KSHV Latent Genes and Their Regulation

Total Page:16

File Type:pdf, Size:1020Kb

KSHV Latent Genes and Their Regulation SPB-141312 20 August 12, 2008 Time: 9:46 Proof 1 1 Chapter 20 2 3 KSHV Latent Genes and Their Regulation 4 5 6 Dirk P. Dittmer 7 8 9 10 11 12 OOF 13 ‘‘Herpesviridae omnia divisa est in partes tres.’’ 14 15 Herpesviridae are divided into three groups: alpha herpesviruses, beta herpes- 16 viruses and gamma herpesviruses. Only the two gammaPR herpesviruses, Kaposi 17 18 sarcoma-associated herpesvirus (KSHV, or human herpesvirus 8) and 19 Epstein–Barr virus (EBV, or human herpesvirus 4) are associated with human 20 cancer. Herpesvirus lytic replication is also customarily divided into three 21 phases: alpha or immediate early (IE), beta or early (E) and gamma or late (L). We base this classification on the temporal order of viral gene expression. 22 ED 23 Here, I propose that KSHV latent genes likewise may be divided into three 24 categories based upon their pattern of transcription in KSHV-associated dis- 25 eases (Table 20.1). 26 KSHV is associated with three proliferative malignancies in immune- 27 compromised patients: Kaposi sarcoma (KS), primary effusion lymphoma 28 (PEL) and the plasmablastic variant of multicentric Castleman disease (MCD) 29 (reviewed in (Antman and Chang 2000; Ablashi et al. 2002; Dourmishev et al. 30 2003)). Overwhelming epidemiological evidence shows that KSHV infection is 31 required for the disease phenotypes. Every tumor cell carries the viral genome 32 and expresses KSHV latent proteins. Every cell that stably maintains the KSHV 33 genome must express the latency-associatedRRECT nuclear antigen (LANA), as this 34 protein is required for latent episome maintenance (see Chapter 19). LANA is 35 expressed in KS as well as in PEL and MCD (Kedes et al. 1997a; Kellam et al. 36 1997a; Dittmer et al. 1998; Dupin et al. 1999a). LANA transcription is regulated 37 by the LANA promoter, which is constitutively active in all cell types. LANA-2/ 38 vIRF-3/K10.2 is only expressed in PEL, a tumor of B cell origin. By contrast K9/ 39 vIRF-1 is seen more prominently in KS, a tumor of endothelial origin. Another 40 KSHV latent gene Kaposin/k12/t0.7 is also constitutively transcribed in all 41 42 D.P. Dittmer (*) 43 University of NorthUNCO Carolina at Chapel Hill, Department of Microbiology 44 and Immunology and Lineberger Comprehensive Cancer Center 45 e-mail: [email protected] B. Damania, J. Pipas (eds.), DNA Tumor Viruses, DOI 10.1007/978-0-387-68945-6_20, Ó Springer ScienceþBusiness Media LLC 2008 SPB-141312 20 August 12, 2008 Time: 9:46 Proof 1 D.P. Dittmer 46 Table 20.1 The three categories of KSHV latency 47 KS (in situ) PEL, MCD (in situ) TPA inducible Genes 48 Type I 100% 100% Minimal LANA*, vFLIP*, vCYC* 49 Type II 0% 100% Not inducible LANA-2/vIRF-3 50 Type III 100% 100% Maximal Kaposin*, K9/vIRF-1* 51 *A second, proximal promoter drives transcription in response to Rta/orf50. 52 53 54 KSHV-associated tumors. K9/vIRF-1 and Kaposin are greatly induced by phor- 55 bol ester (TPA) stimulation of PEL. The transcriptional response to TPA of 56 LANA/orf73, vCYC/orf72 and vFLIP/orf71/K13, which are derived from dif- ferentially spliced, overlapping transcripts, is complex and LANA-2/vIRF-3/ 57 OOF 58 K10.5-10.6/K10.7 mRNA levels are impervious to TPA. 59 In sum, KSHV latent transcription seems divinely designed, or rather evo- 60 lutionarily tuned, to respond to different host environments. This ensures 61 commensal coexistence of virus and host under normal circumstances, but can lead to tumorigenesis in the setting of immune deficiency.PR 62 63 64 65 20.1 Profiling KSHV Transcription in Experimental Models 66 and Primary Tumors 67 ED 68 Herpesvirus transcription is divided into