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Mixed lineage leukemia 1 (MLL1) is a histone methyltransferase. Kaposi's sarcoma-associated herpesvirus (KSHV) is a leading cause of malignancy in AIDS. KSHV latently infects tumor cells and its genome is decorated with epigenetic marks. Here, we show that KSHV latency-associated nuclear antigen (LANA) recruits MLL1 to viral DNA where it establishes H3K4me3 modifications at the extensive KSHV terminal repeat elements during primary infection. LANA interacts with MLL1 complex members, including WDR5, integrates into the MLL1 complex, and regulates MLL1 activity. We describe the 1.5-Å crystal structure of N-terminal LANA peptide complexed with MLL1 complex member WDR5, which reveals a potential regulatory mechanism. Disruption of MLL1 expression rendered KSHV latency establishment highly deficient. This deficiency was rescued by MLL1 but not by catalytically inactive MLL1. Therefore, MLL1 is LANA regulable and exerts a central role in virus infection. These results suggest broad potential for MLL1 regulation, including by non-host factors.

Precision cancer medicine aims to classify tumors by site, histology, and molecular testing to determine an individualized profile of cancer alterations. Viruses are a major contributor to oncogenesis, causing 12% to 20% of all human cancers. Several viruses are causal of specific types of cancer, promoting dysregulation of signaling pathways and resulting in carcinogenesis. In addition, integration of viral DNA into the host (human) genome is a hallmark of some viral species. Tests for the presence of viral infection used in the clinical setting most often use quantitative PCR or immunohistochemical staining. Both approaches have limitations and need to be interpreted/scored appropriately. In some cases, results are not binary (virus present/absent), and it is unclear what to do with a weakly or partially positive result. In addition, viral testing of cancers is performed separately from tests to detect human genomic alterations and can thus be time-consuming and use limited valuable specimen. We present a hybrid-capture and massively parallel sequencing approach to detect viral infection that is integrated with targeted genomic analysis to provide a more complete tumor profile from a single sample.

Kaposi’s sarcoma-associated herpesvirus (KSHV) causes the endothelial tumor KS, a leading cause of morbidity and mortality in sub-Saharan Africa. KSHV-encoded microRNAs (miRNAs) are known to play an important role in viral oncogenesis; however, the role of host miRNAs in KS tumorigenesis remains largely unknown. Here, high-throughput small-RNA sequencing of the cellular transcriptome in a KS xenograft model revealed miR-127-3p as one of the most significantly down-regulated miRNAs, which we validated in KS patient tissues. We show that restoration of miR-127-3p suppresses KSHV-driven cellular transformation and proliferation and induces G(1) cell cycle arrest by directly targeting the oncogene SKP2. This miR-127-3p–induced G(1) arrest is rescued by disrupting the miR-127-3p target site in SKP2 messenger RNA (mRNA) using gene editing. Mechanistically, miR-127-3p–mediated SKP2 repression elevates cyclin-dependent kinase (CDK) inhibitor p21(Cip1) and down-regulates cyclin E, cyclin A, and CDK2, leading to activation of the RB protein tumor suppressor pathway and suppression of the transcriptional activities of E2F and Myc, key oncoprotein transcription factors crucial for KSHV tumorigenesis. Consequently, metabolomics analysis during miR-127-3p–induced cell cycle arrest revealed significant depletion of dNTP pools, consistent with RB-mediated repression of key dNTP biosynthesis enzymes. Furthermore, miR-127-3p reconstitution in a KS xenograft mouse model suppresses KSHV-positive tumor growth by targeting SKP2 in vivo. These findings identify a previously unrecognized tumor suppressor function for miR-127-3p in KS and demonstrate that the miR-127-3p/SKP2 axis is a viable therapeutic strategy for KS.

Primary effusion lymphoma (PEL) has a very poor prognosis. To evaluate the contributions of enhancers/promoters interactions to PEL cell growth and survival, here we produce H3K27ac HiChIP datasets in PEL cells. This allows us to generate the PEL enhancer connectome, which links enhancers and promoters in PEL genome-wide. We identify more than 8000 genomic interactions in each PEL cell line. By incorporating HiChIP data with H3K27ac ChIP-seq data, we identify interactions between enhancers/enhancers, enhancers/promoters, and promoters/promoters. HiChIP further links PEL super-enhancers to PEL dependency factors MYC, IRF4, MCL1, CCND2, MDM2, and CFLAR. CRISPR knock out of MEF2C and IRF4 significantly reduces MYC and IRF4 super-enhancer H3K27ac signal. Knock out also reduces MYC and IRF4 expression. CRISPRi perturbation of these super-enhancers by tethering transcription repressors to enhancers significantly reduces target gene expression and reduces PEL cell growth. These data provide insights into PEL molecular pathogenesis., Primary effusion lymphoma (PEL) has a very poor prognosis. Here, the authors perform H3K27ac HiChIP in PEL cells and generate the PEL enhancer connectome, linking enhancers and promoters in PEL, as well as super-enhancers to dependency factors.

Significance Understanding how viruses modulate host cell function is central to prospects for potential therapies. A recently described mechanism mediates selective protein interactions between acidic domain readers and unacetylated, lysine-rich regions, opposite of bromodomain function. Here we show that the latency-associated nuclear antigen (LANA) encoded by Kaposi´s sarcoma-associated herpesvirus (KSHV) contains a SET oncoprotein homologous acidic domain reader that interacts with p53 in an acetylation-dependent manner. Significantly, the LANA acidic domain reader is critical for viral latency and persistent infection. The finding that KSHV has evolved a mechanism for acetylation-dependent protein interaction underscores the physiological importance of this cellular regulatory posttranslational modification. Furthermore, opportunity may exist to develop inhibitors that disrupt LANA acidic domain reader function for therapy of KSHV-associated tumors., Viruses modulate biochemical cellular pathways to permit infection. A recently described mechanism mediates selective protein interactions between acidic domain readers and unacetylated, lysine-rich regions, opposite of bromodomain function. Kaposi´s sarcoma (KS)-associated herpesvirus (KSHV) is tightly linked with KS, primary effusion lymphoma, and multicentric Castleman’s disease. KSHV latently infects cells, and its genome persists as a multicopy, extrachromosomal episome. During latency, KSHV expresses a small subset of genes, including the latency-associated nuclear antigen (LANA), which mediates viral episome persistence. Here we show that LANA contains two tandem, partially overlapping, acidic domain sequences homologous to the SET oncoprotein acidic domain reader. This domain selectively interacts with unacetylated p53, as evidenced by reduced LANA interaction after overexpression of CBP, which acetylates p53, or with an acetylation mimicking carboxyl-terminal domain p53 mutant. Conversely, the interaction of LANA with an acetylation-deficient p53 mutant is enhanced. Significantly, KSHV LANA mutants lacking the acidic domain reader sequence are deficient for establishment of latency and persistent infection. This deficiency was confirmed under physiological conditions, on infection of mice with a murine gammaherpesvirus 68 chimera expressing LANA, where the virus was highly deficient in establishing latent infection in germinal center B cells. Therefore, LANA’s acidic domain reader is critical for viral latency. These results implicate an acetylation-dependent mechanism mediating KSHV persistence and expand the role of acidic domain readers.

Primary effusion lymphoma (PEL) is a largely incurable malignancy of B cell origin with plasmacytic differentiation. Here, we report the identification of a highly effective inhibitor of PEL. This compound, 6-ethylthioinosine (6-ETI), is a nucleoside analog with toxicity to PEL in vitro and in vivo, but not to other lymphoma cell lines tested. We developed and performed resistome analysis, an unbiased approach based on RNA sequencing of resistant subclones, to discover the molecular mechanisms of sensitivity. We found different adenosine kinase-inactivating (ADK-inactivating) alterations in all resistant clones and determined that ADK is required to phosphorylate and activate 6-ETI. Further, we observed that 6-ETI induces ATP depletion and cell death accompanied by S phase arrest and DNA damage only in ADK-expressing cells. Immunohistochemistry for ADK served as a biomarker approach to identify 6-ETI-sensitive tumors, which we documented for other lymphoid malignancies with plasmacytic features. Notably, multiple myeloma (MM) expresses high levels of ADK, and 6-ETI was toxic to MM cell lines and primary specimens and had a robust antitumor effect in a disseminated MM mouse model. Several nucleoside analogs are effective in treating leukemias and T cell lymphomas, and 6-ETI may fill this niche for the treatment of PEL, plasmablastic lymphoma, MM, and other ADK-expressing cancers.

