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Arvin A, Campadelli-Fiume G, Mocarski E, et al., editors. Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis. Cambridge: Cambridge University Press; 2007.

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Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis.

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Chapter 28KSHV gene expression and regulation

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Introduction

In this chapter, both in vivo and in vitro KSHV viral gene expression patterns are described. Observations in both systems have been critical for the identification of viral proteins contributing to the pathogenic properties of this virus and for our appreciation of how this virus persists and replicates in the course of naturally occurring infections, the vast majority of which are asymptomatic (see Epidemiology). In contrast to other human herpesviruses, cell-free infection with KSHV in vitro is still inefficient and only a few studies have investigated viral gene expression following de novo infection. However, informative studies using in situ hybridization (ISH), immunohistochemistry (IHC), and various methods of transcript analysis have been carried out with stably infected, primary effusion lymphoma (PEL)-derived cell lines and, to a lesser extent, biopsy samples. Gradually, a picture on viral gene expression patterns and their regulation in different cell types is beginning to emerge.

Viral gene expression patterns in culture

PEL derived cell lines

PEL cell lines remain the most tractable system for examining KSHV viral gene expression. The vast majority of cells are infected latently and express a restricted repertoire of genes, while a small percentage (this varies from cell line to cell line, usually in the order of 1%–5%) of cells spontaneously switch into the lytic replication cycle. Lytic reactivation can be enhanced (up to 20% in some cell lines) in this system by chemical treatment with butyrate or phorbol esters. Despite the convenience of working with PEL cell lines, limitations exist: (1) if a bulk analysis of all cells in such a culture is carried out using Northern blots or RT-PCR, both the group of genuinely latent genes and that of strongly expressed, albeit only in a few cells, lytic genes may be detected, (2) chemical manipulation may have extraneous and secondary effects on viral gene expression, and (3) expression patterns in PEL B cell lines may not extend to expression patterns in KSHV infected tissues, in particular, spindle cells. With these caveats in mind, Fig. 28.1 shows an overview of the KSHV viral genome and the position of individual viral genes. The color coding reflects their expression during the different stages of lytic replication or latency. The coding is the result of a comparison of three publications that used gene arrays covering the entire viral genome (Jenner et al., 2001; Paulose-Murphy et al., 2001; Nakamura et al., 2003), and two earlier publications using Northern blots to investigate the basal expression and induction of KSHV genes in PEL cell lines following treatment with butyrate or phorbol esters (Sarid et al., 1998; Sun et al., 1999).

Fig. 28.1. KSHV gene expression in PEL cell lines and biopsy samples.

Fig. 28.1

KSHV gene expression in PEL cell lines and biopsy samples. This Figure summarizes the expression of individual KSHV genes in PEL cells during latency and following reactivation of the lytic cycle by treatment with TPA, Na-butyrate or heterologous expression (more...)

The first report to systematically analyze the expression pattern, by Northern blot, of the vast majority of KSHV genes in PEL cells before and after induction of the lytic cycle (Sarid et al., 1998) distinguished three categories of viral genes: class Ⅰ genes, expressed during latency and not upregulated by chemical induction; class Ⅱ genes, characterized by a baseline expression of variable abundance (high, moderate, and low) before, and increased expression after, chemical induction; class Ⅲ genes that are only expressed after chemical induction. Class Ⅰ genes comprised those marked in red in Fig. 28.1, i.e. ORF73 (encoding LANA1), ORF72 (vCYC), and ORF71/K13 (vFLIP). Transcripts most likely related to ORFs K2 (vIL6), ORF70, K4 (vMIP-Ⅱ), K5 (MIR1), K6 (vMIP-I), K7 (PAN/nut-1/T1.1 RNA), K8 (bZIP), ORF57, the vIRFs locus, and T0.7/Kaposin were classified as class Ⅱ genes, as were at least another five transcripts that could not be unambiguously assigned to individual ORFs. The remainder were considered class Ⅲ genes. The existence of Class Ⅱ transcripts outside of the classical dichotomous lytic-latent classification may, in part, be explain by a recent publication from H. Chang and colleagues which showed that a subset of immunoregulatory and growth promoting KSHV genes can be activated by RTA-independent notch signaling. Further, detection of these transcripts was shown to be dissociated from the full repertoire of lytic viral gene expression (Chang et al. 2005).

A subsequent study (Sun et al., 1999) attempted to relate the pattern of viral gene expression seen following the induction of the lytic cycle by treatment with Na-butyrate or phorbol-12-myristate-13-acetate (TPA) to the conventional cascade of herpesviral gene expression during lytic replication (Fig. 28.2). This conventional classification distinguishes immediate–early, early, and late genes: Immediate–early genes are expressed immediately following viral entry, do not require the de novo expression of viral proteins and are therefore resistant to treatment of the infected cell with cycloheximide. Early genes are sensitive to cycloheximide (i.e., require de novo protein synthesis in the infected cell) but are expressed prior to the replication of the viral genome and therefore are resistant to PAA or PFA.

Fig. 28.2. KSHV lytic replication.

Fig. 28.2

KSHV lytic replication. Herpesvirus lytic replication is characterized by wide-spread gene activation, classically in an ordered cascade of sequential induction. The full expression of this cascade can be chemically manipulated to temporally distinguish (more...)

Sun et al. (1999) classified ORF50 (RTA) as an immediate–early gene on the basis of its early onset of transcription (8 hours) following treatment with Na-butyrate and (only partial) resistance to cycloheximide. In spite of a similar rapid onset of expression seen with several other genes (e.g., h vIL6, vMIP-II, PAN/nut-1, vTS), none of these other genes examined by Sun et al. were resistant to cycloheximide and these therefore represent early genes. Since none of the gene array studies (see below) examined cycloheximide resistance, ORF50/RTA is therefore the only gene for which at least a partial resistance to cycloheximide has been demonstrated. It could be argued that the criterion of cycloheximide resistance should not be over-interpreted since reactivation rather than de novo infection (as in the classical experiments with alpha herpesviruses) was examined in all these studies, and that the designation of “immediate–early” for a KSHV gene on the basis of cycloheximide resistance is therefore problematic. However, ORF50/RTA stands out as the only KSHV gene with rapid expression kinetics for which this property has been demonstrated and is also the first viral gene to be expressed following de novo infection. Functional studies (see below and elsewhere in this book), as well as a systematic survey of viral genes whose expression is induced, directly or indirectly, by RTA (Nakamura et al., 2003), have shown that RTA is the central regulator of the lytic replication cycle. It has therefore been labeled pink in Fig. 28.1.