four stages: the three lytic stages 69 (alpha, beta and gamma) and the latent stage. In KSHV, and in the related 70 71 EBV, latency can be further divided into additional types, based on gene 72 expression and host cell origin. Many excellent studies have profiled KSHV 73 transcription genome-wide (Table 20.2). Although the KSHV transcript map is 74 of yet incomplete (see Chapter 19 for the current transcript map), all gene 75 expression profiling studies concur as to the identity of the KSHV latent 76 genes (Fig. 20.1). These are LANA (orf73), vCYC (orf72), vFLIP (orf71), the 77 viral miR cluster, Kaposin (orf K12) and LANA-2/vIRF-3. Except for LANA- 2/vIRF-3, all other latent genes are clustered in the far right region of the viral 78 RRECT 79 genome. Except for LANA-2/vIRF-3, which is B-cell specific, all other latent 80 genes are expressed in all KSHV-associated cancers and all experimental mod- 81 els of KSHV latency. 82 The kinetics of KSHV latent transcripts outside of long-term latency forms a 83 focus of recent research. Chandran and colleagues found that LANA is 84 expressed as an early gene upon primary infection of a HUVEC and BJAB 85 cells and that there exists a reciprocal relationship between IE transactivator 86 Rta/orf50 message and LANA message (Krishnan et al. 2004). Rta/orf50 87 mRNA peaks within minutes of infection, but then declines as LANA mRNA 88 increases. TheUNCO relative ratios depend on the host propensity to establish latent 89 or lytic infection. In cells that support high-level KSHV lytic replication 90 (HUVEC, HEK293), Rta/orf50 levels stay high as a significant proportion of SPB-141312 20 August 12, 2008 Time: 9:46 Proof 1 20 KSHV Latent Genes and Their Regulation 91 Table 20.2 Genome-wide profiling studies for KSHV viral gene transcription 92 Tissue Platform Citation 93 PEL 94 BCBL-1 Reverse Northern Blot (Zhong et al. 1996) 95 BCBL-1 70-mer array (Wang et al. 2002) 96 BCBL-1 real-time QPCR (Fakhari and Dittmer 2002) 97 BCBL-1 cDNA array (Paulose-Murphy et al. 2001a) 98 BCBL-1/rapamycin real-time QPCR (Sin et al. 2007) BCBL-1/cidofovir cDNA array (Lu et al. 2004) 99 BCBL-1/ganciclovir real-time QPCR (Staudt et al. 2004) 100 BC-1 Northern Blot (Sarid et al. 1998) 101 BC-1 Real-time QPCR (Whitby et al. 2007) 102 BC-3 cDNA array (Jenner et al.OOF 2001a) 103 JSC-1 real-time QPCR Bagni et al. (submitted) 104 JSC-1 cDNA array (Suscovich et al. 2004) 105 BCBL-1/K1 cDNA array (Lee et al. 2002) 106 BCBL-1/Rta/orf50 cDNA array (NakamuraPR et al. 2003) 107 BCBL-1/NotchIC Real-time QPCR (Chang et al. 2005) 108 Endothelial 109 TIVE Real-time QPCR (An et al. 2006) 110 KS Real-time QPCR (Dittmer 2003) 111 Murine endothelial cells Real-time QPCR (Mutlu et al. 2007) 112 HMVEC-d cDNA arrayED (Krishnan et al. 2004) 113 HMVEC-d cDNA array (Moses et al. 1999) HUVEC Real-time QPCR (Yoo et al. 2005) 114 115 116 117 the culture enter complete lytic replication as characterized by subsequent E and 118 L gene transcription. In cells that do not support KSHV lytic replication in the 119 absence of external stimuli such as phorbol ester (BJAB, fibroblasts), Rta/orf50 120 levels rapidly decline as infected cells enter latency or loose the viral genome. 121 At times profiling studies also showed an increase of latent (LANA, vCYC, 122 vFLIP) mRNA at late times after lytic reactivation (Jenner et al. 2001a), which 123 would classify LANA as a gamma2RRECT class mRNA (Fig. 20.1). A similar observa- 124 tion has recently been made in EBV (Yuan et al. 2006). We did not find evidence 125 for significant latent mRNA induction at late times in BCBL-1 cells (Fakhari 126 and Dittmer 2002); significant in as much, as it could not be explained by 127 genome copy number amplification or changes in PEL culture composition. 