Elisa Bergamo

Peripheral Blood Endothelial Progenitors as Potential Reservoirs of Kaposi's Sarcoma-Associated Herpesvirus

Background The cellular reservoirs of Kaposis sarcoma-associated herpesvirus (KSHV) and the exact nature of the putative KSHV-infected circulating precursor of spindle cells of Kaposis sarcoma (KS) still remain poorly defined. Because KS spindle cells are thought to be of endothelial origin, and because mature endothelial cells do not sustain persistent KSHV-infection, our attention was focalized on circulating hematopoietic precursors able to differentiate into endothelial lineage. Methods and Findings Late endothelial progenitor cells (late-EPCs) were cultured from the peripheral blood mononuclear cells of 16 patients with classic KS. The presence and load of KSHV genomes were analyzed by real-time polymerase chain reaction in DNA extracted from cells and supernatants of late-EPC cultures obtained from 7 patients. Endothelial colonies cultured from the peripheral blood of KS patients were found to satisfy all requisites to be defined late-EPCs: they appeared from the CD14-negative fraction of adherent cells after 11–26 days of culture, could be serially expanded in vitro, expressed high levels of endothelial antigens but lacked leukocyte markers. Late-EPC cultures were found to harbor KSHV-DNA at variable levels and to retain the virus after multiple passages in cells as well as in supernatants, suggesting that a quote of KSHV lytic infection may spontaneously occur. Lytic phase induction or hypoxia could amplify virus release in supernatants. Conclusion Our results suggest that circulating endothelial progenitors from KS patients are KSHV-infected and support viral productive replication and may therefore represent potential virus reservoirs and putative precursors of KS spindle cells.

Highlights on distinctive structural and functional properties of HTLV Tax proteins

Human T cell leukemia viruses (HTLVs) are complex human retroviruses of the Deltaretrovirus genus. Four types have been identified thus far, with HTLV-1 and HTLV-2 much more prevalent than HTLV-3 or HTLV-4. HTLV-1 and HTLV-2 possess strictly related genomic structures, but differ significantly in pathogenicity, as HTLV-1 is the causative agent of adult T cell leukemia and of HTLV-associated myelopathy/tropical spastic paraparesis, whereas HTLV-2 is not associated with neoplasia. HTLVs code for a protein named Tax that is responsible for enhancing viral expression and drives cell transformation. Much effort has been invested to dissect the impact of Tax on signal transduction pathways and to identify functional differences between the HTLV Tax proteins that may explain the distinct oncogenic potential of HTLV-1 and HTLV-2. This review summarizes our current knowledge of Tax-1 and Tax-2 with emphasis on their structure, role in activation of the NF-κB (nuclear factor kappa-B) pathway, and interactions with host factors.

In Vitro and In Vivo Human Herpesvirus 8 Infection of Placenta

Herpesvirus infection of placenta may be harmful in pregnancy leading to disorders in fetal growth, premature delivery, miscarriage, or major congenital abnormalities. Although a correlation between human herpesvirus 8 (HHV-8) infection and abortion or low birth weight in children has been suggested, and rare cases of in utero or perinatal HHV-8 transmission have been documented, no direct evidence of HHV-8 infection of placenta has yet been reported. The aim of this study was to evaluate the in vitro and in vivo susceptibility of placental cells to HHV-8 infection. Short-term infection assays were performed on placental chorionic villi isolated from term placentae. Qualitative and quantitative HHV-8 detection were performed by PCR and real-time PCR, and HHV-8 proteins were analyzed by immunohistochemistry. Term placenta samples from HHV-8-seropositive women were analyzed for the presence of HHV-8 DNA and antigens. In vitro infected histocultures showed increasing amounts of HHV-8 DNA in tissues and supernatants; cyto- and syncitiotrophoblasts, as well as endothelial cells, expressed latent and lytic viral antigens. Increased apoptotic phenomena were visualized by the terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end-labeling method in infected histocultures. Ex vivo, HHV-8 DNA and a latent viral antigen were detected in placenta samples from HHV-8-seropositive women. These findings demonstrate that HHV-8, like other human herpesviruses, may infect placental cells in vitro and in vivo, thus providing evidence that this phenomenon might influence vertical transmission and pregnancy outcome in HHV-8-infected women.

Oral human papillomavirus and human herpesvirus-8 infections among human immunodeficiency virus type 1-infected men and women in Italy

Background Oral human papillomavirus (HPV) and human herpesvirus-8 (HHV8) infections are sexually transmitted and respectively associated with the development of oropharyngeal carcinoma and Kaposi sarcoma. The aim of the study was to evaluate HPV prevalence and its possible correlation with HHV8 oral shedding, in relation to sex, human immunodeficiency virus (HIV) behavioral risk factor, and immune function. Methods The study population comprised 100 HIV-infected individuals divided into 3 groups: (1) 38 men who have sex with men (MSM), (2) 24 heterosexual men, and (3) 38 women. DNA was obtained from cells of unstimulated whole saliva. Human papillomavirus sequences were searched for by polymerase chain reaction (PCR) with MY09/MY11 primers or by nested PCR with GP5+/GP6+ primers as the second step. Typing was accomplished by restriction fragment length polymorphism analysis or by direct sequencing or by reverse line blot. Human herpesvirus-8 sequences were detected and quantified by nested PCR and real-time PCR, respectively. Results Oral HPV infection was present in 37 (prevalence, 37%) of 100 (13 with high-risk and 24 with low-risk types) patients; the most frequent types were HPV16, HPV6, HPV10, HPV61, HPV66, and HPV83. Human herpesvirus-8 DNA was detected in 46 (46%) of 100 subjects. Both infections had the highest prevalence among MSM and the lowest among women; women had a lower prevalence of high-risk HPV types than did both male groups (P = 0.05). An inverse correlation was observed with concomitant oral HHV8 infection (P = 0.007). Conclusions High prevalence of oral HPV and HHV8 infections was observed; MSM had the highest figures, despite better control of HIV infection.