In addition to ORF50/RTA, a few other viral genes have been shown to be resistant to cycloheximide in chemically induced PEL cell lines and thus to have immediate–early characteristics (Zhu et al., 1999; Haque et al., 2000; Rimessi et al., 2001). In their study, Zhu et al. (1999) found the transcripts for ORFK8, ORF45 and ORFK4.2, in addition to that for ORF50/RTA, to have immediate-early characteristics. Haque et al. (2000) reported this for the ORFK5 transcript and Rimessi et al. (2001) for the ORFK3 mRNA.

Four groups (Jenner et al., 2001; Paulose-Murphy et al., 2001; Nakamura et al., 2003; Fakhari & Dittmer, 2002; Dittmer, 2003) have recently examined KSHV gene expression patterns in PEL cells by DNA array or real time PCR methods. All studies attempted to group individual viral genes into categories reflecting their onset or rate of expression. The results correlate on the whole, except for a few genes classified differently by several studies. These are indicated by open boxes in Fig. 28.1. All studies group ORFs 73 (LANA1), 72 (vCYC), 71/K13 (vFLIP) together as expressed in PEL cell lines prior to, and increasing only moderately following, induction of the lytic cycle (“latent” or “constitutive” genes). Based on in situ hybridization or immunohistochemistry, these genes, or their proteins, are expressed in the majority of infected cells in vivo (see below). These are therefore, marked in red in Fig. 28.1. As outlined below, these three viral genes are expressed from two alternatively spliced mRNAs (Fig. 28.4), with vCYC and vFLIP expressed from the same bicistronic mRNA. In spite of the general consensus that these are latent transcripts, the study by Nakamura et al. observed an increased expression of these mRNAs early (ORF71/ORF72) or late (ORF73) after triggering the lytic cycle by RTA expression. Since this has not been seen when Na-butyrate or phorbol esters were used to induce the lytic cycle, it may reflect the more “physiological” induction of the lytic cycle by RTA in this experiment and indicate that this group of mRNAs, although expressed during latent persistence, may increase during lytic replication.

Fig. 28.4. Splicing pattern of latency-associated and neighboring transcripts.

Fig. 28.4

Splicing pattern of latency-associated and neighboring transcripts. Two main transcripts, LT1 and LT2, and an additional minor transcript are directed by a latent promoter (horizontally striped box) and are translated to yield LANA1, vCYC and vFLIP. An (more...)

The non-coding nuclear RNA variously referred to as PAN, nut-1 or T1.1, and T0.7 or Kaposin, one of several transcripts derived from ORFK12, were the first abundant transcripts to be identified in KS biopsies (Zhong et al., 1996). On Northern blots from uninduced vs. induced PEL cell lines both mRNAs show basal expression that is increased upon induction of the lytic cycle (Sarid et al., 1998; Sun et al., 1999) and were designated as class Ⅱ transcripts by Sarid et al. In a gene array study, Jenner et al. found that the expression kinetics of T0.7/Kaposin resembled that of ORFK10 (these were designated latent/lytic transcripts), while that of T1.1/PAN clustered with those of ORFK7, ORFK14 and other early genes, grouped together as “primary lytic” genes. However, in a similarly designed study by Paulose-Murphy et al. (2001) and in that by Nakamura et al. (2003) T0.7/Kaposin and K10 did not group together, with T0.7/Kaposin having later expression kinetics. Similarly, T1.1/PAN, ORFK7, ORFK14 did not group together in these two studies. We therefore chose a separate color coding in Fig. 28.1 for T0.7/ Kaposin to reflect its probable latent nature in KS spindle cells in vivo. In spite of the controversial expression kinetics for T 1.1/PAN and K7, reflected in a hatched box in Fig. 28.1, it is clear that both are lytic genes.

Recently, in the region encompassing both T1.1/PAN and K7, Taylor et al. (2005) found a large transcript of approximately 6.1 kb by northern blot hybridization designated T6.1. This transcript is inducible with TPA, but is resistant to PFA. RACE identified the 5′ end of the transcript at nucleotide 23,586, and 5 excised clones from a screening of a PEL cDNA lambda phage library identified the 3′ end of the transcript at nucleotide 29,741 making the transcript co-terminal with T1.1/PAN (Fig. 28.1). This transcript encompasses K7 in the same orientation and ORFs K5 and K6 in the reverse orientation. Thus far, this 6.1 kb transcript is the largest found in the KSHV genome, but the presence of large lytic transcripts is not unique to KSHV and have been reported for other herpesviruses. Smuda et al. (1997) found high molecular mass overlapping early lytic transcripts of 6 kb, 8 kb, 10 kb, and 14 kb within the human cytomegalovirus genome. Wirth et al. (1989) identified a 6 kb immediate–early transcript and six late lytic transcripts ranging from 4.5 kb to >8 kb in bovine herpesvirus 1. It is unclear why these herpesviruses produce such large transcripts, although it has been postulated that their size may lead to RNA stability by the formation of pseudoknots. Regardless of function, the presence of this large transcript has implications in the examination of expression patterns for overlapping ORFs if DNA array and real time PCR methods are used.

The KSHV genome contains a region encoding a series of proteins with homologies to cellular interferon regulatory factors (IRFs). In this region one spliced gene, K10.5, shows latent gene expression in PEL cell lines in vitro (Rivas et al., 2001; Cunningham et al., 2003) and the corresponding protein, LANA2 or vIRF3, has been demonstrated by immunohistochemistry in all infected B cells in MCD and PEL in vivo, suggesting a B-lineage specific latent expression pattern (Rivas et al., 2001). However, the study by Nakamura et al. (2003) indicates that its expression is increased at a late stage following activation of the lytic cycle by RTA in a PEL cell line. Because of its B-cell specific expression pattern, K10.5/vIRF3 has been given its own (light brown) color coding in Fig. 28.1.