A 37-year-old woman with a history of inflammatory bowel disease on mercaptopurine presented with a week of recurrent fever, headache, myalgias and mildly elevated serum transaminases and leucopenia. Her workup revealed primary cytomegalovirus (CMV) infection with atypical lymphocytosis, elevated viral load, positive IgM and negative IgG. Two weeks after her initial presentation, she developed odynophagia and diarrhoea prompting endoscopic evaluation with biopsies, which demonstrated CMV disease of the gastrointestinal tract. Her fever and systemic symptoms improved rapidly with initiation of intravenous ganciclovir. She was transitioned to and maintained on oral valganciclovir until two and half months after discharge when her symptoms and lab abnormalities had fully subsided.

Kaposi’s sarcoma-associated herpesvirus (KSHV) latently infects host cells and establishes lifelong persistence as an extra-chromosomal episome in the nucleus. To persist in proliferating cells, the viral genome typically replicates once per cell cycle and is distributed into daughter cells. This process involves host machinery utilized by KSHV, however the underlying mechanisms are not fully elucidated. In present study, we found that N-Myc downstream regulated gene 1 (NDRG1), a cellular gene known to be non-detectable in primary B cells and endothelial cells which are the major cell types for KSHV infection in vivo, was highly upregulated by KSHV in these cells. We further demonstrated that the high expression of NDRG1 was regulated by latency-associated nuclear antigen (LANA), the major viral latent protein which tethers the viral genome to host chromosome and plays an essential role in viral genome maintenance. Surprisingly, knockdown of NDRG1 in KSHV latently infected cells resulted in a significant decrease of viral genome copy number in these cells. Interestingly, NDRG1 can directly interact with proliferating cell nuclear antigen (PCNA), a cellular protein which functions as a DNA polymerase clamp during DNA replication. Intriguingly, we found that NDRG1 forms a complex with LANA and PCNA and serves as a scaffold protein bridging these two proteins. We further demonstrated that NDRG1 is critical for mediating LANA to recruit PCNA onto terminal repeat (TR) of KSHV genome, and facilitates viral DNA replication and episome persistence. Taken together, our findings suggest that NDRG1 plays an important role in KSHV viral genome replication, and provide new clues for understanding of KSHV persistence., Author summary KSHV latently infects cells and establishes lifelong persistence, but the underlying mechanisms of this process has not been fully elucidated. Here, we find a novel host protein NDRG1 is highly up-regulated by KSHV infection and the viral protein LANA is essential in this process. NDRG1 is a multiple functional protein, but the role in KSHV infection remains unknown. Our findings show that NDRG1 functions as a scaffold protein that forms a complex with PCNA and LANA, thereby helping LANA load PCNA onto the viral genome and facilitating the replication and persistence of KSHV. Since NDRG1 is non-detectable in primary B cells and endothelial cells, which are the major cell types susceptible to KSHV infection in vivo, NDRG1 might be a candidate of therapeutic target for inhibition of KSHV persistence in malignant cells.

Certain viruses, such as herpesviruses, are capable of persistent and latent infection of host cells. Distinguishing and separating live, latently infected cells from uninfected cells is not easily attainable using current approaches. The ability to perform such separation would greatly enhance the ability to study primary, infected cells and potentially enable elimination of latently infected cells from the host. Here, the dielectrophoretic response of B cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV) were investigated and compared to uninfected B cells. We evaluated the effect of applied voltage, signal frequency, and flow rate of the sample on the cell capture efficiency. We achieved 37.1% ± 8.5% difference in capture efficiencies between latently KSHV-infected and uninfected BJAB B lymphoma cells at the chip operational conditions of 1V, 50 kHz and 0.02 μl/min sample flow rate. Our results show that latently infected B lymphoma cells demonstrated significantly different electrical response compared to uninfected B cells and DEP-based microchips can be potentially used for sorting latently infected cells based on their electrical properties.

Kaposi’s sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is a 1,162-amino-acid protein that mediates episome persistence of viral genomes. LANA binds the KSHV terminal-repeat (TR) sequence through its carboxy-terminal domain to mediate DNA replication. LANA simultaneously binds mitotic chromosomes and TR DNA to segregate virus genomes to daughter cell nuclei. Amino-terminal LANA attaches to chromosomes by binding histones H2A/H2B, and carboxy-terminal LANA contributes to mitotic-chromosome binding. Although amino- and carboxy-terminal LANA are essential for episome persistence, they are not sufficient, since deletion of all internal LANA sequence renders LANA highly deficient for episome maintenance. Internal LANA sequence upstream of the internal repeat elements contributes to episome segregation and persistence. Here, we investigate this region with a panel of LANA deletion mutants. Mutants retained the ability to associate with mitotic chromosomes and bind TR DNA. In contrast to prior results, deletion of most of this sequence did not reduce LANA’s ability to mediate DNA replication. Deletions of upstream sequence within the region compromised segregation of TR DNA to daughter cells, as assessed by retention of green fluorescent protein (GFP) expression from a replication-deficient TR plasmid. However, deletion of this upstream sequence did not reduce episome maintenance. In contrast, deletions that included an 80-amino-acid sequence immediately downstream resulted in highly deficient episome persistence. LANA with this downstream sequence deleted maintained the ability to replicate and segregate TR DNA, suggesting a unique role for the residues. Therefore, this work identifies adjacent LANA regions with distinct roles in episome segregation and persistence. IMPORTANCE KSHV LANA mediates episomal persistence of viral genomes. LANA binds the KSHV terminal-repeat (TR) sequence to mediate DNA replication and tethers KSHV DNA to mitotic chromosomes to segregate genomes to daughter cell nuclei. Here, we investigate LANA sequence upstream of the internal repeat elements that contributes to episome segregation and persistence. Mutants with deletions within this sequence maintained the ability to bind mitotic chromosomes or bind and replicate TR DNA. Deletion of upstream sequence within the region reduced segregation of TR DNA to daughter cells, but not episome maintenance. In contrast, mutants with deletions of 80 amino acids immediately downstream were highly deficient for episome persistence yet maintained the ability to replicate and segregate TR DNA, the two principal components of episome persistence, suggesting another role for the residues. In summary, this work identifies adjacent LANA sequence with distinct roles in episome segregation and persistence.

KSHV is a human oncogenic virus for which there is no tractable, immunocompetent animal model of infection. MuHV-4, a related rodent gammaherpesvirus, enables pathogenesis studies in mice. In latency, both viruses persist as extrachromosomal, circular genomes (episomes). LANA proteins encoded by KSHV (kLANA) and MuHV-4 (mLANA) contain a C-terminal DNA binding domain (DBD) that acts on the virus terminal repeats to enable episome persistence. mLANA is a smaller protein than kLANA. Their DBDs are structurally conserved but differ strikingly in the conformation of DNA binding. We report a recombinant, chimeric MuHV-4 which contains kLANA in place of mLANA, but in which the DBD is replaced with that of mLANA. Results showed that kLANA functionally accommodated mLANA's mode of DNA binding. In fact, the new chimeric virus established latency in vivo more efficiently than MuHV-4 expressing full-length kLANA., The latency-associated nuclear antigen from Kaposi's sarcoma-associated herpesvirus (KSHV), kLANA, and its homolog from the murid herpesvirus 4 (MuHV-4), mLANA, are essential for viral latency. kLANA is nearly four times the size of mLANA, mainly due to an extensive central repeat region that is absent in mLANA. Both proteins harbor a C-terminal DNA binding domain (DBD). The DBD binds the terminal repeat (TR) DNA sequences of the viral genome to mediate persistence. Despite structural conservation, the kLANA and mLANA DBDs differ in sequence and mode of oligomerization. kLANA DBD oligomers are flexible and bent, while mLANA DBD oligomers bind DNA in a rigid, linear conformation. We previously reported that kLANA and mLANA acted reciprocally on TR sequences. Furthermore, a MuHV-4 expressing kLANA instead of mLANA (v-kLANA) established latency in mice, albeit at a lower magnitude than the wild-type (WT) virus. Here, we asked if kLANA can accommodate the mLANA DBD and generated a fusion protein which contains kLANA but with the mLANA C-terminal region in place of that of kLANA. We report a recombinant MuHV-4 (v-KM) encoding this LANA fusion protein instead of mLANA. The fusion protein was expressed in lytic infection in vitro and assembled nuclear LANA dots in infected splenocytes. Results demonstrated that kLANA functionally accommodated mLANA's mode of DNA binding, allowing MuHV-4 chimeric virus to establish latency in vivo. Notably, v-KM established latency in germinal center B cells more efficiently than did v-kLANA, although levels were reduced compared to WT MuHV-4. IMPORTANCE KSHV is a human oncogenic virus for which there is no tractable, immunocompetent animal model of infection. MuHV-4, a related rodent gammaherpesvirus, enables pathogenesis studies in mice. In latency, both viruses persist as extrachromosomal, circular genomes (episomes). LANA proteins encoded by KSHV (kLANA) and MuHV-4 (mLANA) contain a C-terminal DNA binding domain (DBD) that acts on the virus terminal repeats to enable episome persistence. mLANA is a smaller protein than kLANA. Their DBDs are structurally conserved but differ strikingly in the conformation of DNA binding. We report a recombinant, chimeric MuHV-4 which contains kLANA in place of mLANA, but in which the DBD is replaced with that of mLANA. Results showed that kLANA functionally accommodated mLANA's mode of DNA binding. In fact, the new chimeric virus established latency in vivo more efficiently than MuHV-4 expressing full-length kLANA.

© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited., Latency-associated nuclear antigen (LANA) is central to episomal tethering, replication and transcriptional regulation of γ2-herpesviruses. LANA binds cooperatively to the terminal repeat (TR) region of the viral episome via adjacent LANA binding sites (LBS), but the molecular mechanism by which LANA assembles on the TR remains elusive. We show that KSHV LANA and MHV-68 LANA proteins bind LBS DNA using strikingly different modes. Solution structure of LANA complexes revealed that while kLANA tetramer is intrinsically bent both in the free and bound state to LBS1-2 DNA, mLANA oligomers instead adopt a rigid linear conformation. In addition, we report a novel non-ring kLANA structure that displays more flexibility at its assembly interface than previously demonstrated. We identified a hydrophobic pivot point located at the dimer-dimer assembly interface, which gives rotational freedom for kLANA to adopt variable conformations to accommodate both LBS1-2 and LBS2-1-3 DNA. Alterations in the arrangement of LBS within TR or at the tetramer assembly interface have a drastic effect on the ability of kLANA binding. We also show kLANA and mLANA DNA binding functions can be reciprocated. Although KSHV and MHV-68 are closely related, the findings provide new insights into how the structure, oligomerization, and DNA binding of LANA have evolved differently to assemble on the TR DNA., Fundação para a Ciência e a Tecnologia (FCT) Investigator Grant [IF/01023/2013 to C.E.M.]; Harvard Medical School Portugal Program in Translational Research and Information [HMSP-ICT/0021/2010 to J.P.S., C.E.M., M.A.C. and K.M.K.]; National Cancer Institute/National Institutes of Health (NCI/NIH) [CA082036 to KMK]; National Institutes of Dental and Craniofacial Research/National Institutes of Health (NIDCR/NIH) [DE025208 to K.M.K.]; European Community's Seventh Framework Programme (FP7/2007–2013) under BioStruct-X [283570]. This work was also supported by Instruct, part of the European Strategy Forum on Research Infrastructures (ESFRI) and through national member agreements. Funding for open access charges: FCT.

CD4+ T cells are critical for the control of virus infections, T cell memory, and immune surveillance. We studied the differentiation and function of murine γ-herpesvirus 68 (MHV-68)–specific CD4+ T cells using gp150-specific TCR-transgenic mice. This allowed a more detailed study of the characteristics of the CD4+ T cell response than did previously available approaches for this virus. Most gp150-specific CD4+ T cells expressed T-bet and produced IFN-γ, indicating that MHV-68 infection triggered differentiation of CD4+ T cells largely into the Th1 subset, whereas some became follicular Th cells and Foxp3+ regulatory T cells. These CD4+ T cells were protective against MHV-68 infection in the absence of CD8+ T cells and B cells, and protection depended on IFN-γ secretion. Marked heterogeneity was observed in the CD4+ T cells, based on lymphocyte Ag 6C (Ly6C) expression. Ly6C expression positively correlated with IFN-γ, TNF-α, and granzyme B production; T-bet and KLRG1 expression; proliferation; and CD4+ T cell–mediated cytotoxicity. Ly6C expression inversely correlated with survival, CCR7 expression, and secondary expansion potential. Ly6C+ and Ly6C− gp150-specific CD4+ T cells were able to interconvert in a bidirectional manner upon secondary Ag exposure in vivo. These results indicate that Ly6C expression is closely associated with antiviral activity in effector CD4+ T cells but is inversely correlated with memory potential. Interconversion between Ly6C+ and Ly6C− cells may maintain a balance between the two Ag-specific CD4+ T cell populations during MHV-68 infection. These findings have significant implications for Ly6C as a surface marker to distinguish functionally distinct CD4+ T cells during persistent virus infection.

Copyright: © 2017 Habison et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited., Many pathogens, including Kaposi's sarcoma herpesvirus (KSHV), lack tractable small animal models. KSHV persists as a multi-copy, nuclear episome in latently infected cells. KSHV latency-associated nuclear antigen (kLANA) binds viral terminal repeat (kTR) DNA to mediate episome persistence. Model pathogen murine gammaherpesvirus 68 (MHV68) mLANA acts analogously on mTR DNA. kLANA and mLANA differ substantially in size and kTR and mTR show little sequence conservation. Here, we find kLANA and mLANA act reciprocally to mediate episome persistence of TR DNA. Further, kLANA rescued mLANA deficient MHV68, enabling a chimeric virus to establish latent infection in vivo in germinal center B cells. The level of chimeric virus in vivo latency was moderately reduced compared to WT infection, but WT or chimeric MHV68 infected cells had similar viral genome copy numbers as assessed by immunofluorescence of LANA intranuclear dots or qPCR. Thus, despite more than 60 Ma of evolutionary divergence, mLANA and kLANA act reciprocally on TR DNA, and kLANA functionally substitutes for mLANA, allowing kLANA investigation in vivo. Analogous chimeras may allow in vivo investigation of genes of other human pathogens., This work was supported in part by National Institutes of Health grants CA082036 (NCI), DE025208, and DE024971 (both NIDCR), to KMK, FCT PTDC/IMI-MIC/0980/2014 to JPS, FCT Harvard Medical School Portugal Program in Translational Research (HMSP-ICT/0021/2010) to JPS, KMK, CEM, Instituto de Medicina Molecular Directors Fund to JPS, and iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344) FCT/FEDER (PT2020 Partnership Agreement) to CEM. M.P.M is supported by a fellowship from Fundação para a Ciência e Tecnologia (FCT), Portugal.

Kaposi's sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies, especially in immunocompromised hosts. KSHV latently infects tumor cells and persists as an extrachromosomal episome (plasmid). KSHV latency-associated nuclear antigen (LANA) mediates KSHV episome persistence. LANA binds specific KSHV sequence to replicate viral DNA. In addition, LANA tethers KSHV genomes to mitotic chromosomes to efficiently segregate episomes to daughter nuclei after mitosis. N-terminal LANA (N-LANA) binds histones H2A and H2B to attach to chromosomes. Currently, there are no specific inhibitors of KSHV latent infection. To enable high-throughput screening (HTS) of inhibitors of N-LANA binding to nucleosomes, here we develop, miniaturize, and validate a fluorescence polarization (FP) assay that detects fluorophore-labeled N-LANA peptide binding to nucleosomes. We also miniaturize a counterscreen to identify DNA intercalators that nonspecifically inhibit N-LANA binding to nucleosomes, and also develop an enzyme-linked immunosorbent assay to assess N-LANA binding to nucleosomes in the absence of fluorescence. HTS of libraries containing more than 350,000 compounds identified multiple compounds that inhibited N-LANA binding to nucleosomes. No compounds survived all counterscreens, however. More complex small-molecule libraries will likely be necessary to identify specific inhibitors of N-LANA binding to histones H2A and H2B; these assays should prove useful for future screens.

