Cristiano Salata

Intracellular Trafficking and Maturation of Herpes Simplex Virus Type 1 gB and Virus Egress Require Functional Biogenesis of Multivesicular Bodies

ABSTRACT The biogenesis of multivesicular bodies (MVBs) is topologically equivalent to virion budding. Hence, a number of viruses exploit the MVB pathway to build their envelope and exit from the cell. By expression of dominant negative forms of Vps4 and Vps24, two components of the MVB pathway, we observed an impairment in infectious herpes simplex virus (HSV) assembly/egress, in agreement with a recent report showing the involvement in HSV envelopment of Vps4, the MVB-specific ATPase (C. M. Crump, C. Yates, and T. Minson, J. Virol. 81:7380-7387). Furthermore, HSV infection resulted in morphological changes to MVBs. Glycoprotein B (gB), one of the most highly conserved glycoproteins across the Herpesviridae family, was sorted to MVB membranes. In cells expressing the dominant negative form of Vps4, the site of intracellular gB accumulation was altered; part of gB accumulated as an endoglycosidase H-sensitive immature form at a calreticulin-positive compartment, indicating that gB traffic was dependent on a functional MVB pathway. gB was ubiquitinated in both infected and transfected cells. Ubiquitination was in part dependent on ubiquitin lysine 63, a signal for cargo sorting to MVBs. Partial deletion of the gB cytoplasmic tail resulted in a dramatic reduction of ubiquitination, as well as of progeny virus assembly and release to the extracellular compartment. Thus, HSV envelopment/egress and gB intracellular trafficking are dependent on functional MVB biogenesis. Our data support the view that the sorting of gB to MVB membranes may represent a critical step in HSV envelopment and egress and that modified MVB membranes constitute a platform for HSV cytoplasmic envelopment or that MVB components are recruited to the site(s) of envelopment.

Involvement of p53 in specific anti-neuroectodermal tumor activity of aloe-emodin

Previously, we have identified aloe‐emodin (AE) as a new type of anticancer agent, with activity that is based on apoptotic cell death promoted by a neuroectodermal tumor‐specific drug uptake. We attempt to clarify the intracellular target of AE and the apoptosis‐signaling pathway activated by AE in neuroblastoma cell lines. Two‐photon excitation microscopy and spectroscopic titrations documented that AE is highly concentrated in susceptible cells and binds to DNA. One of the most important mediators of apoptotic response to genotoxic stimuli, such as anticancer agents, is the p53 tumor suppressor gene. To evaluate the role played by p53 in AE‐induced apoptosis a p53 mutant cell line, which lacks transcriptional activity of p53 targeted genes, was tested. AE displayed a reduced growth inhibitory and pro‐apoptotic activity in p53 mutant cells (SK‐N‐BE(2c)) with respect to the p53 wild‐type line (SJ‐N‐KP). This effect was not caused by a reduced drug uptake in the mutant neuroblastoma cell line but was related to a different apoptotic cell phenotype. Whereas SJ‐N‐KP cells were susceptible to a p53 transcription‐dependent pathway of apoptosis, SK‐N‐BE(2c) cells underwent apoptosis with up‐regulation of p53 expression but not of p53‐target genes. After AE treatment p53 translocates to the mitochondria inter‐membrane space in both neuroblastoma cell lines. Due to its high accumulation in neuroectodermal tumor cells AE could also kill tumor cells harboring p53 mutant genes. This property would further contribute to AE specific anti‐tumor activity and might be exploitable in the clinic.

Human endogenous retroviruses and cancer prevention: evidence and prospects.

