The latency-associated nuclear antigen of Kaposi sarcoma-associated herpesvirus induces B cell hyperplasia and lymphoma

Kaposi sarcoma-associated herpesvirus (KSHV) is a human lymphotropic herpesvirus. It is implicated in B cell neoplasias such as primary effusion lymphoma and multicentric Castleman disease in AIDS patients. The KSHV latency-associated nuclear antigen (LANA) is consistently expressed in all KSHV-associated tumor cells and was shown to bind the tumor suppressor proteins p53 and pRb. To test LANA's contribution to lymphomagenesis in vivo we generated transgenic mice expressing LANA under the control of its own promoter, which is B cell specific. All of the transgenic mice developed splenic follicular hyperplasia due to an expansion of IgM+ IgD+ B cells and showed increased germinal center formation. We also observed lymphomas, implying that LANA can activate B cells and provide the first step toward lymphomagenesis.     

Potent antiviral activity of north-methanocarbathymidine against Kaposi's sarcoma-associated herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) infection is a prerequisite for the development of Kaposi's sarcoma (KS). Blocking lytic KSHV replication may hinder KS tumorigenesis. Here, we report potent in vitro anti-KSHV activity of 2'-exo-methanocarbathymidine [North-methanocarbathymidine (N-MCT)], a thymidine analog with a pseudosugar ring locked in the northern conformation, which has previously been shown to block the replication of herpes simplex virus types 1 and 2. N-MCT inhibited KSHV virion production in lytically induced KSHV-infected BCBL-1 cells with a substantially lower 50% inhibitory concentration (IC50) than those of cidofovir (CDV) and ganciclovir (GCV) (IC50, mean +/- standard deviation: 0.08 +/- 0.03, 0.42 +/- 0.07, and 0.96 +/- 0.49 microM for N-MCT, CDV, and GCV, respectively). The reduction in KSHV virion production was accompanied by a corresponding decrease in KSHV DNA levels in the N-MCT-treated BCBL-1 cells, indicating that the compound blocked lytic KSHV DNA replication. A time- and dose-dependent accumulation of N-MCT-triphosphate (TP) was demonstrated in lytically induced BCBL-1 cells, while uninfected cells showed virtually no accumulation. The levels of N-MCT-TP were significantly decreased in the presence of 5'-ethynylthymidine, a potent inhibitor of herpesvirus thymidine kinase, resulting in the abrogation of anti-KSHV activity of N-MCT. N-MCT-TP more effectively blocked in vitro DNA synthesis by KSHV DNA polymerase with an IC50 of 6.24 +/- 0.08 microM (mean +/- standard deviation) compared to CDV-diphosphate (14.70 +/-2.47 microM) or GCV-TP (24.59 +/- 5.60 microM). Taken together, N-MCT is a highly potent and target-specific anti-KSHV agent which inhibits lytic KSHV DNA synthesis through its triphosphate metabolite produced in KSHV-infected cells expressing a virally encoded thymidine kinase.     

Chemokines activate Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor in mammalian cells in culture.

Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8, a virus that appears to be involved in the pathogenesis of Kaposi's sarcoma and primary effusion lymphomas, encodes a G protein-coupled receptor (KSHV-GPCR) that exhibits constitutive signaling. In this report, we show that two chemokines, interleukin 8 (IL-8) and growth-related protein-alpha, activate KSHV-GPCR over constitutive levels. Moreover, as with human receptors, the integrity of the ELR motif of these chemokines is required for activation of KSHV-GPCR. Other residues that are required for IL-8 binding to human chemokine receptors CXCR1 and CXCR2 are important for KSHV-GPCR activation also. Thus, it appears that the ELR binding site and other key domains of ELR chemokine activation have been preserved in the virus KSHV-GPCR. The results suggest that KSHV-GPCR originated from CXCR1 or CXCR2 and that activation of KSHV-GPCR by endogenous chemokines may affect the pathobiology of KSHV infection in humans.     