Kaposi's Sarcoma-associated herpesvirus (KSHV) is maintained as a stable episome in latently infected pleural effusion lymphoma (PEL) cells. Episome maintenance is conferred by the binding of the KSHV-encoded LANA protein to the viral terminal repeats (TR). Here, we show that DNA replication in the KSHV TR is coupled with DNA recombination and mediated in part through the cellular replication fork protection factors Timeless (Tim) and Tipin. We show by two-dimensional (2D) agarose gel electrophoresis that replication forks naturally stall and form recombination-like structures at the TR during an unperturbed cell cycle. Chromatin immunoprecipitation (ChIP) assays revealed that Tim and Tipin are selectively enriched at the KSHV TR during S phase and in a LANA-dependent manner. Tim depletion inhibited LANA-dependent TR DNA replication and caused the loss of KSHV episomes from latently infected PEL cells. Tim depletion resulted in the aberrant accumulation of recombination structures and arrested MCM helicase at TR. Tim depletion did not induce the KSHV lytic cycle or apoptotic cell death. We propose that KSHV episome maintenance requires Tim-assisted replication fork protection at the viral terminal repeats and that Tim-dependent recombination-like structures form at TR to promote DNA repeat stability and viral genome maintenance.

Cancer cells have been increasingly grown in pharmaceutical research to understand tumorigenesis and develop new therapeutic drugs. Currently, cells are typically grown using two-dimensional (2-D) cell culture approaches, where the native tumor microenvironment is difficult to recapitulate. Thus, one of the main obstacles in oncology is the lack of proper infection models that recount main features present in tumors. In recent years, microtechnology-based platforms have been employed to generate three-dimensional (3-D) models that better mimic the native microenvironment in cell culture. Here, we present an innovative approach to culture Kaposi’s sarcoma-associated herpesvirus (KSHV) infected human B cells in 3-D using a microwell array system. The results demonstrate that the KSHV-infected B cells can be grown up to 15 days in a 3-D culture. Compared with 2-D, cells grown in 3-D had increased numbers of KSHV latency-associated nuclear antigen (LANA) dots, as detected by immunofluorescence microscopy, indicating a higher viral genome copy number. Cells in 3-D also demonstrated a higher rate of lytic reactivation. The 3-D microwell array system has the potential to improve 3-D cell oncology models and allow for better-controlled studies for drug discovery.

Kaposi’s sarcoma-associated herpesvirus (KSHV) has an etiologic role in Kaposi’s sarcoma, primary effusion lymphoma and multicentric Castleman’s disease. These diseases are most common in immunocompromised individuals, especially those with AIDS. Similar to all herpesviruses, KSHV infection is lifelong. KSHV infection in tumor cells is primarily latent, with only a small subset of cells undergoing lytic infection. During latency, the KSHV genome persists as a multiple copy, extrachromosomal episome in the nucleus. In order to persist in proliferating tumor cells, the viral genome replicates once per cell cycle and then segregates to daughter cell nuclei. KSHV only expresses several genes during latent infection. Prominent among these genes, is the latency-associated nuclear antigen (LANA). LANA is responsible for KSHV genome persistence and also exerts transcriptional regulatory effects. LANA mediates KSHV DNA replication and in addition, is responsible for segregation of replicated genomes to daughter nuclei. LANA serves as a molecular tether, bridging the viral genome to mitotic chromosomes to ensure that KSHV DNA reaches progeny nuclei. N-terminal LANA attaches to mitotic chromosomes by binding histones H2A/H2B at the surface of the nucleosome. C-terminal LANA binds specific KSHV DNA sequence and also has a role in chromosome attachment. In addition to the essential roles of N- and C-terminal LANA in genome persistence, internal LANA sequence is also critical for efficient episome maintenance. LANA’s role as an essential mediator of virus persistence makes it an attractive target for inhibition in order to prevent or treat KSHV infection and disease.

Murine gammaherpesvirus 68 (MHV68) ORF73 (mLANA) has sequence homology to Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA). LANA acts on the KSHV terminal repeat (TR) elements to mediate KSHV episome maintenance. Disruption of mLANA expression severely reduces the ability of MHV68 to establish latent infection in mice, consistent with the possibility that mLANA mediates episome persistence. Here we assess the roles of mLANA and MHV68 TR (mTR) elements in episome persistence. mTR-associated DNA persisted as an episome in latently MHV68-infected tumor cells, demonstrating that the mTR elements can serve as a cis- acting element for MHV68 episome maintenance. In some cases, both control vector and mTR-associated DNAs integrated into MHV68 episomal genomes. Therefore, we also assessed the roles of mTRs as well as mLANA in the absence of infection. DNA containing both mLANA and mTRs in cis persisted as an episome in murine A20 or MEF cells. In contrast, mTR DNA never persisted as an episome in the absence of mLANA. mLANA levels were increased when mLANA was expressed from its native promoters, and episome maintenance was more efficient with higher mLANA levels. Increased numbers of mTRs conferred more efficient episome maintenance, since DNA containing mLANA and eight mTR elements persisted more efficiently in A20 cells than did DNA with mLANA and two or four mTRs. Similar to KSHV LANA, mLANA broadly associated with mitotic chromosomes but relocalized to concentrated dots in the presence of episomes. Therefore, mLANA acts on mTR elements to mediate MHV68 episome persistence.

This article reviews the new beta-lactam (beta-lactam) antibiotics doripenem, ceftobiprole, and ceftaroline. It covers pharmacokinetic and pharmacodynamic properties, dosing, in vitro activities, safety, and clinical trial results. Doripenem (Doribax) has been approved by the US Food and Drug Administration (FDA) for the treatment of complicated intra-abdominal and urinary tract infections. At this writing, ceftobiprole is under review by the FDA for approval based on results of phase 3 clinical trials, whereas at least one phase 3 clinical trial of ceftaroline has been completed. The article also reviews recent data suggesting increased overall mortality with Cefepime (Maxipime) use compared with other beta-lactam antibiotics and the potential risk for neurotoxicity in the setting of renal failure.

The cellular immunity against Kaposi's sarcoma-associated herpesvirus (KSHV) is poorly characterized and has not been compared to T-cell responses against other human herpesviruses. Here, novel and dominant targets of KSHV-specific cellular immunity are identified and compared to T cells specific for lytic and latent antigens in a second human gammaherpesvirus, Epstein-Barr virus. The data identify a novel HLA-B57- and HLA-B58-restricted epitope in the Orf57 protein and show consistently close parallels in immune phenotypes and functional response patterns between cells targeting lytic or latent KSHV- and EBV-encoded antigens, suggesting common mechanisms in the induction of these responses.

Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen (LANA) tethers viral terminal repeat (TR) DNA to mitotic chromosomes to mediate episome persistence. The 1,162-amino-acid LANA protein contains both N- and C-terminal chromosome attachment regions. The LANA C-terminal domain self-associates to specifically bind TR DNA and mitotic chromosomes. Here, we used alanine scanning substitutions spanning residues 1023 to 1145 to investigate LANA self-association, DNA binding, and C-terminal chromosome association. No residues were essential for LANA oligomerization, as assayed by coimmunoprecipitation experiments, consistent with redundant roles for amino acids in self-association. Different subsets of amino acids were important for DNA binding, as assayed by electrophoretic mobility shift assay, and mitotic chromosome association, indicating that distinct C-terminal LANA subdomains effect DNA and chromosome binding. The DNA binding domains of LANA and EBNA1 are predicted to be structurally homologous; certain LANA residues important for DNA binding correspond to those with roles in EBNA1 DNA binding, providing genetic support for at least partial structural homology. In contrast to the essential role of N-terminal LANA chromosome targeting residues in DNA replication, deficient C-terminal chromosome association did not reduce LANA-mediated DNA replication.

We report a biosensing platform for viral load measurement through electrical sensing of viruses on a flexible plastic microchip with printed electrodes. Point-of-care (POC) viral load measurement is of paramount importance with significant impact on a broad range of applications, including infectious disease diagnostics and treatment monitoring specifically in resource-constrained settings. Here, we present a broadly applicable and inexpensive biosensing technology for accurate quantification of bioagents, including viruses in biological samples, such as plasma and artificial saliva, at clinically relevant concentrations. Our microchip fabrication is simple and mass-producible as we print microelectrodes on flexible plastic substrates using conductive inks. We evaluated the microchip technology by detecting and quantifying multiple Human Immunodeficiency Virus (HIV) subtypes (A, B, C, D, E, G and panel), Epstein-Barr Virus (EBV) and Kaposi’s Sarcoma-associated Herpes Virus (KSHV) in a fingerprick volume (50 µL) of PBS, plasma, and artificial saliva samples for a broad range of virus concentrations between 102 copies/mL and 107 copies/mL. We have also evaluated the microchip platform with discarded, de-identified HIV-infected patient samples by comparing our microchip viral load measurement results with reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) as the gold standard method using Bland-Altman Analysis.