128 Unlike EBV-infected BL cell lines, in which the majority of cells reactivate from 129 latency upon IgM cross-linking, in any PEL cell line never more than 50% 130 (often less than 20%) of cells reactivate the virus in response to phorbol ester. 131 This poses no problem for the study of lytic transcription, as lytic transcripts are 132 virtually undetectable in latent cells and only cells that do reactivate contribute 133 to the signal.UNCO However, it makes studies of latent gene transcription in response 134 to stimuli difficult to interpret, since these are conducted in a background of 135 uninduced cells. SPB-141312 20 August 12, 2008 Time: 9:46 Proof 1 D.P. Dittmer 136 latent ie/early late 137 138 139 140 141 142 143 144 145 146 147 OOF 148 149 150 Fig. 20.1 Transcription profile of the KSHV latency locus (See Color Insert) TM 151 *Primary data from (Jenner et al. 2001a) were re-analyzed using ArrayMinerPR 152 153 20.2 The KSHV Latency Locus 154 155 The KSHV latent genes LANA, vCYC and vFLIP (and miRs) all cluster 156 together within one transcription unit (Fig. 20.2). At least one nascent tran- 157 script exists that traverses the entire locus (PearceED et al. 2005; Samols et al. 2005; 158 Cai and Cullen 2006). It initiates at the constitutive LANA promoter start site 159 at nt 127,880 and terminates at the Kaposin-distal poly-A site at nt 117,432. 160 Transcription across this locus proceeds only in one direction: from right to left. 161 To date no evidence for mRNAs originating at the opposite strand has been 162 found. Furthermore, latent transcription was polymerase-II dependent.
Recommended publications
  • ORIGINAL ARTICLE Short Telomeres and High Telomerase Activity in T
    Leukemia (2007) 21, 2456–2462 & 2007 Nature Publishing Group All rights reserved 0887-6924/07 $30.00 www.nature.com/leu ORIGINAL ARTICLE Short telomeres and high telomerase activity in T-cell prolymphocytic leukemia ARo¨th1,JDu¨rig1, H Himmelreich2,3, S Bug4, R Siebert4,UDu¨hrsen1, PM Lansdorp5,6 and GM Baerlocher2,3 1Department of Hematology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany; 2Department of Hematology, University Hospital, Bern, Switzerland; 3Department of Clinical Research, University Hospital, Bern, Switzerland; 4Institute of Human Genetics, Christian-Albrechts-University Hospital Schleswig-Holstein, Kiel, Germany; 5Terry Fox Laboratory, BC Cancer Research Centre, Vancouver, Canada and 6Department of Medicine, University of British Columbia, Vancouver, Canada 4 5 To test the role of telomere biology in T-cell prolymphocytic repeats of the sequence T2AG3 and associated proteins folded leukemia (T-PLL), a rare aggressive disease characterized by into a telomere loop structure.6 Telomeres are required to the expansion of a T-cell clone derived from immuno-compe- tent post-thymic T-lymphocytes, we analyzed telomere length maintain chromosomal integrity and prevent end-to-end fusions and telomerase activity in subsets of peripheral blood leuko- of chromosomes. When telomeric ends become too short, DNA cytes from 11 newly diagnosed or relapsed patients with damage signals from telomeres can induce apoptosis or a state sporadic T-PLL. Telomere length values of the leukemic T cells of replicative senescence.7,8 Telomeres shorten with each round (mean7s.d.: 1.5370.65 kb) were all below the 1st percentile of of cell division as a result of failure to completely replicate the 30 telomere length values observed in T cells from healthy age- end of chromosomes9,10 as well as other causes.11 The average matched controls whereas telomere length of normal T- and telomere length in cells from most human tissues decreases with B cells fell between the 1st and 99th percentile of the normal 12,13 distribution.