Three other genes in this locus, ORFs K9, K10, K11, are induced after activation of the lytic cycle (Cunningham et al., 2003; Nakamura et al., 2001; Paulose-Murphy et al., 2001; Jenner et al., 2001). Jenner et al. (2001) classified ORFK10, encoding vIRF4, as a “latent/lytic” gene, since its expression kinetics was similar to that of the T0.7/Kaposin transcript. The two other published gene array studies (Paulose-Murphy et al., 2001; Nakamura et al., 2003) concur to the extent that the ORFK10 transcript increases early after induction of the lytic cycle but group it with several other early transcripts. We have based our color coding of this gene on their results. In a similar manner, ORFK9 appears to be expressed relatively early, while in comparison ORFK11 expression appears to come on somewhat later. The upstream exon of K11, termed K11.1 in the study by Nakamura et al. (2003) and referred to as vIRF2 by Jenner et al. (2001), was classified in an earlier expression group than the second K11 exon by Nakamura et al. (2003), but in the same group as the second exon by Jenner et al. (2001).

Although the three gene array studies published so far do not always concur on the expression kinetics of individual viral genes, as illustrated by the above examples, there is broad agreement that viral proteins required for DNA replication or gene expression are produced earlier in the lytic cycle than viral structural proteins necessary for assembly of new virions. Likewise, lytic viral proteins known to be expressed in a slightly higher number of productively infected cells in vivo such as vIL6 or PF8 (see below) appear to be encoded by transcripts expressed in PEL cells early during the lytic cycle, whereas a structural glycoprotein encoded by ORFK8.1 and expressed only in few cells in vivo has been grouped with late transcripts. However, it needs to be emphasized that other factors than the stage of the lytic cycle may affect the expression of certain genes, e.g., vIL6, in vivo. However, in the case of vGPCR, the viral homologue of a G-coupled receptor that has been proposed to play an important role in KS pathogenesis by virtue of its ability to induce the secretion of VEGF and other paracrine factors from infected cells, the available data are controversial. While its expression kinetics in PEL cells resembled that of an early gene in the study by Jenner et al. and Nakamura et al., Paulose-Murphy et al. found a delayed onset and Sun et al. (1999) showed that its transcript was at least partially sensitive to PAA, suggesting late viral gene expression. However, using Northern blots Kirshner et al. (1999) did not see an inhibition of the vGPCR mRNA by PAA and classified it as an early lytic gene.

Viral gene expression in newly infected cultured endothelial, epithelial or fibroblast cultures

Experiments with KSHV released from PEL cell lines after chemical induction, or with recombinant KSHV preparations induced in Vero or 293 cells, have shown that KSHV can infect a wide variety of cultured cells belonging to several lineages (endothelial, epithelial, fibroblast) with the notable exception of lymphoid cell lines (Bechtel et al., 2003; Lagunoff et al., 2002; Moses et al., 1999; Vieira et al., 1997; Renne et al., 1998; Gao et al., 2003; Ciufo et al., 2001). It is not know at this time why B lymphocytes are resistant to de novo infection, however, Chen and Lagumoff have shown that naked DNA of a KSHV BAC, engineered with a selectable element, can be introduced into BJAB cells such that virus can be maintained in latency as well as undergo lytic reactivation (Chen and Lagunoff, 2005). In the majority of these reports, KSHV quickly established a latent infection, as defined by the expression of LANA1, in most infected cells. Only a small proportion of infected cells express viral transcripts or proteins characteristic of the lytic replication cycle such as ORF59/PFA, the K8.1 envelope glycoprotein, or the minor capsid protein mCP/SCIP encoded by ORF65 (Renne et al., 1998; Lagunoff et al., 2002; Ciufo et al., 2001; Vieira et al., 1997; Moses et al., 1999; Bechtel et al., 2003). In these latently KSHV-infected cells the lytic replication cycle can be reactivated using phorbol esters (as in PEL cell lines), or by superinfection with CMV (Vieira et al., 1997), and expression of RTA by transfection or transduction can do so in all cell types examined (Bechtel et al., 2003). A possible interpretation of these findings is that events upstream and controlling the expression of RTA are responsible for the blocked lytic replication and default latency in these cells. As discussed above, methylation of CpG residues in the ORF50/RTA promoter may represent such an event (Chen et al., 2001).

In one experimental system, which used a recombinant KSHV carrying a bacterial artificial chromosome and GFP cassette between ORFs 18 and 19, spontaneous transient lytic activation of KSHV in newly infected endothelial cells was observed during the first week of culture which subsided subsequently, giving way to long-term latent infection (Gao et al., 2003). Lytic reactivation in this experimental system was measured by the expression of the minor capsid protein SCIP, encoded by ORF65, and latent infection by expression of LANA1 (Gao et al., 2003).

Investigating early events following virus entry Dezube et al. (2002) found that rapid circularization of the viral genome occurred within 8 hours of infecting cultured endothelial cells with PEL-line derived KSHV. This was followed by the appearance of linear genomes, indicating lytic replication, approximately 72 hours after infection. Expression of the lytic transcripts T1.1/PAN and K8.1 could be detected by Northern blot at 3–6 days, and increased up to days 8–10, after infection. Latent transcripts for ORF72 (vCYC) and ORF73 (LANA1) were first detected on day 8. Interestingly, ORF74 (vGPCR) mRNA expression oscillated between days 1–8 (Dezube et al., 2002).

Viral gene expression early after de novo infection of endothelial cell and fibroblast cultures has also been investigated using a KSHV microarray (Krishnan et al., 2004). Following de novo infection, a limited number of KSHV genes are initially expressed (Fig. 28.3, Krishnan et al., 2004). However, with the exception of the latent transcripts for LANA1, vCYC and vFLIP, all mRNAs are down-regulated over the following 24 hours and KSHV therefore quickly adopts a transcriptional latency pattern. The first viral transcript to be expressed following de novo infection encodes ORF 50/RTA, the central regulator of the viral lytic replication cycle (Krishnan et al., 2004). However, only a limited number of RTA-activated viral genes (see below) show a transient expression. These include the genes for vIL6 (ORFK2), vMIP-Ⅱ (ORFK4), MIR2 (ORFK5), vMIP-I (ORFK6), vIRF2 (ORF11) and the survivin homologue (ORFK7), which are all thought to play a role in modulating the response to interferon, cytotoxic T-lymphocytes, NK cells or apoptosis. In contrast, many KSHV genes involved in DNA synthesis or encoding structural proteins were not expressed. This pattern of viral gene expression suggests that, unlike α- or β-herpesviruses, KSHV quickly adopts a latent program of gene expression and only transiently expresses a set of genes that counteract the effects of the interferon system – which is induced in endothelial and fibroblast cells very early following KSHV infection (Naranatt et al., 2004) – or other components of the innate or adaptive immune system. Which viral or cellular factors determine this switch into latency and interfere with the completion of the full lytic transcription program is not understood at present and remains one of the most interesting aspects of the control of KSHV gene expression.