© 2015 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology., Kaposi sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies. KSHV latency-associated nuclear antigen (LANA) mediates persistence of viral episomes in latently infected cells. LANA mediates KSHV DNA replication and segregates episomes to progeny nuclei. The structure of the LANA DNA binding domain was recently solved, revealing a positive electrostatic patch opposite the DNA binding surface, which is the site of BET protein binding. Here we investigate the functional role of the positive patch in LANA-mediated episome persistence. As expected, LANA mutants with alanine or glutamate substitutions in the central, peripheral, or lateral portions of the positive patch maintained the ability to bind DNA by EMSA. However, all of the substitution mutants were deficient for LANA DNA replication and episome maintenance. Mutation of the peripheral region generated the largest deficiencies. Despite these deficiencies, all positive patch mutants concentrated to dots along mitotic chromosomes in cells containing episomes, similar to LANA. The central and peripheral mutants, but not the lateral mutants, were reduced for BET protein interaction as assessed by co-immunoprecipitation. However, defects in BET protein binding were independent of episome maintenance function. Overall, the reductions in episome maintenance closely correlated with DNA replication deficiencies, suggesting that the replication defects account for the reduced episome persistence. Therefore, the electrostatic patch exerts a key role in LANA-mediated DNA replication and episome persistence and may act through a host cell partner(s) other than a BET protein or by inducing specific structures or complexes.

The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is required for viral episome maintenance in host cells during latent infection. Two regions of the protein have been implicated in tethering LANA/viral episomes to the host mitotic chromosomes, and LANA chromosome-binding sites are subjects of high interest. Because previous studies had identified bromodomain protein Brd4 as the mitotic chromosome anchor for the bovine papillomavirus E2 protein, which tethers the viral episomes to host mitotic chromosomes (J. You, J. L. Croyle, A. Nishimura, K. Ozato, and P. M. Howley, Cell 117: 349-360, 2004, and J. You, M. R. Schweiger, and P. M. Howley, J. Virol. 79: 14956-14961, 2005), we examined whether KSHV LANA interacts with Brd4. We found that LANA binds Brd4 in vivo and in vitro and that the binding is mediated by a direct protein-protein interaction between the ET (extraterminal) domain of Brd4 and a carboxyl-terminal region of LANA previously implicated in chromosome binding. Brd4 associates with mitotic chromosomes throughout mitosis and demonstrates a strong colocalization with LANA and the KSHV episomes on host mitotic chromosomes. Although another bromodomain protein, RING3/Brd2, binds to LANA in a similar fashion in vitro, it is largely excluded from the mitotic chromosomes in KSHV-uninfected cells and is partially recruited to the chromosomes in KSHV-infected cells. These data identify Brd4 as an interacting protein for the carboxyl terminus of LANA on mitotic chromosomes and suggest distinct functional roles for the two bromodomain proteins RING3/Brd2 and Brd4 in LANA binding. Additionally, because Brd4 has recently been shown to have a role in transcription, we examined whether Brd4 can regulate the CDK2 promoter, which can be transactivated by LANA.

Kaposi's sarcoma-associated herpesvirus (KSHV) latently infects tumor cells and has an etiologic role in Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Survival in rapidly dividing cells depends on a carefully orchestrated chain of events. The viral genome, or episome, must replicate in concert with cellular genetic material, and then efficiently segregate to progeny nuclei. KSHV achieves this through its latency associated nuclear antigen (LANA), which simultaneously binds to viral DNA and mitotic chromosomes to efficiently partition episomes. LANA's N-terminal region has been shown to be essential for efficient KSHV DNA replication and tethering to mitotic chromosomes. The precise mechanism by which LANA attaches to host chromosomes has been an area of active investigation. We recently reported that this association is mediated by the chromatin components histones H2A and H2B. Binding between LANA and these proteins was demonstrated in vivo and in vitro, and use of an H2A-H2B depleted system demonstrated their central role in LANA's chromosome binding. Further, we provided a structural description of the interaction of LANA's N-terminal chromosome association region with the nucleosome using x-ray crystallography. Our data offer further insight into the mechanism of KSHV latency, and also reveal a new concept for a role of the nucleosome as a docking site for other proteins.

To persist in latently infected, proliferating cells, Kaposi's sarcoma-associated herpesvirus (KSHV) episomes must replicate and efficiently segregate to progeny nuclei. Episome persistence in uninfected cells requires latency-associated nuclear antigen 1 (LANA1) in trans and cis -acting KSHV terminal repeat (TR) DNA. The LANA1 C terminus binds TR DNA, and LANA1 mediates TR-associated DNA replication in transient assays. LANA1 also concentrates at sites of KSHV TR DNA episomes along mitotic chromosomes, consistent with a tethering role to efficiently segregate episomes to progeny nuclei. LANA1 amino acids 5 to 22 constitute a chromosome association region (Piolot et al., J. Virol. 75: 3948-3959, 2001). We now investigate LANA1 residues 5 to 22 with scanning alanine substitutions. Mutations targeting LANA1 5 GMR 7 , 8 LRS 10 , and 11 GRS 13 eliminated chromosome association, DNA replication, and episome persistence. LANA1 mutated at 14 TG 15 retained the ability to associate with chromosomes but was partially deficient in DNA replication and episome persistence. These results provide genetic support for a key role of the LANA1 N terminus in chromosome association, LANA1-mediated DNA replication, and episome persistence.

A number of nucleoside analogues are used successfully for the treatment of several cancers, and in particular leukemias and lymphomas, but these have distinct efficacies for different tumor types, and many malignancies do not respond to currently available nucleoside analogues or other forms of chemotherapy. A high throughput screen conducted in our lab to search for inhibitors of primary effusion lymphoma (PEL) identified the nucleoside analog 6-ethylthioinosine (6-ETI) as a potent and selective inhibitor of PEL, a largely incurable malignancy of B cell origin with plasmacytic differentiation. 6-ETI induced necrosis and ATP-depletion accompanied by S-phase arrest, DNA damage and inhibition of DNA synthesis. To understand 6-ETI mechanism of selectivity, RNA-seq analysis of in vitro generated drug-resistant PEL clones revealed inactivating mutations and loss of expression of adenosine kinase (ADK) as the mechanism of resistance. In vitro assays showed that 6-ETI is a pro-drug that gets phosphorylated and activated by adenosine kinase (ADK) into its active form. We found high ADK expression in PEL cell lines and primary specimens of PEL, multiple myeloma (MM) and plasmablastic lymphoma (PBL) patient samples. 6-ETI was effective at killing multiple myeloma cell lines, primary MM specimens, and had a remarkable anti-tumor response in a disseminated multiple myeloma and PEL xenograft mouse models. Thus, ADK expression can serve as a predictive biomarker to help identify patients that are most likely to respond to 6-ETI treatment. To further assess the spectrum of activity and sensitivity of 6-ETI, we examined ADK expression in other cancer subtypes and found that colorectal and pancreatic adenocarcinomas also overexpress ADK and are highly sensitive to killing by 6-ETI at the low nanomolar concentration. We also found high ADK expression in primary colon and pancreatic adenocarcinoma patient specimens. We compared 6-ETI to other FDA-approved purine analogs and failed to find other compounds with similar potency or selectivity profile. Herein, we report the identification of a novel purine analog, 6-ethylthioinosine, as an effective therapeutic with exquisite sensitivity to plasma cell malignancies and other ADK-expressing cancers. We have successfully used RNASeq-based “resistome” analysis to identify its mechanism of specificity and discovered a new biomarker that can potentially impact patient care and the treatment of some of the most aggressive tumors. Citation Format: Jouliana Sadek, Utthara Nayar, Jonathan Reichel, Jennifer Totonchy, Shizuko Sei, Robert Shoemaker, David Warren, Olivier Elemento, Kenneth Kaye, Ethel Cesarman. A novel nucleoside analog therapeutically active against plasma cell malignancies and other ADK-expressing cancers including colon and pancreatic adenocarcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5110. doi:10.1158/1538-7445.AM2017-5110

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) (also known as human herpesvirus 8) latently infects KS tumors, primary effusion lymphomas (PELs), and PEL cell lines. In latently infected cells, KSHV DNA is maintained as circularized, extrachromosomal episomes. To persist in proliferating cells, KSHV episomes must replicate and efficiently segregate to progeny nuclei. In uninfected B-lymphoblastoid cells, KSHV latency-associated nuclear antigen (LANA1) is necessary and sufficient for persistence of artificial episomes containing specific KSHV DNA. In previous work, the cis -acting sequence required for episome persistence contained KSHV terminal-repeat (TR) DNA and unique KSHV sequence. We now show that cis -acting KSHV TR DNA is necessary and sufficient for LANA1-mediated episome persistence. Furthermore, LANA1 binds TR DNA in mobility shift assays and a 20-nucleotide LANA1 binding sequence has been identified. Since LANA1 colocalizes with KSHV episomes along metaphase chromosomes, these results are consistent with a model in which LANA1 may bridge TR DNA to chromosomes during mitosis to efficiently segregate KSHV episomes to progeny nuclei.