    [Show full text]
  • S Sarcoma-Associated Herpesvirus Lana2 Protein Interacts with the Pocket Proteins and Inhibits Their Sumoylation
    Oncogene (2014) 33, 495–503 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc ORIGINAL ARTICLE Kaposi’s sarcoma-associated herpesvirus lana2 protein interacts with the pocket proteins and inhibits their sumoylation L Marcos-Villar1,5, P Gallego1,5, C Mun˜ oz-Fontela2, CF de la Cruz-Herrera1, M Campagna1, D Gonza´lez1, F Lopitz-Otsoa3, MS Rodrı´guez3,4 and C Rivas1 The pocket proteins retinoblastoma protein (pRb), p107 and p130 are the key targets of oncoproteins expressed by DNA tumor viruses. Some of these viral proteins contain an LXCXE motif that mediates the interaction with the three pocket proteins and the inhibition of the pRb SUMOylation. Kaposi’s sarcoma herpesvirus (KSHV) contains at least two proteins that can regulate pRb function but, so far, a KSHV-encoded protein targeting p107 and p130 has not been identified. Here, we show that the KSHV latent protein LANA2 binds to pRb, p107 and p130. LANA2 contains an LXCXE motif that is required for bypassing pRb-mediated cell-cycle arrest and for inhibiting pRb SUMOylation. Finally, we demonstrate that, in addition to pRb, both p107 and p130 can be SUMOylated, and this modification is also inhibited by LANA2 in an LXCXE-dependent manner. These results demonstrate, for the first time, the SUMOylation of p107 or p130 and, so far, they represent the first example of a KSHV protein able to interact with the three pocket proteins and to inhibit their conjugation to SUMO. Oncogene (2014) 33, 495–503; doi:10.1038/onc.2012.603; published online 14 January 2013 Keywords: pocket proteins; KSHV; LANA2; pocket proteins; sumoylation; LXCXE domain INTRODUCTION vIRF3, interacts with pRb, p130 and p107 in vitro and in vivo.
    [Show full text]
  • THE NANOARCHITECTURE of the KSHV LANA TETHER and APPROACHES to ITS DISRUPTION Margaret Josephine Grant Shelton, CT B.S., Brown U
    THE NANOARCHITECTURE OF THE KSHV LANA TETHER AND APPROACHES TO ITS DISRUPTION Margaret Josephine Grant Shelton, CT B.S., Brown University, 2007 A Dissertation presented to the Graduate Faculty of the University of Virginia in Candidacy for the degree of Doctor of Philosophy Department of Microbiology, Immunology, and Cancer Biology University of Virginia May 2018 _____________________________ _____________________________ _____________________________ _____________________________ _____________________________ _____________________________ ii ABSTRACT Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of three human malignancies and presents a special concern to immunocompromised individuals. Its latency-associated nuclear antigen protein (LANA) tethers latent viral genomes to host chromatin, thereby maintaining viral infection, and as such, is an attractive target for therapeutic intervention. In an effort to better understand this protein and its tethering function, we have used super-resolution microscopy to examine LANA tethers, obtaining information that remained obscured in earlier studies using standard epifluorescence microscopy. We have determined several characteristics of these tethers, including the folding properties of the underlying viral DNA and occupancy of LANA on its available viral binding sites. Quantitative data support the prediction of a coiled-coil feature in LANA dimers, and computer modeling of a minimal LANA tether illustrates the importance of viral DNA bending and nucleosome positioning on tether structure. Preliminary data examining LANA tethers during cellular mitosis suggest a potential role for mitotic machinery in manipulating tether positioning and condensation. This work also begins to address the relative timing of host chromosome condensation and LANA tether formation. These promising early results compel further study of the mitotic LANA tether and its dynamics.