Fig. 28.3. KSHV gene expression pattern in endothelial and fibroblast cells following de novo infection.

Fig. 28.3

KSHV gene expression pattern in endothelial and fibroblast cells following de novo infection. This figure summarizes the expression of individual KSHV genes following de novo infection of endothelial and fibroblast cultures reported in Ciufo et al., 2001; (more...)

Viral gene expression in vivo

Despite the relative ease of working with KSHV infected PEL-derived cell lines, a major problem of studying viral gene expression in such a system is that expression patterns may not reflect that found in infected tissues. Initial results from immunohistochemistry suggest that some KSHV genes can become dysregulated in tissue culture and that tissue-specific patterns of expression exist. The lack of a KSHV-infected spindle cell line raises additional concerns regarding generalizing findings from infected B cell lines to endothelial-derived KS lesions.

KS lesions

The first studies on viral expression in KS lesions examined the abundance of the T1.1/PAN and T0.7/Kaposin transcripts by in situ hybridization. The vast majority of spindle cells comprising KS lesions expressed T0.7/Kaposin in a cytoplasmic distribution with a predicted membrane proclivity (Zhong et al., 1996). By colocalization, a subpopulation (1%–10%) of T0.7/Kaposin positive cells also expressed T1.1/PAN transcripts and is thought to represent cells supporting lytic viral replication. The T1.1/PAN transcripts, although expressed in few cells, were present in high abundance ranging from an estimated 10 000 to 25 000 transcript copies per cell and were targeted to the nuclear compartment (Zhong et al., 1996). Consistent with a transcript expressed in lytic replication, T1.1/PAN also co-localized to the same cell population with probes to the major capsid protein (ORF25) (Staskus et al., 1997) and to the viral GPCR (ORF74) (Kirschner et al., 1999). The transcripts of two other genes, K3, K8 were shown to have a similar distribution to T1.1/Kaposin in KS lesions (Rimessi et al., 2001).

By immunohistochemistry, LANA1 protein is expressed in almost all tumor spindle and endothelial cells (Rainbow et al., 1997; Dupin et al., 1999; Katano et al., 2000). This is consistent with in vivo hybridization studies using the T0.7/Kaposin riboprobe as a surrogate marker for viral latent replication. Although vCYC and vFLIP proteins have not been formally shown to be expressed in a similar manner as LANA1, in situ hybridization studies of their transcripts in KS lesions demonstrate their presence in the majority of spindle cells (Reed et al., 1998; Dittmer et al., 1998; Sturzl et al.,1999). Reed and colleagues additionally found vCYC transcripts in epithelial cells of eccrine ducts and scattered epidermal cells (Reed et al., 1998). In contrast to the expression of latent genes, only a few cells within KS tumors express proteins associated with lytic replication indicating a relatively tight regulation of lytic viral reactivation. The low number of cells hosting viral lytic reactivation in KS lesions is reflected in the numerous reports where lytic viral proteins are detected not at all or in only a few cells (<1%) when a series of KS lesions are examined. Viral proteins found to be expressing in rare cells of KS lesions include PF-8 (ORF59), (Katano et al., 1999a,b; Parravicini et al., 2000) and ORF50 (Katano et al., 2001). The expression of vIL6 protein was found to be highly variable. Parravicini and colleagues were unable to detect the protein in 15 KS lesions they examined, while Cannon and colleagues found its expression in one out of 13 KS cases in one series but 7 out of 7 KS cases in another series deliberately selected for the presence of lytic foci (Parravicini et al., 2000; Cannon et al., 1999). The vIRF1 protein has not been detected in KS lesions by immunohistocheminstry thus far (Parravicini et al., 2000). This is most likely due to antibody sensitivity, since cells hosting lytic replication would be expected to express the full spectrum of encoded proteins.

Primary effusion lymphomas

Due to the scarcity of these lymphomas, extensive sampling of in vivo viral gene expression has not been done in this disorder. In a pattern similar to that found in KS lesions, tumor cells in PEL all express LANA1 protein (Dupin et al., 1999; Katano et al., 1999a,b) and only rarely express ORF50 (Katano et al., 2001). The major difference detected between PEL and KS lesions at this point is the expression of LANA2 which appears to be lymphoid specific. In contrast to ORF50 protein which is rarely detected, vIL6 is present in up to 5% of the PEL tumor cells. This partial uncoupling of vIL6 gene expression from RTA activation has been confirmed by in vitro studies on PEL cell lines showing that the vIL6 gene has a promoter containing two interferon stimulated response elements (ISRE) with the ability to induce vIL6 expression in cells treated with IFN-α. Additional evidence that transcriptional regulation of vIL6 is unique comes from microarray studies showing that only vIL6 transcripts in PEL cell lines were upregulated in response to IFN-α in the presence of cycloheximide (Chatterjee et al., 2002). These results show that additional pathways, beyond the dichotomous latency-lytic pathways traditionally described for herpesviruses, regulate vIL6 transcription.

The LANA2/vIRF3 gene (K10.5) is one of the few KSHV proteins which has been found to be latently expressed in PEL and MCD cells in vivo; however, LANA2/vIRF3 is not expressed in the vast majority of KS spindle cells. This finding reinforces the concept that KSHV is capable of multiple latency expression programs, and genes that are expressed in some tissues or cell lines may be silenced or absent in others. LANA2/vIRF3 differs from vIL6 in that vIL6 is expressed in a minority population of PEL tumor cells. Since vIL6 is a secreted cytokine, limited expression of vIL6 may nonetheless contribute to the pathogenesis of PEL tumors. In contrast, LANA2/vIRF3 expression is uniformly present in PEL tumor cells, indicating that it may have a critical role in maintaining the PEL tumor cell phenotype. These patterns of expression could be expected if the vIL6 promoter is activated by cytokine signaling pathways that are dependent on the local cellular milieu, whereas the LANA2/vIRF3 promoter is activated by B-cell transcription factors. It bears repeating at this point that although PEL-derived cell lines also express LANA2/vIRF3 and vIL6 there is a greater percentage of cells positive for class Ⅱ and class Ⅲ proteins (see above) in vitro suggesting that regulation of KSHV protein expression may be different in culture (Parravicini et al., 2000).

