Beta1 integrins mediate tubule formation induced by supernatants derived from KSHV-infected cells.

OBJECTIVE: Angiogenesis is defined as the formation of new blood vessels. In a recently concluded study, we identified Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells derived from primary effusion lymphoma (PEL) to overexpress vascular endothelial growth factor (VEGF) that had the propensity to mediate tubule formation on a Matrigel, an indicator of angiogenesis. The objective of this study was to determine the receptor molecules that mediate the tubule formation induced by the supernatant derived from KSHV-infected PEL cells. METHODS: The identity of receptor(s) that play a role in mediating tubule formation driven by PEL supernatant was determined by the classical in vitro angiogenesis assay conducted on a Matrigel. RESULTS: RGD peptides, antibodies, and siRNA specific to beta1 integrins significantly lowered the ability of the PEL supernatants to induce tubule formation by endothelial cells. beta1 Integrins mediated tubule formation to comparable levels in endothelial cells that were incubated with supernatants derived from uninduced or TPA-induced PEL cells. Interestingly, the beta1 integrins did not seem to have a major role in cellular attachment. CONCLUSION: We report for the first time a critical role for beta1 integrins in angiogenesis supported by the supernatant from KSHV-infected PEL cells.

Raf/MEK/ERK signalling triggers reactivation of Kaposi's sarcoma-associated herpesvirus latency.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease. KSHV infection of cells produces both latent and lytic cycles of infection. In vivo, the virus is found predominantly in the latent state. In vitro, a lytic infection can be induced in KSHV-infected cells by treating with phorbol ester (TPA). However, the exact signalling events that lead to the reactivation of KSHV lytic infection are still elusive. Here, a role is demonstrated for B-Raf/MEK/ERK signalling in TPA-induced reactivation of KSHV latent infection. Inhibiting MEK/ERK signalling by using MEK-specific inhibitors decreased expression of the TPA-induced KSHV lytic-cycle gene ORF8. Transfection of BCBL-1 cells with B-Raf small interfering RNA inhibited TPA-induced KSHV lytic infection significantly. Additionally, overexpression of MEK1 induced a lytic cycle of KSHV infection in BCBL-1 cells. The significance of these findings in understanding the biology of KSHV-associated pathogenesis is discussed.

Identifying cellular genes crucial for the reactivation of Kaposi's sarcoma-associated herpesvirus latency.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the latest addition to the long list of human herpesviruses. Reactivation of latent herpesvirus infections is still a mystery. It was demonstrated recently that the phorbol ester TPA was efficient in inducing a reactivation of KSHV infection in the S phase of the cell cycle. In the present study, flow cytometry-sorted, TPA-induced, KSHV-infected haematopoietic cells (BCBL-1) were used to analyse the expression profiles of cancer-related cellular genes in the S phase of the cell cycle compared with the G0/1 phase by using microarrays. Overall, the S phase of the cell cycle seems to provide KSHV with an apt environment for a productive lytic cycle of infection. The apt conditions include cellular signalling that promotes survivability, DNA replication and lipid metabolism, while blocking cell-cycle progression to M phase. Some of the important genes that were overexpressed during the S phase of the cell cycle compared with the G0/1 phase of TPA-induced BCBL-1 cells are v-myb myeloblastosis (MYBL2), protein kinase-membrane associated tyrosine/threonine 1 (PKMYT1), ribonucleotide reductase M1 polypeptide (RRM1) and peroxisome proliferator-activated receptors delta (PPARD). Inhibition of PKMYT1 expression by the use of specific short interfering RNAs significantly lowered the TPA-induced KSHV lytic cycle of infection. The significance of these and other genes in the reactivation of KSHV is discussed in the following report. Taken together, a flow cytometry-microarray-based method to study the cellular conditions critical for the reactivation of KSHV infection is reported here for the first time.

AIDS related viruses, their association with leukemia, and Raf signaling.