The aminoglycosides are a class of bactericidal antibiotics characterized by the presence of a six-carbon aminocyclitol ring covalently bonded to multiple amino sugar groups. 24 Aminoglycosides commonly used for the treatment of serious bacterial infections in the United States include gentamicin, tobramycin, amikacin, netilmicin, and streptomycin. 18 These drugs act in part by impairing bacterial protein synthesis through irreversible binding to the 30S subunit of the bacterial ribosome. 24 Since the introduction of streptomycin in the 1940s, aminoglycosides have proved extremely useful in the treatment of infections caused by gram-negative bacilli, including Pseudomonas aeruginosa, and infections caused by staphylococci, mycobacteria, and several other pathogens. 18 The Food and Drug Administration (FDA) approved dosing regimens for aminoglycoside antibiotics require multiple daily doses in individuals with normal renal function. Improvements in the understanding of the pharmacodynamics of aminoglycoside efficacy and mechanisms of toxicity, however, have prompted the evaluation of once-daily dosing regimens in clinical studies. In the pages that follow, we review the rationale behind once-daily dosing of aminoglycoside therapy as well as clinical data on the efficacy and toxicity of once-daily dosing of aminoglycosides. We also review practical aspects of dosing and monitoring once-daily aminoglycoside therapy and issues complicating the use of these regimens in special populations (including children, adults with an altered volume of distribution for aminoglycosides, and individuals with renal dysfunction) and in certain illnesses (including bacterial endocarditis, neutropenia and fever, and cystic fibrosis).

Expression of the Epstein-Barr virus (EBV) transforming LMP1 in B cells activates the transcription factor NF-kappaB and induces phenotypic changes through two distinct domains in the cytoplasmic C-terminus of the protein. The aa 187-231 domain of LMP1, which is important for growth transformation, binds tumour necrosis factor (TNF) receptor associated factor (TRAF) 1 and TRAF3 and this interaction mediates subsequent signalling events. The TRAFs also associate with CD40, a member of the TNFR family, which upon ligation activates NF-kappaB and induces phenotypic changes similar to those mediated by LMP1. This study demonstrates that LMP1 expression in carcinoma cell lines and SV40-transformed keratinocytes results in induction of the pleiotropic cytokine interleukin 6 (IL6), an effect which is also observed upon CD40 ligation. The mechanism by which either LMP1 expression or CD40 ligation induces IL6 production was found to be NF-kappaB-dependent. Mutational analysis identified domains in the C-terminus of LMP1 which are important for NF-kappaB activation and IL6 secretion. LMP1 and CD40 share a common PxQxT core TRAF binding motif and mutations in or adjacent to this sequence impaired the ability of LMP1 or CD40 to induce NF-kappaB activation and IL6 secretion. The importance of TRAF interactions in mediating these effects was confirmed using dominant negative TRAF2 and TRAF3 mutants which also identified differences in the signalling events mediated by the two NF-kappaB activating domains of LMP1. A20, an anti-apoptotic protein which interacts with TRAF2 and blocks CD40-mediated NF-kappaB activity, also blocked NF-kappaB and IL6 secretion in LMP1-transfected epithelial cells. These results suggest that LMP1 regulates IL6 production in epithelial cells in a manner similar to CD40 ligation and implicate TRAFs as common mediators in the transduction of signals generated via the CD40 and LMP1 pathways. As a role for IL6 in regulating epithelial cell growth has previously been suggested, the control of IL6 secretion via the CD40 and LMP1 pathways may have implications for the growth of both normal and transformed epithelial cells.

Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1) is essential for transforming primary B lymphocytes into lymphoblastoid cell lines. EBV recombinants with LMP1 genes truncated after the proximal 45 codons of the LMP1 carboxyl terminus are adequate for transformation. The proximal 45 residues include a domain that engages the tumor necrosis factor receptor associated factors (TRAFs). We investigated the importance of the TRAF binding domain by assaying the transforming ability of recombinant EBV genomes with a deletion of LMP1 codons 185–211. This mutation eliminates TRAF association in yeast and in lymphoblasts but does not affect LMP1 stability or localization. Specifically mutated recombinant EBV genomes were generated by transfecting P3HR-1 cells with overlapping EBV cosmids. Infection of primary B lymphocytes resulted in cell lines that were coinfected with an LMP1Δ185–211 EBV recombinant and P3HR-1 EBV, which has a wild-type LMP1 gene but is transformation defective due to another deletion. Despite the equimolar mixture of wild-type and mutated LMP1 genes in virus preparations from five coinfected cell lines, only the wild-type LMP1 gene was found in 412 cell lines obtained after transformation of primary B lymphocytes. No transformed cell line had only the LMP1Δ185–211 gene. An EBV recombinant with a Flag-tagged LMP1 gene passaged in parallel segregated from the coinfecting P3HR-1. These data indicate that the LMP1 TRAF binding domain is critical for primary B lymphocyte growth transformation.

Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is a 1,162-amino-acid protein that mediates the maintenance of episomal viral genomes in latently infected cells. The two central components of episome persistence are DNA replication with each cell division and the segregation of DNA to progeny nuclei. LANA self-associates to bind KSHV terminal-repeat (TR) DNA and to mediate its replication. LANA also simultaneously binds to TR DNA and mitotic chromosomes to mediate the segregation of episomes to daughter nuclei. The N-terminal region of LANA binds histones H2A and H2B to attach to mitotic chromosomes, while the C-terminal region binds TR DNA and also associates with chromosomes. Both the N- and C-terminal regions of LANA are essential for episome persistence. We recently showed that deletion of all internal LANA sequences results in highly deficient episome maintenance. Here we assess independent internal LANA regions for effects on episome persistence. We generated a panel of LANA mutants that included deletions in the large internal repeat region and in the unique internal sequence. All mutants contained the essential N- and C-terminal regions, and as expected, all maintained the ability to associate with mitotic chromosomes in a wild-type fashion and to bind TR DNA, as assessed by electrophoretic mobility shift assays (EMSA). Deletion of the internal regions did not reduce the half-life of LANA. Notably, deletions within either the repeat elements or the unique sequence resulted in deficiencies in DNA replication. However, only the unique internal sequence exerted effects on the ability of LANA to retain green fluorescent protein (GFP) expression from TR-containing episomes deficient in DNA replication, consistent with a role in episome segregation; this region did not independently associate with mitotic chromosomes. All mutants were deficient in episome persistence, and the deficiencies ranged from minor to severe. Mutants deficient in DNA replication that contained deletions within the unique internal sequence had the most-severe deficits. These data suggest that internal LANA regions exert critical roles in LANA-mediated DNA replication, segregation, and episome persistence, likely through interactions with key host cell factors.

Latency-associated nuclear antigen (LANA) mediates γ2-herpesvirus genome persistence and regulates transcription. We describe the crystal structure of the murine gammaherpesvirus-68 LANA C-terminal domain at 2.2 A resolution. The structure reveals an alpha-beta fold that assembles as a dimer, reminiscent of Epstein-Barr virus EBNA1. A predicted DNA binding surface is present and opposite this interface is a positive electrostatic patch. Targeted DNA recognition substitutions eliminated DNA binding, while certain charged patch mutations reduced bromodomain protein, BRD4, binding. Virus containing LANA abolished for DNA binding was incapable of viable latent infection in mice. Virus with mutations at the charged patch periphery exhibited substantial deficiency in expansion of latent infection, while central region substitutions had little effect. This deficiency was independent of BRD4. These results elucidate the LANA DNA binding domain structure and reveal a unique charged region that exerts a critical role in viral latent infection, likely acting through a host cell protein(s).