Blockade of viral interleukin-6 expression of Kaposi's sarcoma-associated herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8, is associated with several malignant disorders, including Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. An early lytic gene of KSHV encodes viral interleukin-6 (vIL-6), a viral homologue of the proinflammatory cytokine and an autocrine/paracrine growth factor human IL-6. In this study, we examined the effects of suppressing vIL-6 expression in PEL cells with antisense peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO). PPMO are ssDNA-analogues that have a modified backbone and enter cells readily. Treatment of PEL cells with a PPMO designed against vIL-6 mRNA led to a marked reduction in the proportion of vIL-6-positive cells detected by immunofluorescence assay. Analysis by Western blot confirmed a specific reduction in the vIL-6 protein level and showed that the reduction was dependent on the dose of vIL-6 PPMO. PEL cells treated with the vIL-6 PPMO exhibited reduced levels of cellular growth, IL-6 expression and KSHV DNA, and an elevated level of p21 protein. Treatment of PEL cells with a combination of two vIL-6 PPMO compounds targeting different sequences in the vIL-6 mRNA led to an inhibitory effect that was greater than that achieved with either PPMO alone. These results show that PPMO targeting vIL-6 mRNA can potently reduce vIL-6 protein translation and indicate that further exploration of these compounds in an animal model for potential clinical application is warranted.     

Induction of B cell hyperplasia in simian immunodeficiency virus-infected rhesus macaques with the simian homologue of Kaposi's sarcoma-associated herpesvirus.

A simian homologue of Kaposi's sarcoma-associated herpesvirus (KSHV), the eighth human herpesvirus (HHV8), was isolated from a simian immunodeficiency virus (SIV)-infected rhesus macaque (Macaca mulatta) that developed a multicentric lymphoproliferative disorder (LPD). This simian rhadinovirus is genetically similar to a recently described rhesus rhadinovirus (RRV) (Desrosiers, R.C., V.G. Sasseville, S.C. Czajak, X. Zhang, K.G. Mansfield, A. Kaur, R.P. Johnson, A.A. Lackner, and J.U. Jung. 1997. J. Virol. 71:9764-9769) and is designated RRV 17577. RRV 17577 was experimentally inoculated into rhesus macaques with and without SIV(mac239) infection to determine if RRV played a role in development of the LPD observed in the index case. In contrast to control animals inoculated with SIV(mac239) or RRV alone, two animals coinfected with SIV(mac239) and RRV 17577 developed hyperplastic LPD resembling the multicentric plasma cell variant of Castleman's disease, characterized by persistent angiofollicular lymphadenopathy, hepatomegaly, splenomegaly, and hypergammaglobulinemia. Hypergammaglobulinemia was associated with severe immune-mediated hemolytic anemia in one RRV/SIV-infected macaque. Both RRV/SIV-infected macaques exhibited persistent RRV viremia with little or no RRV-specific antibody response. The macaques inoculated with RRV alone displayed transient viremia followed by a vigorous anti-RRV antibody response and lacked evidence of LPD in peripheral blood and lymph nodes. Infectious RRV and RRV DNA were present in hyperplastic lymphoid tissues of the RRV/SIV-infected macaques, suggesting that lymphoid hyperplasia is associated with the high levels of replication. Thus, experimental RRV 17577 infection of SIV-infected rhesus macaques induces some of the hyperplastic B cell LPDs manifested in AIDS patients coinfected with KSHV.     

Prostratin and bortezomib are novel inducers of latent Kaposi's sarcoma-associated herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes latent infections in lymphocytes and endothelial cells, and latent infection is closely linked to tumorigenesis. As few viral markers are expressed during latency, compounds that can safely and efficiently increase lytic gene expression in vivo have been sought. We have found that the non-tumour-promoting phorbol ester prostratin and the proteasome inhibitor bortezomib induce immediate-early, early and late KSHV gene expression from two lymphoma cell lines in vitro. Their ability to induce lytic gene expression supports a role for phorbol-ester and proteasome-regulated signalling pathways in KSHV reactivation and prompts further investigation of prostratin and bortezomib as therapeutic agents for KSHV-associated malignancies.     