    [Show full text]
  • Transcription Profile of Kaposi's Sarcoma-Associated Herpesvirus in Primary Kaposi's Sarcoma Lesions As Determined by Real-Time PCR Arrays
    [CANCER RESEARCH 63, 2010–2015, May 1, 2003] Advances in Brief Transcription Profile of Kaposi’s Sarcoma-associated Herpesvirus in Primary Kaposi’s Sarcoma Lesions as Determined by Real-Time PCR Arrays1 Dirk P. Dittmer2 Department of Microbiology and Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104 Abstract tumor cell expresses at least one viral protein: the LANA/orf73 (2). Antibodies to LANA exist in virtually all HIV-infected as well as Kaposi’s sarcoma (KS) is the signature pathology of AIDS. KS-associ- non-HIV-infected KS patients, and other viral antigens have also been ated herpesvirus (KSHV/HHV-8) is causally linked to KS. Here, we report identified as the targets of this response. Prospective, longitudinal the first complete profile of KSHV transcription in primary KS lesions studies found that increases in peripheral blood viral load as well as using a novel, real-time PCR array. The KSHV latency I mRNAs [latency- associated nuclear antigen (LANA)/orf73, v-cyclin/orf72, and v-FLIP/ KSHV-specific antibody titers precede the onset of disease and cor- orf71] were invariably present in all biopsies. Yet, viral lytic mRNAs were relate with increased risk for KS. In addition, two lymphoproliferative detectable in only a subset of tumors. Interestingly, there was a difference disorders, primary effusion lymphoma (PEL) and multicentric Castle- in the expression pattern of the viral IFN-regulatory factors (vIRFs) man’s disease (MCD), are associated with KSHV (3, 4). These ob- encoded by KSHV. The vIRF-1/K9 clustered with LANA in KS, in con- servations imply (a) that KSHV viral oncogenes are required for KS trast to its homologue, vIRF-3/LANA-2, which is transcribed only in development and (b) that the identification of these viral genes may KSHV-associated lymphomas.
    [Show full text]
  • Visualization of Molecular Biology: the LANA Tether Vaibhav Jaina and Rolf Rennea,B,C,1
    COMMENTARY COMMENTARY Visualization of molecular biology: The LANA tether Vaibhav Jaina and Rolf Rennea,b,c,1 The latency-associated nuclear antigen (LANA) plays we focus on the role of LANA with respect to ge- a central role in the biology and pathogenesis of nome persistence during latency. The first evidence Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV). that KSHV LANA, like EBNA1 from the related hu- Both classical and endemic KS in HIV-infected individ- man tumor virus Epstein–Barr virus (EBV), is re- uals and two lymphoproliferative diseases are associ- sponsibleforgenomesegregationcamein1999, ated with KSHV. During the latent phase of the viral when it was demonstrated that plasmids containing TR life cycle in dividing tumor cells, the LANA protein en- sequences were stably segregated in cells expressing sures that viral genomes persist by supporting both LANA (9, 10). Multiple groups identified the TR se- the initiation of DNA replication and segregation of quences as cis-regulatory elements essential for both viral episomes (nonintegrated circular viral genomes) the initiation of DNA replication and the segregation into daughter cells. Arguably, interrupting these com- of TR-containing plasmids during mitosis. The LANA plex LANA-dependent processes could be one of the C-terminal domain was mapped and shown to bind most promising antiviral and antitumor therapeutic to two LANA binding sites (LBS1 and LBS2) in a co- strategies. Hence, studying the molecular and cell bio- operative manner (11). Next, elegant structural and ge- logical details of LANA’s host/viral protein and chro- netic approaches demonstrated that an 18-aa-long matin interactions has been a focus in a number of N-terminal peptide specifically interacts with the H2A/ laboratories since 1996.