Leukemia is characterized by the production of an excessive number of abnormal white blood cells. Over time, this expanding population of poorly/non- functional white blood cells overwhelms the normal function of the body's blood and immune systems. DNA translocations have been found common to leukemia, including Raf mutations. While the cause of leukemia is not known, several risk factors have been identified. In this review, we present an update on the role of AIDS related viruses as an etiology for leukemia. Human immunodeficiency virus-1 and -2 (HIV-1; -2) are the cause for the development of acquired immune deficiency syndrome (AIDS). Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), Human papillomavirus (HPV), and Kaposi's sarcoma-associated herpesvirus (KSHV) are specifically implicated in AIDS associated malignancies. However, there are other viruses that are associated to a lesser extent with the AIDS condition and they are Human T-cell leukemia virus-1 (HTLV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), and human herpesvirus-6 (HHV-6). Of these viruses, HTLV-1 has been etiologically associated with leukemia. Recent evidence suggests that EBV, HBV, HCV, and KSHV may also play a role in the development of some types of leukemia. Raf signaling has been shown to aid in the infection and pathogenesis of many of these viruses, making Raf pathway components good potential targets for the treatment of leukemia induced by AIDS related viruses.

Cigarette smoke concentrate inhibits Kaposi's sarcoma-associated herpesvirus infection.

Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma (KS), primary effusion lymphoma(PEL), multicentric Castleman disease, and other tumors. Progression of KS is dictated by an aberrant production of inflammatory cytokines and increase in KSHV infection of cells. In this study, we analyzed the effect of cigarette smoke concentrate (CSC) on KSHV infection of human foreskin fibroblasts (HFF) using real time quantitative RT-PCR. Our results demonstrated that the CSC-treated cells supported 50% lower infection of KSHV when compared to the untreated cells. Radiolabeled-binding assays indicated that CSC inhibited KSHV infection of cells at a post attachment stage of entry. Taken together, we report for the first time the ability of CSC to specifically inhibit KSHV infection of cells.

Spectroscopic analysis of Kaposi's sarcoma-associated herpesvirus infected cells by Raman tweezers.

Kaposi's sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus-8 (HHV-8), is a tumor causing virus. KSHV is the cause of several disease conditions known as Kaposi's sarcoma, multicentric Castleman disease, and primary effusion lymphoma. Cell culture supernatants from KSHV infected hematopoietic cells induced angiogenic tubule formation to a significantly greater extent than uninfected hematopoietic cells. Raman spectrum profiles were generated to differentiate the uninfected from KSHV infected cells. In general, profiles from all the hematopoietic cells shared similar peaks; however, the relative abundance of specific components varied significantly between the cells. Subsequent use of the multivariate analysis of the Raman spectra revealed significant differences between the uninfected and the KSHV infected cells. Taken together, this study reports the use of Raman tweezers to distinguish and analyze the biological relevance of KSHV infected cell signaling.

Biology of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus-8 (HHV-8) is a newly identified herpesvirus. KSHV is an important pathogen capable of causing disease that affects all age groups worldwide. KSHV is etiologically associated with all forms of Kaposi's sarcoma (KS), body cavity lymphomas, and multicentric Castleman disease (MCD). The use of highly active antiretroviral therapy (HAART) since 1996 has markedly reduced the prevalence of KS in western countries, but because 99% of the 40 million patients with AIDS in the world cannot afford HAART, KSHV pathogenesis is still a very common problem. In this chapter, we delineate some of the latest findings about KSHV infection and pathogenesis.

B-Raf-dependent expression of vascular endothelial growth factor-A in Kaposi sarcoma-associated herpesvirus-infected human B cells.

Kaposi sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8) is etiologically linked to Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease. Vascular endothelial growth factor-A (VEGF-A) is one of the essential factors required in KSHV pathogenesis, mainly due to its ability to mediate angiogenesis. In this report we analyzed the relationship between Raf and VEGF-A expression in KSHV-infected hematopoietic cells. All of the KSHV-infected cell lines (derived from PEL) expressed higher levels of B-Raf and VEGF-A when compared with uninfected cells. Inhibition of Raf to mitogen-induced extracellular kinase (MEK) to extracellular signal-related kinase (ERK) signaling, either by the use of MEK inhibitor (PD98059) or by siRNA specific to B-Raf, significantly lowered VEGF-A expression. In addition, B-Raf-induced VEGF-A expression was demonstrated to be sufficient to enhance tubule formation in endothelial cells. Interestingly, we did not observe mutation in the B-Raf gene of the KSHV-infected PEL cell lines. Taken together, we report for the first time the ability of Raf-associated signaling to play a role in the expression of VEGF-A in KSHV-infected hematopoietic cells.