Human herpesvirus-8/Kaposi's sarcoma-associated herpesvirus is a new transmissible virus that infects B cells

Herpesviral DNA fragments isolated from AIDS-associated Kaposi's sarcoma (KS) tissue (KSHV-DNA) share homology with two lymphotropic oncogenic gamma-herpesviruses, Epstein-Barr virus and Herpesvirus saimiri, and are present in the lesions of more than 95% of HIV and non- HIV-associated forms of KS, AIDS-related body cavity-based lymphomas, and AIDS-related multicentric Castleman's disease. Here we show that BC- 1, a KSHV-DNA-positive, body cavity-based lymphoma cell line, produces infective herpesviral particles carrying a linear 270-kb genome that specifically transmits KSHV-DNA to CD19+ B cells. Transmission of KSHV- DNA is dependent upon a biologically active, replicating virus, since it is blocked by UV irradiation and foscarnet, an inhibitor of viral DNA-polymerase. This study represents the first isolation and transmission of the human herpesvirus-8/KS-associated herpesvirus.     

Ugandan Kaposi's sarcoma-associated herpesvirus phylogeny: evidence for cross-ethnic transmission of viral subtypes

OBJECTIVE: The aim of this study was to test the relationship between Kaposi's sarcoma-associated herpesvirus (KSHV) phylogeny and host ethnicity at the within-country scale. METHODS: KSHV genomic DNA samples were isolated from 31 patients across eleven Ugandan ethnic groups. Amino acid sequences of the ORF-K1 gene were used to construct a neighbor-joining phylogenetic tree. RESULTS: A5 and B1 variants predominated with no evidence of distinct ethnic or geographic distribution. A new K1 subtype (F) was identified in a member of the Bantu Gisu tribe and a new subtype B variant (B3) among members of the Bantu Ganda tribe. CONCLUSIONS: The phylogeny may yet be structured by host ethnicity if members of Ugandan groups have convoluted biological origins, even as they identify with single tribes. An alternative possibility is that KSHV subtype evolution may have preceded major diversification of sub-Saharan Africans into ethnicities as we know them today, with ethnic groups beginning their histories already hosting multiple subtypes. A third alternative is that horizontal transmission of multiple KSHV subtypes may have broken up vertical lineages of the virus passed down within Ugandan populations.     

Potent antiviral activity of topoisomerase I and II inhibitors against Kaposi's sarcoma-associated herpesvirus

The lytic DNA replication of Kaposi's sarcoma-associated herpesvirus (KSHV) initiates at an origin (ori-Lyt) and requires trans-acting elements, both viral and cellular. We recently demonstrated that several host cellular proteins, including topoisomerases I and II (Topo I and II), are involved in KSHV lytic DNA replication (Y. Wang, H. Li, Q. Tang, G. G. Maul, and Y. Yuan. J. Virol. 82: 2867-2882, 2008). To assess the importance of these topoisomerases in viral lytic replication, shRNA-mediated gene silencing was used. Depletion of Topo I and II severely inhibited viral lytic DNA replication as well as virion production, suggesting essential roles of these cellular proteins in viral DNA replication. The discovery of Topo I and II as enzymes indispensable for KSHV DNA replication raises a possibility that these cellular proteins could be new targets of therapeutic approaches to halt KSHV replication and treat KSHV-associated diseases. In this report, we examined one Topo I inhibitor and several Topo II inhibitors (inclusive of Topo II poison and catalytic inhibitors) as potential therapeutic agents for blocking KSHV replication. The Topo II catalytic inhibitors in general exhibited marked inhibition on KSHV replication and minimal cytotoxicity. In particular, novobiocin, with the best selectivity index (SI = 31.62) among the inhibitors tested in this study, is effective in inhibiting KSHV DNA replication and virion production but shows little adverse effect on cell proliferation and cycle progression in its therapeutic concentration, suggesting its potential to become an effective and safe drug for the treatment of human diseases associated with KSHV infection.     