Host colonization by lymphotropic γ-herpesviruses depends critically on expansion of viral genomes in germinal center (GC) B-cells. Myc is essential for the formation and maintenance of GCs. Yet, the role of Myc in the pathogenesis of γ-herpesviruses is still largely unknown. In this study, Myc was shown to be essential for the lymphotropic γ-herpesvirus MuHV-4 biology as infected cells exhibited increased expression of Myc signature genes and the virus was unable to expand in Myc defficient GC B-cells. We describe a novel strategy of a viral protein activating Myc through increased protein stability resulting in increased progression through the cell cycle. This is acomplished by modulating a physiological post-translational regulatory pathway of Myc. The molecular mechanism involves Myc heterotypic poly-ubiquitination mediated via the viral E3 ubiquitin-ligase mLANA protein. EC5SmLANA modulates cellular control of Myc turnover by antagonizing SCFFbw7 mediated proteasomal degradation of Myc, mimicking SCFβ-TrCP. The findings here reported reveal that modulation of Myc is essential for γ-herpesvirus persistent infection, establishing a link between virus induced lymphoproliferation and disease., Author Summary Being obligatory intracellular parasites, it is not surprising that viruses have evolved mechanisms to induce cellular proliferation to promote their own life cycle. This is notorious in the case of γ-herpesviruses, such as Epstein-Barr virus (EBV) and Kaposi's sarcoma virus (KSHV), which are human pathogens associated with lymphoproliferative disease and several tumors. Host colonization by γ-herpesviruses is critically dependent on the ability to expand latent infection in proliferating B-cells. Virus-induced cellular proliferation is a process mediated by the expression of specific viral proteins. One of such proteins is the latency-associated protein (LANA) of KSHV. In this study, we use murid herpesvirus-4 (MuHV-4) as a mouse model of γ-herpesvirus pathogenesis. We show that the MuHV-4 LANA (mLANA) stabilizes the cellular oncogene Myc, increasing its half-life, thus promoting its activity as a potent inducer of cellular proliferation. The molecular mechanism involves heterotypic poly-ubiquitination of Myc mediated via mLANA. The findings here reported demonstrate that modulation of Myc is essential for γ-herpesvirus persistent infection, establishing a link between virus induced lymphoproliferation and disease. The implication is that revealing a critical function of a viral protein possibly allows the development of small molecule probes to disrupt mLANA-Myc interaction, therefore inhibit virus induced lyhophoproliferative disease.

Recombinant Epstein-Barr viruses (EBVs) were made with mutated latent membrane protein 1 (LMP1) genes that express only the LMP1 amino-terminal cytoplasmic and six transmembrane domains (MS187) or these domains and the first 44 amino acids of the 200-residue LMP1 carboxy-terminal domain (MS231). After infection of primary B lymphocytes with virus stocks having small numbers of recombinant virus and large numbers of P3HR-1 EBV which is transformation defective but wild type (WT) for LMP1, all lymphoblastoid cell lines (LCLs) that had MS187 or MS231 LMP1 also had WT LMP1 provided by the coinfecting P3HR-1 EBV. Lytic virus infection was induced in these coinfected LCLs, and primary B lymphocytes were infected. In over 200 second-generation LCLs, MS187 LMP1 was never present without WT LMP1. Screening of over 600 LCLs infected with virus from MS231 recombinant virus-infected LCLs identified two LCLs which were infected with an MS231 recombinant without WT LMP1. The MS231 recombinant virus could growth transform primary B lymphocytes when cells were grown on fibroblast feeders. Even after 6 months on fibroblast feeder layers, cells transformed by the MS231 recombinant virus died when transferred to medium without fibroblast feeder cells. These data indicate that the LMP1 carboxy terminus is essential for WT growth-transforming activity. The first 44 amino acids of the carboxy-terminal cytoplasmic domain probably include an essential effector of cell growth transformation, while a deletion of the rest of LMP1 can be complemented by growth on fibroblast feeder layers. LMP1 residues 232 to 386 therefore provide a growth factor-like effect for the transformation of B lymphocytes. This effect may be indicative of the broader role of LMP1 in cell growth transformation.

Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is a 1,162-amino-acid protein that acts on viral terminal repeat (TR) DNA to mediate KSHV episome persistence. The two essential components of episome persistence are DNA replication prior to cell division and episome segregation to daughter nuclei. These functions are located within N- and C-terminal regions of LANA. N- and C-terminal regions of LANA are sufficient for TR DNA replication. In addition, N- and C-terminal regions of LANA tether episomes to mitotic chromosomes to segregate episomes to progeny cell nuclei. To generate a tethering mechanism, N-terminal LANA binds histones H2A/H2B to attach to mitotic chromosomes, and C-terminal LANA binds TR DNA and also associates with mitotic chromosomes. Here, we test the importance of the internal LANA sequence for episome persistence. We generated LANA mutants that contain N- and C-terminal regions of LANA but have most of the internal sequence deleted. As expected, the LANA mutants bound mitotic chromosomes in a wild-type pattern and also bound TR DNA as assayed by electrophoretic mobility shift assays (EMSA). The mutants mediated TR DNA replication, although with reduced efficiency compared with LANA. Despite the ability to replicate DNA and exert the chromosome and DNA binding functions necessary for segregating episomes to daughter nuclei, the mutants were highly deficient for the ability to mediate both short- and long-term episome persistence. These data indicate that internal LANA sequence exerts a critical effect on its ability to maintain episomes, possibly through effects on TR DNA replication.

The gene encoding latent-infection membrane protein 1 (LMP1) was specifically mutated in Epstein-Barr virus (EBV) recombinants by inserting a nonsense linker after codon 9 or codon 84 or into an intron 186 bp 3' to the latter insertion site. EBV recombinants with the LMP1 intron mutation were wild type for LMP1 expression and for growth transformation of primary B lymphocytes. In contrast, EBV recombinants with the mutations in the LMP1 open reading frame expressed N-terminally truncated crossreactive proteins and could initiate or maintain primary B-lymphocyte transformation only when wild-type LMP1 was provided in trans by a coinfecting, transformation-defective EBV, P3HR-1. These data indicate that LMP1 is essential for EBV-mediated transformation of primary B lymphocytes, that the first 43 amino acids are critical for LMP1's function, and that codon 44-initiated LMP1 does not have a dominant negative effect on transformation.

Signaling through the transforming growth factor-beta (TGF-beta) pathway results in growth inhibition and induction of apoptosis in various cell types. We show that this pathway is blocked in Kaposi sarcoma herpesvirus (KSHV)-infected primary effusion lymphoma through down-regulation of the TGF-beta type II receptor (TbetaRII) by epigenetic mechanisms. Our data also suggest that KSHV infection may result in lower expression of TbetaRII in Kaposi sarcoma and multicentric Castleman disease. KSHV-encoded LANA associates with the promoter of TbetaRII and leads to its methylation and to the deacetylation of proximal histones. Reestablishment of signaling through this pathway reduces viability of these cells, inferring that KSHV-mediated blockage of TGF-beta signaling plays a role in the establishment and progression of KSHV-associated neoplasia. These data suggest a mechanism whereby KSHV evades both the antiproliferative effects of TGF-beta signaling by silencing TbetaRII gene expression and immune recognition by suppressing TGF-beta-responsive immune cells through the elevated secretion of TGF-beta1.