    [Show full text]
  • Herpesviral Latency—Common Themes
    pathogens Review Herpesviral Latency—Common Themes Magdalena Weidner-Glunde * , Ewa Kruminis-Kaszkiel and Mamata Savanagouder Department of Reproductive Immunology and Pathology, Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Tuwima Str. 10, 10-748 Olsztyn, Poland; [email protected] (E.K.-K.); [email protected] (M.S.) * Correspondence: [email protected] Received: 22 January 2020; Accepted: 14 February 2020; Published: 15 February 2020 Abstract: Latency establishment is the hallmark feature of herpesviruses, a group of viruses, of which nine are known to infect humans. They have co-evolved alongside their hosts, and mastered manipulation of cellular pathways and tweaking various processes to their advantage. As a result, they are very well adapted to persistence. The members of the three subfamilies belonging to the family Herpesviridae differ with regard to cell tropism, target cells for the latent reservoir, and characteristics of the infection. The mechanisms governing the latent state also seem quite different. Our knowledge about latency is most complete for the gammaherpesviruses due to previously missing adequate latency models for the alpha and beta-herpesviruses. Nevertheless, with advances in cell biology and the availability of appropriate cell-culture and animal models, the common features of the latency in the different subfamilies began to emerge. Three criteria have been set forth to define latency and differentiate it from persistent or abortive infection: 1) persistence of the viral genome, 2) limited viral gene expression with no viral particle production, and 3) the ability to reactivate to a lytic cycle. This review discusses these criteria for each of the subfamilies and highlights the common strategies adopted by herpesviruses to establish latency.
    [Show full text]
  • Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated
    JOURNAL OF VIROLOGY, Oct. 2004, p. 10348–10359 Vol. 78, No. 19 0022-538X/04/$08.00ϩ0 DOI: 10.1128/JVI.78.19.10348–10359.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved. Latency-Associated Nuclear Antigen of Kaposi’s Sarcoma-Associated Herpesvirus Up-Regulates Transcription of Human Telomerase Reverse Transcriptase Promoter through Interaction with Transcription Factor Sp1 Subhash C. Verma, Sumit Borah, and Erle S. Robertson* Department of Microbiology and Abramson Comprehensive Cancer Center, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania Received 20 March 2004/Accepted 11 May 2004 Telomerase is required for the maintenance of telomere length and is an important determinant for cell immortalization. In human cells, telomerase activity is due to the expression of its enzymatic subunit, human telomerase reverse transcriptase (hTERT). The expression of hTERT is not typically detectable in healthy somatic human cells but is present in cancerous tissues and immortalized cells. We have previously shown that hTERT promoter activity is up-regulated by the Kaposi’s sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA). LANA is expressed in all forms of human malignancies associated with KSHV. The hTERT promoter sequence located at positions ؊130 to ؉5 contains several Sp1 binding motifs and was shown be important for up-regulation by LANA. In this report, we demonstrate that hTERT promoter activity is due to the direct interaction of LANA with Sp1. The interaction of LANA with Sp1 was demonstrated through in vitro binding experiments and coimmunoprecipitation and is supported by the colocalization of these two molecules in the nuclei of KSHV-infected cells.
    [Show full text]
  • Deregulation of C-Myc in Primary Effusion Lymphoma by Kaposi's
    Oncogene (2007) 26, 4979–4986 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE Deregulation of c-Myc in primary effusion lymphoma by Kaposi’s sarcoma herpesvirus latency-associated nuclear antigen D Bubman1, I Guasparri2 and E Cesarman3 1Department of Pathology and Laboratory Medicine, Weill Graduate School of Medical Sciences of Cornell University, New York, NY, USA; 2Department of Pathology and Laboratory Medicine, Weill Graduate School of Medical Sciences of Cornell University, New York, NY, USA and 3Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, New York, NY, USA Primary effusion lymphoma (PEL) is a rare subtype of Introduction non-Hodgkin’s lymphoma, which is associated with infec- tion by Kaposi’s sarcoma herpesvirus (KSHV)/human Kaposi’s sarcoma herpesvirus (KSHV), or human herpesvirus-8. The c-Myc transcription factor plays an herpesvirus-8, is a lymphotrophic g2-herpesvirus that is important role in cellular proliferation, differentiation the etiologic agent of Kaposi’s sarcoma (Moore and and apoptosis. Lymphomas frequently have deregulated Chang, 1995), a subset of multicentric Castleman’s c-Myc expression owing to chromosomal translocations, disease (Soulier et al., 1995) and primary effusion amplifications or abnormal stabilization. However, no lymphoma (PEL) (Cesarman et al., 1995a). PEL is a structural abnormalities were found in the c-myc oncogene rare subtype of non-Hodgkin’s lymphoma that is most in PEL. Given that c-Myc is often involved in lympho- common in human immunodeficiency virus-infected magenesis, we hypothesized that it is deregulated in PEL. individuals, usually presenting as lymphomatous effu- We report that PEL cells have abnormally stable c-Myc sions in serous cavities.