BackgroundCancer is a significant and growing problem worldwide. While this increase may, in part, be attributed to increasing longevity, improved case notifications and risk-enhancing lifestyle (such as smoking, diet and obesity), hygiene-related factors resulting in immuno-regulatory failure may also play a major role and call for a revision of vaccination strategies to protect against a range of cancers in addition to infections.DiscussionHuman endogenous retroviruses (HERVs) are a significant component of a wider family of retroelements that constitutes part of the human genome. They were originated by the integration of exogenous retroviruses into the human genome millions of years ago. HERVs are estimated to comprise about 8% of human DNA and are ubiquitous in somatic and germinal tissues.Physiologic and pathologic processes are influenced by some biologically active HERV families. HERV antigens are only expressed at low levels by the host, but in circumstances of inappropriate control their genes may initiate or maintain pathological processes. Although the precise mechanism leading to abnormal HERVs gene expression has yet to be clearly elucidated, environmental factors seem to be involved by influencing the human immune system.HERV-K expression has been detected in different types of tumors.Among the various human endogenous retroviral families, the K series was the latest acquired by the human species. Probably because of its relatively recent origin, the HERV-K is the most complete and biologically active family.The abnormal expression of HERV-K seemingly triggers pathological processes leading to melanoma onset, but also contributes to the morphological and functional cellular modifications implicated in melanoma maintenance and progression.The HERV-K-MEL antigen is encoded by a pseudo-gene incorporated in the HERV-K env-gene. HERV-K-MEL is significantly expressed in the majority of dysplastic and normal naevi, as well as other tumors like sarcoma, lymphoma, bladder and breast cancer. An amino acid sequence similar to HERV-K-MEL, recognized to cause a significant protective effect against melanoma, is shared by the antigenic determinants expressed by some vaccines such as BCG, vaccinia virus and the yellow fever virus.HERV-K are also reactivated in the majority of human breast cancers. Monoclonal and single-chain antibodies against the HERV-K Env protein recently proved capable of blocking the proliferation of human breast cancer cells in vitro, inhibiting tumor growth in mice bearing xenograft tumors.SummaryA recent epidemiological study provided provisional evidence of how melanoma risk could possibly be reduced if the yellow fever virus vaccine (YFV) were received at least 10 years before, possibly preventing tumor initiation rather than culling melanoma cells already compromised. Further research is recommended to confirm the temporal pattern of this protection and eliminate/attenuate the potential role of relevant confounders as socio-economic status and other vaccinations.It appears also appropriate to examine the potential protective effect of YFV against other malignancies expressing high levels of HERV-K antigens, namely breast cancer, sarcoma, lymphoma and bladder cancer.Tumor immune-therapy, as described for the monoclonal antibodies against breast cancer, is indeed considered more complex and less advantageous than immune-prevention. Cellular immunity possibly triggered by vaccines as for YFV might also be involved in anti-cancer response, in addition to humoral immunity.

Role of multivesicular bodies and their components in the egress of enveloped RNA viruses.

As an enveloped virus buds, the nascent viral capsid becomes wrapped in a plasma membrane‐derived lipid envelope, and a membrane fission event is thus necessary to separate the virion from the host cell. This membrane fission event is well characterised in the case of enveloped RNA viruses, where it is promoted by late assembly domains (L‐domains) present at the level of specific viral structural proteins. Research conducted over the past 10 years has demonstrated that L‐domains represent docking sites for cellular proteins essential for the biogenesis of a cellular organelle, the multivesicular body (MVB). In this way, enveloped RNA viruses hijack the MVB components to the cellular site where the budding is executed. This review will focus on the cellular machinery exploited by enveloped RNA viruses in order to be released from infected cells. The role of ubiquitin and lipids in viral budding will also be discussed. Copyright

vOX2 glycoprotein of human herpesvirus 8 modulates human primary macrophages activity.