Multicentric Castleman’s disease

In MCD, the majority of cells in affected lymphoid tissues are not infected with KSHV: the LANA1 positive cells are largely confined to the mantle zone of lymphoid follicles. These KSHV-infected cells are negative for T-cell or monocytic markers and are felt to be B-cells, although only a minority express CD20 or CD79 B-cell markers. A subset of these LANA1 positive mantle zone cells also express vIL6, K8, K10, PF-8, and ORF65 proteins. However, in contrast to both PEL and KS lesions there are a higher percentage of LANA1 positive cells in MCD that express protein associated with lytic activation. Of these lytic cycle-associated genes, vIL6 appears to be expressed in a larger percentage of KSHV-infected cells. KSHV infected mantle zone cells therefore reproduce the pattern of viral gene expression observed in TPA-stimulated PEL-derived cell lines, in which a small, but significant subset of cells expresses class Ⅱ and Ⅲ genes (Parravicini et al., 2000; Katano et al., 2000).

Immunohistochemical techniques address critical aspects of virus behavior in infected tissue culture cells and pathological lesions that cannot be explored by mRNA studies. Although extensive mRNA mapping of viral gene expression has been performed in KSHV infected cell lines, tissue localization studies show that KS, PEL, and MCD are characterized by differing and unique patterns of KSHV protein expression.

Regulation of gene expression

Splicing

Genomic region containing KSHV latent genes and the viral chemokine receptor homologue

Gene expression in the locus encoding the major latent genes of KSHV, ORF73 (LANA1), ORF72 (vCYC), ORF71/K13 (vFLIP) is controlled by a constitutively active promoter located between nucleotides 127 935 and 129 370, with a minimal promoter region mapped to 127 935–127 968 of the prototypic KSHV sequence (Russo et al., 1996), as shown in Fig. 28.4. (Jeong et al., 2001). This promoter has the characteristics of a latent promoter, i.e. is not upregulated by treatment with phorbol esters or butyrate (Jeong et al., 2004) however, it may be regulated in a cell cycle specific manner (Sarid et al., 1999). It directs the expression of two more abundant and one rare mRNAs. The first transcript, latent transcript 1 (LT1; Fig. 28.4) encodes LANA1, while the second, LT2 represents a bicistronic mRNA from which both vCYC and vFLIP are translated. To enable efficient translation of the downstream reading frame for vFLIP, this mRNA contains an internal ribosomal entry site (IRES) located within ORF72 between nucleotides 122 973 and 123 206 (Bieleski & Talbot, 2001; Grundhoff & Ganem, 2001; Low et al., 2001). In addition, Grundhoff & Ganem (2001) reported the existence of a further spliced mRNA from which most of the ORF72 coding sequence was removed and only vFLIP could be translated (see Fig. 28.4). However, this doubly spliced mRNA was of low abundance and only detected by RT-PCR after induction of the lytic cycle suggesting a mechanism for increasing the expression of this normally latent (Low et al., 2001) anti-apoptotic protein during lytic replication. Low et al. (2001) have also suggested that the IRES dependent translation of vFLIP may allow its expression during apoptosis when normal cap-dependent translation is less efficient due to cleavage of eIF4G by caspase 3 (Low et al., 2001). A further bicistonic mRNA fo vcyc and vFLIP is directed by an additional latent promoter located in the 3′ end of the ORF73/LANA1 gene (Pearce et al., 2005; Cai et al., 2006). This promoter also directs the expression of a spliced mRNA, from which the Kaposin proteins are translated (see below and Fig. 28.8). In addition, un unspliced mRNA transcribed from his promoter serves as the precursor RNA for the viral miRNAs (see below and Fig 28.8).

Fig. 28.8. Splicing patterns and translation products in the K12 region of the KSHV genome.

Fig. 28.8

Splicing patterns and translation products in the K12 region of the KSHV genome. Transcripts in the K12 region appear to originate upstream of K12. In the case of a primary PEL tumor, a promoter has been identified in the latent region of the genome, (more...)

Upregulation of LANA1 by the activator of the lytic cycle RTA shortly after infection of a cell by KSHV is directed by a lytic promoter (127,807–127,620) located downstream of the major constitutive (latent) LANA1 promoter. This lytic mRNA starts at position 127,611, i.e. within the intron in the LT1 latent mRNA (Matsumara et al., 2005).

A recent report (Canham and Talbot, 2004) suggests the existence of a further mRNA which is prematurely polyadenylated within ORF73 and would be predicted to encode a truncated variant of LANA1 lacking the 76 c-terminal amino acids. Based on a deletion analysis of the c-terminal end of LANA1 (Viejo-Borbolla et al., 2003) such a truncated version of LANA1 would be expected to be deficient in its interaction with the nuclear matrix and in its ability to activate heterologous promoters and to replicate viral episomal DNA (Canham and Talbot, 2004; Viejo-Borbolla et al., 2003).

The region upstream of ORF73 contains a second promoter that directs the expression of another bicistronic mRNA, expressed during the lytic replication cycle and oriented in the opposite direction (Fig. 28.4). This mRNA contains the reading frames K14 (vOX2) and 74 (vGPCR) (Talbot et al., 1999; Kirshner et al., 1999; Jeong et al., 2001; Nador et al., 2001; Chiou et al., 2002; see Fig. 28.4). This promoter is activated by RTA, the central regulator of the lytic cycle and the minimal region response to RTA has been mapped to nucleotides 127 297–127 394 (Jeong et al., 2004; Chiou et al., 2002). At this promoter, activation by RTA is mediated through its interaction with the cellular transcriptional repressor RBP-Jκ, rather than by direct DNA binding (Liang and Ganem 2004). The bicistronic ORFK14/ORF74 mRNA results from splicing out an intron located between these two viral genes (Kirshner et al., 1999; Talbot et al., 1999; Nador et al., 2001; Chiou et al., 2002; Fig. 28.4). Although there is clear evidence from immunohistochemical staining of KS, PEL and MCD samples, as well as of TPA-induced PEL cell lines in vitro, that the vGPCR protein is expressed in cells undergoing lytic replication (Chiou et al., 2002), it is not yet clear how this protein is translated from its downstream position in a bicistronic mRNA. Internal ribosomal entry, translational reinitiation, modified ribosomal scanning have been suggested as possible mechanisms (Kirshner et al., 1999). In addition, Nador et al. (2001) found an additional monocistronic mRNA encoding only ORF74/vGPCR which was however much less abundant than the bicistronic mRNA for ORFs K14 and 74. The significance of this monocistronic mRNA and its contribution to vGPCR translation is not clear at present.

