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.