Kaposi's sarcoma-associated herpesvirus/human herpesvirus type 8-positive solid lymphomas: a tissue-based variant of primary effusion lymphoma

Kaposi's sarcoma-associated herpesvirus (KSHV), also termed human herpesvirus type 8, is consistently identified in Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. Here we report four cases of KSHV-bearing solid lymphomas that occurred in AIDS patients (cases 1 to 3) and in a human immunodeficiency virus (HIV)-seronegative person (case 4). The patients presented extranodal masses in the abdomen (cases 1, 3, and 4) or skin (case 2), and nodal involvement, together with Kaposi's sarcoma (case 3). The gastrointestinal tract was involved in two patients (cases 1 and 3). The patients did not develop a lymphomatous effusion. KSHV was detected in the tumor cells of all cases by immunohistochemistry and by polymerase chain reaction. Epstein-Barr virus was detected in two of the HIV-related cases. All KSHV-positive solid lymphomas exhibited PEL-like cell morphology. To investigate the relationship of these disorders to PEL and to other AIDS-associated diffuse large cell lymphomas, KSHV-positive solid lymphomas were tested for the expression of a set of genes that were previously shown by gene profiling analysis to define PEL tumor cells. The results showed that expression of this set of genes in KSHV-positive lymphomas is similar to that of PEL but distinct from KSHV-negative AIDS-associated diffuse large cell lymphomas. Because pathobiological features of KSHV-positive solid lymphomas closely mimic those of PEL, our results suggest that KSHV-positive solid lymphomas should be considered as a tissue-based variant of classical PEL, irrespective of HIV status.     

Sensitivity of Kaposi's sarcoma-associated herpesvirus replication to antiviral drugs. Implications for potential therapy.

Using a cell line (termed BCBL-1) derived from a peripheral effusion (body cavity-based) lymphoma latently infected with Kaposi's sarcoma-associated herpesvirus (KSHV), we recently reported the successful induction of KSHV replication in culture (Renne, R., W. Zhong, B. Herndier, M. McGrath, N. Abbey, D. Kedes, and D. Ganem. 1996. Nat. Med. 2:342-346). Here we report the first use of this system for establishing the susceptibility of KSHV to available antiviral drugs. Latently infected BCBL-1 cells were induced to lytic replication with phorbol esters; such cells secrete large numbers of KSHV virions into the culture medium. We assayed the ability of the antivirals to block KSHV production, as measured by the release of encapsidated viral DNA. The results show that KSHV replication is insensitive to acyclovir (9-[(2-hydroxyethoxy)-methyl]guanine) (50% inhibitory concentration [IC50] = 60-80 microM), but sensitive to ganciclovir (9-[1,3-dihydroxy-2-propoxymethyl]guanine) (IC50 = 2.7-4 microM), foscarnet (trisodium phosphonoformate hexahydrate) (IC50 = 80-100 microM), and cidofovir (1-[(S)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine) (IC50 = 0.5-1 microM).     

RNA-associated autoantigens activate B cells by combined B cell antigen receptor/Toll-like receptor 7 engagement