Genomic region containing immediate–early genes

The immediate–early gene ORF50, encoding the activator of the lytic cycle RTA, is located next to the multiply spliced early gene ORFK8, which encodes another player in lytic cycle regulation, KbZIP or RAP (Fig. 28.5). RTA is translated from an mRNA which incorporates a small exon located upstream of ORF49 in addition to the originally predicted reading frame 50 (Russo et al., 1996; Lukac et al., 1999; Zhu et al., 1999; Saveliev et al., 2002). The immediate–early expression kinetics of ORF50 mRNA, which has been reported to be upregulated by some groups as early as 1 hour following TPA treatment of PEL cell lines, has been discussed above. Expression of this mRNA is directed from a promoter that is autonomous and dependent on cellular factors (Seaman et al., 1999; Deng et al., 2000). In transient transfection assays using constructs in which the RTA promoter was placed upstream of a reporter gene, its activity can be further upregulated by RTA itself, but also by Na-butyrate or phorbol ester treatment and by co-transfection of some other viral proteins, e.g., vGPCR (Deng et al., 2000; Chiou et al., 2002). The autoactivation of the RTA promoter by RTA itself was also seen in persistently infected PEL cell lines (Deng et al., 2000).

Fig. 28.5. Splicing pattern of transcripts originating in the immediate-early region of the KSHV genome.

Fig. 28.5

Splicing pattern of transcripts originating in the immediate-early region of the KSHV genome. The region between ORFs 48 and 52 (see Fig. 28.1) encodes the immediate-early ORF50 transcript, as well as an early transcript for the ORFK8-derived (more...)

In latently infected cells the RTA promoter is partially silenced by methylation and it has been suggested that this provides a mechanism by which KSHV latency could be controlled (Chen et al., 2001). Methylation of CpG islands in the RTA promoter region is seen in persistently infected PEL cell lines in vitro, as well as in samples from KSHV-associated diseases (KS, PEL, MCD) or KSHV-infected cells in vivo (Chen et al., 2001). Treatment of PEL cell lines in vitro with 5-azacytidine, an inhibitor of methyltransferase, activates the lytic cycle (Chen et al., 2001). Activation of the lytic cycle in PEL cell lines by TPA involves demethylation of the ORF50 promoter (Chen et al., 2001). It has therefore been suggested that methylation of the ORF50 promoter is one of the mechanisms by which KSHV establishes latency in vivo (Chen et al., 2001).

The mRNA encoding ORF50 extends through two neighboring downstream genes, K8 and K8.1 (Fig. 28.5) but it is not clear whether there is any translation of these genes from this mRNA. However, a promoter located between ORF50 and K8 directs the expression of three alternatively spliced mRNAs, expressed at the ratio of 16:4:1 (Lin et al., 1999; Fig. 28.5). One of these contains three splice events and consequently joins two exons from the originally predicted K8 reading frame to a downstream third exon which contains a leucine zipper region (Lin et al., 1999; Gruffat et al., 1999). Together with a basic region found in the second exon (Fig. 28.5) the resulting protein resembles members of the basic leucine-zipper transcription factors (bZIP) which includes jun, fos and the activator of the EBV lytic cycle, Zta or BZLF-1 (Sun et al., 1998; Lin et al., 1999; Gruffat et al., 1999; Zhu et al., 1999; Seaman et al., 1999; further references in Sinclair, 2003). This protein, KbZIP or RAP is the predominant protein expressed in PEL cell lines after induction of the lytic cycle (Polson et al., 2001). In contrast to Zta, KbZIP cannot on its own activate the lytic cycle (Lukac et al., 1999; Polson et al., 2001). On the contrary, it has been suggested that KbZIP, by binding to RTA, could repress the ability of RTA to activate some (e.g., ORF57), but not all (e.g., PAN/nut-1), lytic cycle genes (Izumiya et al., 2003a). The basic region of KbZIP is required for the interaction with RTA (Izumiya et al., 2003a), as well as for the more recently described binding to cyclin A/E-cdk2 and its consequent phosphorylation by cdks (Polson et al., 2001) and the resulting slowing of the cell cycle (Izuimya et al., 2003b). The expression kinetics of KbZIP indicates that it is an early gene, since the corresponding mRNAs are sensitive to treatment with cycloheximide, but not phosphonoacetic acid (Lin et al., 1999).

The fourth exon of the three K8/KbZIP mRNAs (Fig. 28.5) overlaps with another reading frame, ORFK8.1, encoding a structural glycoprotein incorporated into the KSHV virion and of importance for the binding of virions to glycosaminoglycans (Birkmann et al., 2001; Wang et al., 2001a, b). However, as shown in Fig. 28.5, this protein is translated from two alternatively spliced mRNAs, whose expression is controlled by a different promoter and which use the same splice acceptor at nt 76,433 as the K8/KbZIP mRNAs, but two different splice donors at nt 76,155 and nt 76,338 (Gruffat et al., 1999; Raab et al., 1998; Chandran et al., 1998). This results in two isoforms of the K8.1 glycoprotein which run at molecular weights of 35 and 38 kDa on SDS gels (Raab et al., 1998; Chandran et al., 1998; Birkmann et al., 2001; Wang et al., 2001). As befits a structural virion glycoprotein, the K8.1 mRNAs have late expression kinetics, with doubling of expression between 16 and 24 hrs after treatment of PEL cell lines with TPA or overexpression of RTA (Nakamura et al., 2003; Jenner et al., 2001; Gradoville et al., 2000) and sensitivity of the 0.9 kb K8.1 transcript to PAA (Gradoville et al., 2000).

vIRF locus

A gene locus located between nt 83,500 and 94,200 of the KSHV genome contains several homologues of cellular interferon regulatory factors, termed vIRFs (Russo et al., 1996; Neipel et al., 1997). Although initially controversial, the gene expression and splicing patterns, as well as the proteins encoded by the different ORFs in this locus, have recently become clearer. A current consensus view is summarized in Fig. 28.6 (Cunningham et al., 2003). The first vIRF to be studied in detail, vIRF1 is encoded by an unspliced gene, ORFK9 (Gao et al., 1997; Chen et al., 2000; Wang et al., 2001a, b; Cunningham et al., 2003). ORFK9 shows expression kinetics compatible with its classification as an early gene: on Northern blots its expression is detected 8–12 hours after induction of PEL cell lines by TPA and is sensitive to treatment with cycloheximide but resistant to PAA (Wang et al., 2001a, b). In DNA array studies ORFK9 expression peaked at 24 hours (Paulose-Murphy et al., 2001; Nakamura et al., 2003) and was classified as a secondary lytic gene by Jenner et al. (2001). Immunofluorescence studies with an antibody to vIRF1 have also indicated a marked increase in vIRF1 expression following TPA stimulation of PEL cell lines (Parravicini et al., 2000).