    [Show full text]
  • Ribosomal Protein S6 Interacts with the Latency-Associated Nuclear Antigen of Kaposi’S Sarcoma-Associated Herpesvirusᰔ Wuguo Chen and Dirk P
    JOURNAL OF VIROLOGY, Sept. 2011, p. 9495–9505 Vol. 85, No. 18 0022-538X/11/$12.00 doi:10.1128/JVI.02620-10 Copyright © 2011, American Society for Microbiology. All Rights Reserved. Ribosomal Protein S6 Interacts with the Latency-Associated Nuclear Antigen of Kaposi’s Sarcoma-Associated Herpesvirusᰔ Wuguo Chen and Dirk P. Dittmer* Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center, Center for AIDS Research (CfAR), University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7290 Received 16 December 2010/Accepted 28 June 2011 The latency-associated nuclear antigen (LANA) is central to the maintenance of Kaposi’s sarcoma-associ- ated herpesvirus (KSHV) and to the survival of KSHV-carrying tumor cells. In an effort to identify interaction partners of LANA, we purified authentic high-molecular-weight complexes of LANA by conventional chroma- tography followed by immunoprecipitation from the BC-3 cell line. This is the first analysis of LANA- interacting partners that is not based on forced ectopic expression of LANA. Subsequent tandem mass spectrometry (MS/MS) analysis identified many of the known LANA-interacting proteins. We confirmed LANA’s interactions with histones. Three classes of proteins survived our stringent four-step purification procedure (size, heparin, anion, and immunoaffinity chromatography): two heat shock proteins (Hsp70 and Hsp96 precursor), signal recognition particle 72 (SRP72), and 10 different ribosomal proteins. These proteins are likely involved in structural interactions within LANA high-molecular-weight complexes. Here, we show that ribosomal protein S6 (RPS6) interacts with LANA. This interaction is mediated by the N-terminal domain of LANA and does not require DNA or RNA.
    [Show full text]
  • CRISPR Interference Efficiently Silences Latent and Lytic Viral Genes in Kaposi’S Sarcoma-Associated Herpesvirus-Infected Cells
    viruses Article CRISPR Interference Efficiently Silences Latent and Lytic Viral Genes in Kaposi’s Sarcoma-Associated Herpesvirus-Infected Cells Kevin Brackett 1, Ameera Mungale 1, Mary Lopez-Isidro 1, Duncan A. Proctor 1, Guillermo Najarro 1 and Carolina Arias 1,2,3,* 1 Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA 93106, USA; [email protected] (K.B.); [email protected] (A.M.); [email protected] (M.L.-I.); [email protected] (D.A.P.); [email protected] (G.N.) 2 Neuroscience Research Institute, University of California, Santa Barbara, CA 93106, USA 3 Center for Stem Cell Biology and Engineering, University of California, Santa Barbara, CA 93106, USA * Correspondence: [email protected] Abstract: Uncovering viral gene functions requires the modulation of gene expression through overexpression or loss-of-function. CRISPR interference (CRISPRi), a modification of the CRISPR- Cas9 gene editing technology, allows specific and efficient transcriptional silencing without genetic ablation. CRISPRi has been used to silence eukaryotic and prokaryotic genes at the single-gene and genome-wide levels. Here, we report the use of CRISPRi to silence latent and lytic viral genes, with an efficiency of ~80–90%, in epithelial and B-cells carrying multiple copies of the Kaposi’s sarcoma-associated herpesvirus (KSHV) genome. Our results validate CRISPRi for the analysis of KSHV viral elements, providing a functional genomics tool for studying virus–host interactions. Citation: Brackett, K.; Mungale, A.; Keywords: KSHV; CRISPR-interference; dCas9-KRAB; Kaposi’s sarcoma-associated herpesvirus; Lopez-Isidro, M.; Proctor, D.A.; gene expression; gene silencing Najarro, G.; Arias, C.