Previous studies (Leadbetter, E.A., I.R. Rifkin, A.H. Hohlbaum, B. Beaudette, M.J. Shlomchik, and A. Marshak-Rothstein. 2002. Nature. 416:603-607; Viglianti, G.A., C.M. Lau, T.M. Hanley, B.A. Miko, M.J. Shlomchik, and A. Marshak-Rothstein. 2003. Immunity. 19:837-847) established the unique capacity of DNA and DNA-associated autoantigens to activate autoreactive B cells via sequential engagement of the B cell antigen receptor (BCR) and Toll-like receptor (TLR) 9. We demonstrate that this two-receptor paradigm can be extended to the BCR/TLR7 activation of autoreactive B cells by RNA and RNA-associated autoantigens. These data implicate TLR recognition of endogenous ligands in the response to both DNA- and RNA-associated autoantigens. Importantly, the response to RNA-associated autoantigens was markedly enhanced by IFN-alpha, a cytokine strongly linked to disease progression in patients with systemic lupus erythematosus (SLE). As further evidence that TLRs play a key role in autoantibody responses in SLE, we found that autoimmune-prone mice, lacking the TLR adaptor protein MyD88, had markedly reduced chromatin, Sm, and rheumatoid factor autoantibody titers.     

Inhibition of interleukin 7 receptor signaling by antigen receptor assembly

After the productive rearrangement of immunoglobulin (Ig) heavy chain genes, precursor (pre-)B lymphocytes undergo a limited number of cell divisions in response to interleukin (IL)-7. Here, we present evidence that this phase of IL-7-dependent expansion is constrained by an inhibitory signal initiated by antigen receptor assembly. A line of pre-B cells from normal murine bone marrow that expresses a mu heavy chain with a D-proximal V(H)7183.2 region divides continuously in IL-7. IL-7 responsiveness ceases upon differentiation to the mu(1), kappa(1) stage, despite continuing expression of the IL-7 receptor (IL-7R), suggesting that antigen receptor assembly inhibits IL-7 responsiveness. This is confirmed by introduction of a rearranged lambda light chain gene, which inhibits proliferative signaling through the IL-7R. Inhibition is specific to the IL-7R, because it is overcome by replacement of the IL-7R cytoplasmic domain with corresponding sequences from the closely related IL-2Rbeta chain. Alteration of a single tyrosine residue, Tyr410, in the IL-7R cytoplasmic domain to phenylalanine also prevents the inhibition of proliferation after antigen receptor assembly. Thus, the loss of IL-7 responsiveness after antigen receptor assembly may be mediated through the recruitment of an inhibitory molecule to this residue. Our findings identify a novel mechanism that limits cytokine-dependent proliferation during B lymphopoiesis. This mechanism may be essential for the proper regulation of peripheral B lymphocyte numbers.     

Inhibition of B Virus (Macacine herpesvirus 1) by Conventional and Experimental Antiviral Compounds