Human herpesvirus 8 (HHV‐8) is a lymphotropic herpesvirus linked to several disorders such as Kaposis sarcoma, primary effusion lymphoma and multicentric Castlemans disease. Several HHV‐8 proteins regulate host innate and adaptive immune response; in particular, orfK14 is expressed as an immediate early gene during the viral lytic cycle and encodes a surface glycoprotein (vOX2), significantly homologous to the cellular OX2, which delivers inhibitory signals to macrophages. Although it has been suggested that vOX2 may down‐regulate basophil and neutrophil functions, its role in macrophages, a cell type lytically infected by HHV‐8 in vivo, is still controversial. Therefore, we investigated the effect of vOX2 expression in human primary monocyte‐derived macrophages (MDMs). In this report, we demonstrate that vOX2‐expressing MDMs in basal conditions are induced to produce inflammatory cytokines and display higher phagocytic activity with respect to mock cells. By contrast, an opposite effect is exhibited by vOX2 in MDMs undergoing IFN‐γ‐activation, with a down‐modulation of the cytokine production and phagocytic activity. Moreover, we observed that, when MDMs are co‐cultured with vOX2‐expressing cells, the inflammatory cytokine release is increased, independently from the MDM activation state. Interestingly, we could correlate our results with the mRNA transcript level of the vOX2 cellular CD200R receptor. Finally, we demonstrate a down‐regulation of the MHC class I and class II molecules on the cell surface of vOX2‐transduced MDMs. Our results provide new insights into the immunomodulatory effects of HHV‐8 vOX2 protein. J. Cell. Physiol. 219: 698–706, 2009.

Feline Tetherin Is Characterized by a Short N-Terminal Region and Is Counteracted by the Feline Immunodeficiency Virus Envelope Glycoprotein

ABSTRACT Tetherin (BST2) is the host cell factor that blocks the particle release of some enveloped viruses. Two putative feline tetherin proteins differing at the level of the N-terminal coding region have recently been described and tested for their antiviral activity. By cloning and comparing the two reported feline tetherins (called here cBST2504 and cBST2*) and generating specific derivative mutants, this study provides evidence that feline tetherin has a shorter intracytoplasmic domain than those of other known homologues. The minimal tetherin promoter was identified and assayed for its ability to drive tetherin expression in an alpha interferon-inducible manner. We also demonstrated that cBST2504 is able to dimerize, is localized at the cellular membrane, and impairs human immunodeficiency virus type 1 (HIV-1) particle release, regardless of the presence of the Vpu antagonist accessory protein. While cBST2504 failed to restrict wild-type feline immunodeficiency virus (FIV) egress, FIV mutants, bearing a frameshift at the level of the envelope-encoding region, were potently blocked. The transient expression of the FIV envelope glycoprotein was able to rescue mutant particle release from feline tetherin-positive cells but did not antagonize human BST2 activity. Moreover, cBST2504 was capable of specifically immunoprecipitating the FIV envelope glycoprotein. Finally, cBST2504 also exerted its function on HIV-2 ROD10 and on the simian immunodeficiency virus SIVmac239. Taken together, these results show that feline tetherin does indeed have a short N-terminal region and that the FIV envelope glycoprotein is the predominant factor counteracting tetherin restriction.

Amiodarone impairs trafficking through late endosomes inducing a Niemann-Pick C-like phenotype

Abstract Patients treated with amiodarone accumulate lysobisphosphatidic acid (LBPA), also known as bis(monoacylglycero)phosphate, in airway secretions and develop in different tissues vacuoles and inclusion bodies thought to originate from endosomes. To clarify the origin of these changes, we studied in vitro the effects of amiodarone on endosomal activities like transferrin recycling, Shiga toxin processing, ESCRT-dependent lentivirus budding, fluid phase endocytosis, proteolysis and exosome secretion. Furthermore, since the accumulation of LBPA might point to a broader disturbance in lipid homeostasis, we studied the effect of amiodarone on the distribution of LBPA, unesterified cholesterol, sphingomyelin and glycosphyngolipids. Amiodarone analogues were also studied, including the recently developed derivative dronedarone. We found that amiodarone does not affect early endosomal activities, like transferrin recycling, Shiga toxin processing and lentivirus budding. Amiodarone, instead, interferes with late compartments of the endocytic pathway, blocking the progression of fluid phase endocytosis and causing fusion of organelles, collapse of lumenal structures, accumulation of undegraded substrates and amassing of different types of lipids. Not all late endocytic compartments are affected, since exosome secretion is spared. These changes recall the Niemann-Pick type-C phenotype (NPC), but originate by a different mechanism, since, differently from NPC, they are not alleviated by cholesterol removal. Studies with analogues indicate that basic pKa and high water-solubility at acidic pH are crucial requirements for the interference with late endosomes/lysosomes and that, in this respect, dronedarone is at least as potent as amiodarone. These findings may have relevance in fields unrelated to rhythm control.