HTLV-1 Tax protein recruitment into IKKε and TBK1 kinase complexes enhances IFN-I expression.

The Tax protein expressed by human T-cell leukemia virus type 1 (HTLV-1) plays a pivotal role in the deregulation of cellular pathways involved in the immune response, inflammation, cell survival, and cancer. Many of these effects derive from Tax multiple interactions with host factors, including the subunits of the IKK-complex that are required for NF-κB activation. IKKɛ and TBK1 are two IKK-related kinases that allow the phosphorylation of interferon regulatory factors that trigger IFN type I gene expression. We observed that IKKɛ and TBK1 recruit Tax into cellular immunocomplexes. We also found that TRAF3, which regulates cell receptor signaling effectors, forms complexes with Tax. Transactivation analyses revealed that expression of Tax, in presence of IKKɛ and TBK1, enhances IFN-β promoter activity, whereas the activation of NF-κB promoter is not modified. We propose that Tax may be recruited into the TBK1/IKKɛ complexes as a scaffolding-adaptor protein that enhances IFN-I gene expression.

Identification of nuclear retention domains in the RBM20 protein.

RBM20 is a nuclear protein which regulates alternative splicing of expressed genes that have a key role in cardiac function. By cloning the human and mouse RBM20 cDNA, producing expressing vectors for truncated proteins, and comparing their sub‐cellular distribution in transfected cells, we have identified the sequences necessary for RBM20 full nuclear retention. The region overlaps an RNA binding motif and a serine–arginine domain. The sequence is conserved in many species but belongs only to RBM20 orthologs. The RMB20 tissue specificity, together with the properties of its nuclear localization determinant, demonstrates a specific evolutionary selection of post‐transcriptional regulation factors.

Evaluation of fast PCR reagents for rapid and sensitive detection of human herpesvirus 8

The potential advantage of using fast PCR to detect human herpesvirus 8 (HHV-8) was tested by running a rapid cycling protocol (5s-steps) in one standard and two fast ramping thermal cyclers to evaluate the performance of 8 different fast reagents. Under this extremely short time profile, assay sensitivity comparable to that of the original protocol was maintained using fast reagents from five suppliers. Reproducibility was higher using fast ramping thermal cyclers, suggesting that fast chemistry may be better matched with advanced instruments. Few fast reagents showed a 2-log-increase in sensitivity and good consistency, that allowed the substitution of the standard nested PCR method with the fast, single round technique. Overall, these studies indicate that some of the fast reagents tested may be used to perform a highly sensitive and reproducible HHV-8 detection with a considerable time saving.

A Luciferase Functional Quantitative Assay for Measuring NF-ĸB Promoter Transactivation Mediated by HTLV-1 and HTLV-2 Tax Proteins

HTLV-1 and HTLV-2 viruses express Tax transactivator proteins required for viral genome transcription and capable of transforming cells in vivo and in vitro. Although Tax oncogenic potential needs to be further elucidated, it is well established that Tax proteins activate, among others, transcription factors of the NF-ĸB family, which are involved in immune and inflammatory responses, cell growth, apoptosis, stress responses and oncogenesis. Here, we describe a reporter gene assay applied for quantitative analysis of Tax-dependent NF-ĸB activation. The procedure is based on co-transfection of two individual vectors containing the cDNA for firefly and Renilla luciferase enzymes and vectors expressing Tax proteins. The luciferase expression is driven by cis-NF-ĸB promoter regulatory elements responsive to Tax transactivating factor. This assay is particularly useful to investigate Tax influence on NF-ĸB activation mediated by viral or host factors.

HTLV Tax-1 and Tax-2 proteins enhance interferon regulatory factor 3 dependent promoter activation

The HTLV-1 infection is known to induce an alteration of type I interferon (IFN-I) signaling since it is capable of escaping IFN-mediated immune response in vitro and Tax-1 protein modulates the expression of factors involved in the interferon signaling. In the present study we have investigated the effect of Tax-1 and Tax-2 expression on the activation of an IFN-regulatory factor 3 (IRF3) regulated promoter through the recruitment of the IFN-I upstream IKKe and TANK-binding kinase 1 (TBK1) factors, two IkB-related kinase homologues, which are essential for the activation of IRF3 pathway. We have demonstrated that both Tax-1 and Tax-2 were detectable in immuno-complexes formed by IKKe and TBK1 in HEK 293T transfected cells, but did not interact with the IRF3 factor. The presence of Tax-1 and IKKe in transfected cells resulted in a significant activation of the IRF3 regulated promoter. A similar effect was measured in the presence of Tax-1 and TBK1. We have also observed that Tax-1 mutants defective in sumoylation and ubiquitination post-translational modification were impaired in their ability to form complexes with IKKe or TBK1 and in the transactivating activity on IRF3 dependent promoter.These data provide evidence for a role of Tax proteins in the activation of IFN-I pathway, mediated by interaction with IKKe and TBK1 kinases. The effects of the Tax interaction with factors that act upstream of interferon regulatory factor IRF3 should be taken into account to further explain the IFN-mediated immune response to HTLV-1 infection. This study is funded by AIRC-Cariverona.