B virus infection of humans results in high morbidity and mortality in as many as 80% of identified cases. The main objective of this study was to conduct a comparative analysis of conventional and experimental antiviral drug susceptibilities of B virus isolates from multiple macaque species and zoonotically infected humans. We used a plaque reduction assay to establish the effective inhibitory doses of acyclovir, ganciclovir, and vidarabine, as well as those of a group of experimental nucleoside analogs with known anti-herpes simplex virus activity. Four of the experimental drugs tested were 10- to 100-fold more potent inhibitors of B virus replication than conventional antiviral agents. Drug efficacies were similar for multiple B virus isolates tested, with variations within 2-fold of the median effective concentration (EC₅₀) for each drug, and each EC₅₀ was considerably lower than those for B virus thymidine kinase (TK) mutants. We observed no differences in the viral TK amino acid sequence between B virus isolates from rhesus monkeys and those from human zoonoses. Differences in the TK protein sequence between cynomolgus and pigtail macaque B virus isolates did not affect drug sensitivity except in the case of one compound. Taken together, these data suggest that future B virus zoonoses will respond consistently to conventional antiviral treatment. Further, the considerably higher potency of FEAU (2′-fluoro-5-ethyl-Ara-U) than of conventional antiviral drugs argues for its compassionate use in advanced human B virus infections.In its natural host, macaque monkeys, B virus (Macacine herpesvirus 1; Simplexvirus, Herpesviridae) causes lesions on epithelial surfaces (2, 33) and establishes reactivatable latent infection in sensory neurons (30, 37), like herpes simplex virus (HSV) in humans. B virus often results in severe pathogenesis, including paralysis, encephalitis, and in many cases, rapid death, following infection of humans (reviewed in reference 22). Nearly all reported cases of B virus zoonosis have been associated with individuals handling macaques during the course of research or technology development (33). Five fatalities, along with at least 23 cases in which the patient survived, have occurred in the past 20 years, underscoring that zoonotic infections remain a problem in the laboratory animal environment (7-9).The CDC''s B Virus Working Group currently recommends treatment of confirmed zoonotic infections with herpesvirus-specific antiviral drugs, including acyclovir (ACV) and ganciclovir (GCV) (10). Both agents in this class of compounds are phosphorylated to active form by virus-encoded thymidine kinase (TK). The resulting nucleoside triphosphate analog inhibits viral DNA replication by termination of chain elongation and by direct inhibition of herpesvirus DNA polymerase (24). While ACV is effective against B virus both in cell culture and in animal models (6), the dose required for 50% plaque reduction is more than 10-fold higher for B virus than for HSV type 1 (HSV-1) (38). GCV is twice as potent as ACV in B virus plaque reduction, yet the median effective concentration (EC₅₀) of GCV for B virus is almost 10 times higher than that for HSV-1 (38). In some, but not all, cases of zoonotic B virus infection, acyclovir and ganciclovir have proven to be effective at curtailing disease progression (7, 8). Zoonotic infections that have progressed to extensive central nervous system (CNS) involvement, including respiratory arrest, appear to be refractory to conventional antiviral intervention (12, 15; J. Hilliard, unpublished data).Vidarabine (9-β-d-arabinofuranosyladenine [ara-A, or VDB]), an antiviral agent effective against HSV, may be useful for the treatment of early stages of zoonotic B virus infections, but it has not been used alone in previous cases. Prior to the use of ACV for treatment of HSV, intravenous VDB was used for treatment of encephalitic infections (34). In cell culture, it has been shown to be equally potent against B virus and HSV-1 (5). VDB does not require selective phosphorylation by the viral TK for activity (4); however, since VDB can be converted to its active form by host enzymes, it has potential toxicity in humans (29).Experimental drugs effective in cell culture against HSV include the β-d-2′-fluoro-5-substituted arabinosyl pyrimidines FMAU (2′-fluoro-5-methyl-Ara-U) and FEAU (2′-fluoro-5-ethyl-Ara-U), both of which require phosphorylation by viral thymidine kinase for activation (19). FMAU and FEAU have potencies similar to that of ACV for inhibition of HSV-1 in cell culture, but FMAU has 10-fold greater potency than ACV against HSV-2 (19). Unfortunately, FMAU has been reported to be toxic to humans at elevated doses used for cancer chemotherapy (1, 13); FMAU can be incorporated into uninfected cell DNA by host DNA polymerase, suggesting the basis of its toxicity in vivo (11). FEAU has been shown to be a selective inhibitor of HSV DNA synthesis in cell culture (7), but its toxicity in humans has not been investigated. Other β-d-2′-fluoro-5-substituted arabinosyl pyrimidines, such as FMAC (2′-fluoro-5-methyl-Ara-C) and FBrAC (2′-fluoro-5-bromyl-Ara-C), have been synthesized and tested against HSV (17, 32; R. F. Schinazi, unpublished results). No information on FBrAC toxicity is known.The goal of this study was to determine the general variability of drug susceptibility and the efficacy of a class of experimental antiviral agents by using a panel of B virus isolates from multiple macaque species and humans. The results presented here suggest that B virus isolates in the wild are susceptible to antiviral agents that require the viral TK for activity. Further, our findings that specific experimental nucleoside analogs are appreciably more effective than conventionally used antiviral agents at blocking B virus replication suggest that these drugs may benefit the treatment of high-morbidity human cases.