Fig. 28.6. Splicing patterns in the vIRF region of the viral genome.

Fig. 28.6

Splicing patterns in the vIRF region of the viral genome. There are 4 KSHV proteins with sequence homology to cellular interferon regulatory factors (IRFs). The region of IRF homology is located at the amino terminal end of the viral proteins and encoded (more...)

In cells undergoing lytic viral replication a major transcriptional start site has been mapped to two adjacent nucleotides 74 and 77 bp upstream of the translational start codon by one group (Chen et al., 2001; Inagi et al., 1999) and to a neighboring nucleotide (–76 bp) by two other groups (Wang et al., 2001a, b; Cunningham et al., 2003). This start site is located 20–25 bp downstream of a TATA box. In addition, Chen et al. (2001) reported another transcriptional start site, found only in latently infected cells, approx. 84 bp upstream of the lytic start site. In contrast, in spite of finding 5′ RACE products with varying start sites upstream of position – 77, Cunningham et al. (Cunningham et al., 2003) could not confirm the existence of a defined latent start site and the evidence for the existence of an additional latent promoter for vIRF1 must therefore be seen as inconclusive. In vivo, vIRF1 expression could only be seen by immunohistochemistry in MCD tissue, but not in KS or PEL tissues, in keeping with the notion, derived from the in vitro studies, that vIRF-1 is expressed early during the lytic cycle (Parravicini et al., 2000).

The neighboring viral gene, ORFK10, is also inducible in PEL cell lines (Jenner et al., 2001; Paulose-Murphy et al., 2001; Nakamura et al., 2003; Cunningham et al., 2003). Jenner et al. and Nakamura et al. noted the very rapid onset of expression following chemical induction of PEL cell lines and Jenner et al. classified ORFK10 as a “latent/lytic” gene because of the basal expression seen in uninduced PEL cell lines. The ORF K10 transcript is spliced (Fig. 28.6). The open reading frame originally designated as K10 (Russo et al., 1996) started at an ATG at position 88,164, i.e., within the larger downstream exon depicted in Fig. 28.6. For this reason, some authors refer to the smaller upstream exon as K10.1 (Jenner et al., 2001; Neipel et al., 1997). This upstream exon contains the protein regions with homology to the DNA binding domains of cellular IRFs (Jenner et al., 2001; Cunningham et al., 2003). However, analysis of the splicing patterns in the K10 region has shown that the original “K10” is expressed as part of a spliced mRNA that includes “K10.1” and consequently the inclusion of the IRF homology domains justifies the designation vIRF4 for the corresponding protein (Cunningham et al., 2003; Jenner et al., 2001; see Fig. 28.6). Cunningham et al. (2003) concluded that this spliced mRNA is inducible in PEL cells, while Jenner et al. (2001) considered this mRNA as “latent/lytic” but reported the existence of an additional alternatively spliced mrna, found only in induced PEL cells, which eliminates the first 111 bp of the coding region in the upstream region. This would theoretically lead to a protein of 767 amino acids and a predicted molecular weight of 82 kDa, initiated at an internal ATG, which lacks the IRF homology region. However, Cunningham et al. (2003) found the alternative splice acceptor to be used by several mRNAs spliced to upstream viral regions as well as cellular sequences and queried whether this alternative transcript would be relevant physiologically. Using an antibody to K10, Katano et al. (2000) detected a dominant band of 100 kDa on Western Blots of induced but not uninduced PEL cell lines, in reasonable agreement with the predicted protein size of 98 kDa for the 905 aa translated from the singly spliced mRNA (Fig. 28.6). Although the existence of an additional minor protein cannot be completely ruled out, vIRF4, derived from the singly spliced mRNA, appears to be the dominant protein and expressed during the lytic cycle. By immunohistochemistry of pathology sections, vIRF4 was found to be expressed in 5% of KSHV-infected cells in MCD, but in less than 1% of cells in KS and PEL samples (Katano et al., 2000). This staining pattern is compatible with the expression of K10/vIRF4 during lytic viral replication.

ORFK10.5 is contained in a spliced mRNA (Lubyova and Pitha, 2000; Rivas et al., 2001; Jenner et al., 2001; Cunningham et al., 2003). In contrast to the original assignation of this ORF, the resulting protein (vIRF3, LANA2) contains sequences from both exons (Rivas et al., 2001; Cunningham et al., 2003; see Fig. 28.6). As in the case of the other vIRFs, a region with homology to cellular IRFs is located at the N-terminal end of vIRF3/LANA2 (Rivas et al., 2001; Cunningham et al., 2003; see Fig. 28.6) and derived from this first exon. While Lubyova and Pitha characterized this gene as inducible and Jenner et al. classified it as “secondary lytic” on their KSHV microarray using a probe for the upstream exon, Rivas et al. and Fakhari and Dittmer found it to be constitutively expressed in B-cells by northern blots or real time PCR. Using an antibody to vIRF3/LANA2, Rivas et al. could show its constitutive expression in latently infected PEL cell lines, as well as in PEL cell tumors and MCD specimens, but not in the endothelial and spindle cells of KS lesions. Cunningham et al. found several transcriptional start sites for the vIRF3/LANA2 transcript, none of which is in close proximity to a TATA box (Cunningham et al., 2003). The TATA box noted by Lubyova and Pitha is located approximately 500 bp further upstream and thus unlikely to direct the transcriptional start of the vIRF3/LANA2 mRNA. However, Cunningham et al. noted the sequence element AAGGTAATGAGGT approx. 250 bp upstream of most 5′ RACE products in their study. This element is closely related to a motif AAGGTAATGAAAT in the latent LANA1 promoter (Talbot et al., 1999) and the Oct-1/TAATGARAT element of immediate–early promoters in other herpesviruses (O’Hare, 1993).