    [Show full text]
  • Superresolution Microscopy Reveals Structural Mechanisms Driving the Nanoarchitecture of a Viral Chromatin Tether Margaret J
    Superresolution microscopy reveals structural mechanisms driving the nanoarchitecture of a viral chromatin tether Margaret J. Granta, Matthew S. Loftusa, Aiola P. Stojaa, Dean H. Kedesa,b,1, and M. Mitchell Smitha,1 aDepartment of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA 22908; and bDepartment of Medicine, Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, VA 22908 Edited by Donald E. Ganem, Novartis Institutes for Biomedical Research, Inc., Emeryville, CA, and approved March 2, 2018 (received for review December 13, 2017) By tethering their circular genomes (episomes) to host chromatin, qPCR to demonstrate a direct proportionality between the num- DNA tumor viruses ensure retention and segregation of their ber of LANA punctae and the amount of viral DNA, leading to genetic material during cell divisions. Despite functional genetic the conclusion that each nuclear dot represented a single viral and crystallographic studies, there is little information address- episome. Previous studies examining these punctae showed asso- ing the 3D structure of these tethers in cells, issues critical for ciation of LANA with mitotic chromosomes at the resolution understanding persistent infection by these viruses. Here, we of epifluorescence microscopy (12, 27). Kelley-Clarke et al. (28) have applied direct stochastic optical reconstruction microscopy later suggested preferential localization of these LANA punc- (dSTORM) to establish the nanoarchitecture of tethers within cells tae to centromeric and telomeric regions on metaphase chromo- latently infected with the oncogenic human pathogen, Kaposi’s some. Such studies have provided a solid foundation for further sarcoma-associated herpesvirus (KSHV). Each KSHV tether com- inquiry into the nature of this tethering mechanism.
    [Show full text]
  • Cell Cycle Regulatory Functions of the KSHV Oncoprotein LANA
    fmicb-07-00334 March 25, 2016 Time: 17:58 # 1 REVIEW published: 30 March 2016 doi: 10.3389/fmicb.2016.00334 Cell Cycle Regulatory Functions of the KSHV Oncoprotein LANA Fang Wei1, Jin Gan2, Chong Wang2, Caixia Zhu2 and Qiliang Cai2* 1 ShengYushou Center of Cell Biology and Immunology, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China, 2 MOE & MOH Key Laboratory of Medical Molecular Virology, School of Basic Medicine, Shanghai Medical College, Fudan University, Shanghai, China Manipulation of cell cycle is a commonly employed strategy of viruses for achieving a favorable cellular environment during infection. Kaposi’s sarcoma-associated herpesvirus (KSHV), the primary etiological agent of several human malignancies including Kaposi’s sarcoma, and primary effusion lymphoma, encodes several oncoproteins that deregulate normal physiology of cell cycle machinery to persist with endothelial cells and B cells and subsequently establish a latent infection. During latency, only a small subset of viral proteins is expressed. Latency-associated nuclear antigen (LANA) is one of the latent antigens shown to be essential for transformation of endothelial cells in vitro. It has been well demonstrated that LANA is critical for the maintenance of latency, episome DNA replication, segregation and gene transcription. In this review, we summarize recent studies and address how LANA Edited by: functions as an oncoprotein to steer host cell cycle-related events including proliferation Erle S. Robertson, and apoptosis by interacting with various cellular and viral factors, and highlight the University of Pennsylvania, USA potential therapeutic strategy of disrupting LANA-dependent signaling as targets in Reviewed by: Masahiro Fujimuro, KSHV-associated cancers.
    [Show full text]