear membrane localization is not associated with viral production, but requires the expression of EBV genes, and suggest that additional proteins, expressed early during viral lytic infection, might be necessary to target the protein to the nuclear membrane. Immunogold electron microscopy on ultrathin cryosections of induced B95-8 cells showed that BFRF1 on the nuclear membranes was concentrated over multilayered domains representing areas of active viral replication or at the sites of viral budding, suggesting that BFRF1 is involved in the process of viral assembly.

A biologic, immunologic and molecular characterization of an HHV-6 isolate (BA92) rescued by the peripheral blood mononuclear cells of a child affected by Exanthem subitum is reported. The comparison with the known HHV-6 prototype strains showed that BA92 is indistinguishable from the Z29 isolate, and can be included in the variant B group of HHV-6. A seroepidemiologic analysis of the antibody response to BA92 of normal individuals as well as patients affected by diseases potentially associated to HHV-6 infection has shown an overall seropravalence of 81%, and that no variations in seroprevalence or in antibody geometric mean titer are observed assaying the sera also against G.S., U1102, or Z29 infected cells, respectively. These findings indicate: (1) HHV-6 infection is widely diffuse in Italy; (2) it is not possible to discriminate between the viral variants by the currently available IF assays, and (3) no conclusions can be drawn on the potential association of HHV-6 with any of the diseases examined.