Herpes simplex virus type 2 infection increases human immunodeficiency virus type 1 entry into human primary macrophages.

Epidemiological and clinical data indicate that genital ulcer disease (GUD) pathogens are associated with an increased risk of human immunodeficiency virus type 1 (HIV-1) acquisition and/or transmission. Among them, genital herpes simplex virus type 2 (HSV-2) seems to play a relevant role. Indeed, the ability of HSV-2 to induce massive infiltration at the genital level of cells which are potential targets for HIV-1 infection may represent one of the mechanisms involved in this process. Here we show that infection of human primary macrophages (MDMs) by HSV-2 results in an increase of CCR5 expression levels on cell surface and allows higher efficiency of MDMs to support entry of R5 HIV-1 strains. This finding could strengthen, at the molecular level, the evidence linking HSV-2 infection to an increased susceptibility to HIV-1 acquisition.

View and review on viral oncology research

To date, almost one and a half million cases of cancer are diagnosed every year in the US and nearly 560,000 Americans are expected to die of cancer in the current year, more than 1,500 people a day (data from the American Cancer Society at http://www.cancer.org/). According to the World Health Organization (WHO), roughly 20% of all cancers worldwide results from chronic infections; in particular, up to 15% of human cancers is characterized by a viral aetiology with higher incidence in Developing Countries. The link between viruses and cancer was one of the pivotal discoveries in cancer research during the past Century. Indeed, the infectious nature of specific tumors has important implications in terms of their prevention, diagnosis, and therapy. In the 21st Century, the research on viral oncology field continues to be vigorous, with new significant and original studies on viral oncogenesis and translational research from basic virology to treatment of cancer. This review will cover different viral oncology aspects, starting from the history of viral oncology and moving to the peculiar features of oncogenic RNA and DNA viruses, with a special focus on human pathogens.

Role of the feline immunodeficiency virus L-Domain in the presence or absence of gag processing: Involvement of ubiquitin and nedd4-2s ligase in viral egress

RNA‐enveloped viruses bud from infected cells by exploiting the multivesicular body (MVB) pathway. In this context, ubiquitination of structural viral proteins and their direct interaction with cellular factors involved in the MVB biogenesis through short proline rich regions, named late domains (L‐domains), are crucial mechanisms. Here we report that, in contrast with the human immunodeficiency virus (HIV), the feline immunodeficiency virus (FIV), a non‐primate lentivirus, is strictly dependent for its budding on a “PSAP”‐type L‐domain, mapping in the carboxy‐terminal region of Gag, irrespective of a functional viral protease. Moreover, we provide evidence that FIV egress is related to Gag ubiquitination, that is, linked to the presence of an active L‐domain. Finally, although FIV Gag does not contain a PPxY motif, we show that the Nedd4‐2s ubiquitin ligase enhances FIV Gag ubiquitination and it is capable to rescue viral mutants lacking a functional L‐domain. In conclusion, our data bring to light peculiar aspects of FIV egress, but we also demonstrate that a non‐primate lentivirus shares with HIV‐1 a novel mechanism of connection to the cellular budding machinery. J. Cell. Physiol. 218: 175–182, 2009.