Primary effusion lymphoma (PEL) is a largely incurable malignancy of B cell origin, driven by infection with KSHV/HHV-8, and with notable plasmacytic differentiation. PEL carries an extremely poor prognosis with a median survival time of less than 6 months and is known to be largely resistant to conventional chemotherapy. Therefore, we conducted high throughput screens for effective inhibitors using PEL cell lines. We discovered a compound, 6-ethylthioinosine (6-ETI), a nucleoside analog with selective toxicity to PEL and multiple myeloma (MM) at nanomolar concentrations, but not to other lymphoma cell lines tested. 6-ETI also showed remarkable responses in a mouse xenograft model of PEL. To understand the molecular mechanism(s) of selectivity towards PEL, we developed and performed resistome analysis, an unbiased approach based on RNA sequencing of in vitro and in vivo generated resistant subclones. We found adenosine kinase (ADK) inactivating genomic alterations in all resistant clones as the mechanism of resistance. Concordantly, we found that plasma cells express ADK, as well as sensitive PELs and multiple myeloma cell lines, while resistant lymphoma cell lines including those with EBV infection had lower levels (if any); interestingly, the latter could be sensitized by cell crowding-induced ADK upregulation. Like other nucleoside analogues, 6-ETI induces ATP-depletion and cell death accompanied by S-phase arrest and DNA damage, but only in ADK-expressing cells. Immunohistochemistry for ADK served as a new biomarker approach to identify tumors that may be sensitive to 6-ETI, which we documented for primary specimens of PEL, multiple myeloma and other lymphoid malignancies with plasmacytic features, namely plasmablastic lymphoma. A number of nucleoside analogues have been reported to be effective in treating leukemias and B and T cell lymphomas. We performed structure-activity relationship studies and tested a number of nucleoside analogs that are in preclinical or clinical development for other hematological malignancies to identify and better understand the structural requirements for 6-ETIÕs biological activity. We successfully demonstrated that 6-ETI was more potent and selective at killing PEL and MM cell lines than other studied nucleoside analogs suggesting that this compound possesses unique and distinct features that are clinically promising. Despite the presence of treatment approaches that can greatly extended the survival of MM patients, resistance remains an issue, warranting the need for new effective agents and combinations. We identified 6-ETI as a novel and clinically viable nucleoside analog for the treatment of PEL, immunoblastic lymphoma, plasmablastic lymphoma, multiple myeloma and other ADK-expressing cancers. Figure 1. Expression of ADK and sensitivity to 6-ETI in plasma cell tumors. (A) BC3 cells ADK expression was evaluated by immunohistochemistry in the BC3 cell line, hyperplastic tonsils and PEL, multiple myeloma and plasmablastic lymphoma primary tumors. 60X original magnification is shown. In the image of a tonsil section, a positive cell with morphological features of a plasma cell is enlarged in the insert. Original magnification 60X. (B) LC50s for multiple myeloma cell lines treated with 6-ETI were determined by CellTiter-Glo assay. BC3 was used as a positive control and IBL1 as a negative control for drug sensitivity. Shown are the average of two independent experiments, where each condition was performed in duplicate in each experiment. (C) Model for 6-ETIÕs mechanism of action within the cell is illustrated, where 6-ETI competes with adenosine (ADO) and other nucleosides for binding to and phosphorylation by ADK, which inhibits ATP-dependent metabolic processes. This also allows 6-ETI to be phosphorylated and activated by ADK, with subsequent phosphorylation steps that allow the compound to be incorporated into DNA and possibly RNA, leading to DNA synthesis inhibition, DNA damage response, and cell death. Figure 1. Expression of ADK and sensitivity to 6-ETI in plasma cell tumors. (A) BC3 cells ADK expression was evaluated by immunohistochemistry in the BC3 cell line, hyperplastic tonsils and PEL, multiple myeloma and plasmablastic lymphoma primary tumors. 60X original magnification is shown. In the image of a tonsil section, a positive cell with morphological features of a plasma cell is enlarged in the insert. Original magnification 60X. (B) LC50s for multiple myeloma cell lines treated with 6-ETI were determined by CellTiter-Glo assay. BC3 was used as a positive control and IBL1 as a negative control for drug sensitivity. Shown are the average of two independent experiments, where each condition was performed in duplicate in each experiment. (C) Model for 6-ETIÕs mechanism of action within the cell is illustrated, where 6-ETI competes with adenosine (ADO) and other nucleosides for binding to and phosphorylation by ADK, which inhibits ATP-dependent metabolic processes. This also allows 6-ETI to be phosphorylated and activated by ADK, with subsequent phosphorylation steps that allow the compound to be incorporated into DNA and possibly RNA, leading to DNA synthesis inhibition, DNA damage response, and cell death. Disclosures Nayar: Weill Cornell Medical College: Patents & Royalties: Submitted patent applicatio for 6-ETI. Cesarman:Weill Cornell Medical College: Patents & Royalties: applied for patent for 6-ETI.

We used KSHV-infected primary effusion lymphoma (PEL) cells as a model to search for novel drugs through a combination of screening and genomic approaches. We discovered 6-ethylthioinosine (6-ETI), a nucleoside analog, as a highly effective and selective inhibitor of PEL. This compound induces necrosis accompanied by S-phase arrest without affecting known oncogenic viral proteins. To understand the selectivity towards PEL, we performed unbiased genomic analysis of 6-ETI-resistant subclones using RNASeq, which revealed that 6-ETI is activated through phosphorylation by cellular adenosine kinase (ADK). PEL cell lines have higher basal levels of ADK than resistant lymphoma cell lines, and resistant lymphoma cell lines could be sensitized by cell crowding-induced ADK upregulation. Finally, 6-ETI was shown to be highly efficacious against incipient and established tumors in a mouse model of PEL, with no apparent toxicity. Thus, we have successfully used RNASeq-based “resistome” analysis to identify the mechanism of specificity for a new and preclinically effective nucleoside analog. Citation Format: Utthara Nayar, Jonathan Reichel, Jouliana Sadek, Denise Hernandez-Hopkins, Gunkut Akar, Hufeng Zhou, Michelle A. Sahai, Peter Barelli, Ilaria Guasparri, Jennifer Totonchy, Duane Hassane, Shizuko Sei, Robert H. Shoemaker, J. David Warren, Olivier Elemento, Kenneth M. Kaye, Ethel Cesarman. Genomics-based resistome analysis revealed endogenous adenosine kinase levels as a chief determinant of specificity for a novel nucleoside analog lymphoma inhibitor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4496. doi:10.1158/1538-7445.AM2015-4496

The Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) tethers KSHV terminal repeat (TR) DNA to mitotic chromosomes to efficiently segregate episomes to progeny nuclei. LANA contains N- and C-terminal chromosome binding regions. We now show that C-terminal LANA preferentially concentrates to paired dots at pericentromeric and peri-telomeric regions of a subset of mitotic chromosomes through residues 996-1139. Deletions within C-terminal LANA abolished both self-association and chromosome binding, consistent with a requirement for self-association to bind chromosomes. A deletion abolishing TR DNA binding did not affect chromosome targeting, indicating LANA's localization is not due to binding its recognition sequence in chromosomal DNA. LANA distributed similarly on human and non-human mitotic chromosomes. These results are consistent with C-terminal LANA interacting with a cell factor that concentrates at pericentromeric and peri-telomeric regions of mitotic chromosomes.

Kaposi's sarcoma–associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) mediates viral genome attachment to mitotic chromosomes. We find that N-terminal LANA docks onto chromosomes by binding nucleosomes through the folded region of histones H2A-H2B. The same LANA residues were required for both H2A-H2B binding and chromosome association. Further, LANA did not bind Xenopus sperm chromatin, which is deficient in H2A-H2B; chromatin binding was rescued after assembly of nucleosomes containing H2A-H2B. We also describe the 2.9-angstrom crystal structure of a nucleosome complexed with the first 23 LANA amino acids. The LANA peptide forms a hairpin that interacts exclusively with an acidic H2A-H2B region that is implicated in the formation of higher order chromatin structure. Our findings present a paradigm for how nucleosomes may serve as binding platforms for viral and cellular proteins and reveal a previously unknown mechanism for KSHV latency.

In latent infection, Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen 1 (LANA1)-specific binding to KSHV terminal repeat DNA mediates multicopy episome persistence. We now use electrophoretic mobility shift assays to investigate LANA1 binding to its 20-bp cognate sequence. Mutations at positions 6, 7, and 8 ( 6 CCC 8 ) severely reduced LANA1 binding, whereas mutations at other positions only modestly reduced binding. Since 6 CCC 8 is in the 5′ half of an inverted repeat sequence, these results are consistent with an asymmetric role for the inverted repeat in LANA1 binding.

beta-Lactam antibiotics share a common structure and mechanism of action, although they differ in their spectrum of antimicrobial activity and utility in treating different infections. The current classes include the penicillins, the penicillinase-resistant penicillins, the extended- spectrum penicillins, the cephalosporins, the carbapenems, and the monobactams. This article discusses some of the newest beta-lactams available for use in the United States: ertapenem, cefditoren, and cefepime. A new formulation of amoxicillin-clavulanate, which contains higher doses of amoxicillin, is also discussed.