ORFK11, as originally assigned by Russo et al. (1996), is also part of a larger coding region generated by a splice event that joins it to an upstream exon. As in the case of vIRF3/LANA2 and vIRF4, this upstream exon contains the region showing homology with cellular IRFs (Cunningham et al., 2003). ORFK11/vIRF2 is an inducible gene in PEL cells (Sarid et al., 1998; Cunningham et al., 2003) which doubles its basic expression level at about 20–24 hours after TPA treatment when measured on gene arrays (Jenner et al., 2001; Paulose-Murphy et al., 2001). In induced PEL cell lines, the vIRF2 mRNA has a single transcriptional start site located 23 nucleotides downstream from a TATA box (Cunningham et al., 2003). Using an antibody raised against ORFK11, Katano et al. (2000) could show that expression of the 110 kDa vIRF2 protein was only seen in TPA-induced PEL cells and that it is only rarely seen in KSHV-infected cells in KS, PEL or MCD, in keeping with its classification as a lytic gene product (Katano et al., 2000).

Terminal membrane proteins

Two viral genes, K1 and K15, located at either end of the virus genome, encode membrane-associated proteins, VIP and TMP, respectively, that can trigger several cellular signal transduction pathways (Lee et al., 1998a,b; Lee et al., 2000, 2002; Lagunoff et al., 1999, 2001; Glenn et al., 1999; Poole et al., 1999; Choi et al., 2000; Brinkmann et al., 2003). Both have no, or minimal, expression in uninduced PEL cells and mRNAs can be detected by northern blot, RT-PCR, RNAse protection or gene array following treatment with TPA or Na-butyrate (Lagunoff and Ganem, 1997; Sarid et al., 1998; Glenn et al., 1999; Choi et al., 2000; Jenner et al., 2001; Paulose-Murphy et al., 2001; Fakhari and Dittmer, 2002; Nakamura et al., 2003). ORFK1 encodes a type I transmembrane protein, containing two hypervariable extracellular domains and an ITAM (immunoreceptor tyrosine activation motif) in its cytoplasmic domain (Lagunoff and Ganem, 1997; Lee et al., 1998; Lagunoff et al., 1999; Poole et al., 1999; Cook et al., 1999). The K1 encoded protein has therefore been termed VIP for variable ITAM containing protein.

ORFK1 gene expression was reported to increase significantly 8–10 hrs following TPA addition and to peak after 24 –72 hours. Lagunoff and Ganem (1997) found that the increase in its mRNA is not sensitive to PAA and therefore classified K1 as an early gene. The rate of increase of K1 gene expression led Jenner et al. (2001) to classify it as a ‘tertiary lytic’ gene, expressed with the same kinetics as many structural viral proteins. Similarly, Paulose-Murphy et al. (2001) placed it among the lytic genes with slightly delayed expression kinetics (peak expression after 36 hours). Nakamura et al. (2003) grouped K1 together with some structural proteins (ORF65/SCIP, a capsid protein; ORF47/gL, a virion glycoprotein; ORF62, a tegument gene), but also the ORF56 DNA replication protein and the ORF74/vGPCR chemokine receptor homologue.

The promoter for ORFK1 has been mapped to a region in the long unique region (LUR) of the viral genome immediately adjacent to the terminal repeats (Fig. 28.7). A 100 bp fragment corresponding to nucleotides 210–310 of the partial BCBL-1 sequence reported by Lagunoff and Ganem (1997), but located largely outside the prototypic KSHV genome sequence reported by Russo et al. (1996), can confer promoter activity to a heterologous indicator gene and has moderate but significant constitutive activity in B cells, epithelial cells and endothelial cells (Bowser et al., 2002). The ORFK1 promoter is activated directly by RTA and TPA in B cells and epithelial cells; however, in SLK endothelial cells this effect is only weak (Bowser et al., 2002). These results are in keeping with the reported lytic cycle expression kinetics of ORFK1. Lee et al. (2003) used monoclonal antibodies to the extracellular domain of K1 to demonstrate its expression early after induction of the lytic cycle in PEL cells and in a small proportion of KSHV-infected cells in MCD biopsies.

Fig. 28.7. Splicing pattern of transcripts and location of promoters at either end of the viral genome.

Fig. 28.7

Splicing pattern of transcripts and location of promoters at either end of the viral genome. The K1 gene at the “left” end of the viral genome is controlled by a promoter located next to the terminal repeat region. At the opposite end, (more...)

ORFK15, at the other end of the genome, consists of 8 exons, which are multiply and alternatively spliced (see Fig. 28.7) to give rise to a family of proteins that share a common c-terminal cytoplasmic domain encoded by exon 8 but vary in the number of membrane anchor domains encoded by exons 1–7 (Glenn et al., 1999; Poole et al., 1999; Choi et al., 2000). The reading frame originally designated as ORFK15 by Russo et al. (1996) represents only a small part of this gene and overlaps with exon 2 (see Fig. 28.7). The designation of TMP (for terminal membrane protein) has recently been adopted for the K15 proteins. The largest of the K15/TMP proteins has an apparent molecular weight of 45 kDa on SDS-PAGE, is predicted to contain 12 such transmembrane segments in addition to the c-terminal cytoplasmic domain, and has recently been shown to be a potent activator of the Ras/ERK, JNK and NF-κB pathways (Brinkmann et al., 2003).

Like K1, K15 is inducible in PEL cells and has been classified as a class Ⅲ gene on the basis of its inducibility but lack of basal expression in PEL cells (Sarid et al., 1998). One of the three published gene array studies classified K15 as “tertiary lytic” (Jenner et al., 2001), another (Nakamura et al., 2003) reported a relatively early (8 hours) onset of K15 transcription which continued to increase up to 48 hours, whereas a third (Paulose-Murphy et al., 2001) observed peak expression at 24 hours with a subsequent decrease. In all three gene array studies (Nakamura et al. 2003; Jenner et al., 2001; Paulose-Murphy et al., 2001) K15 and K1 were grouped close to each other on cluster analysis. A promoter element directing the expression of the K15 gene has recently been identified in the long unique region between the first K15 exon and the terminal repeat region (Wong and Damania, 2006). A region derived from a terminal repeat subunit has promoter activity in vitro but is not responsive to RTA (Henke-Gendo, Rainbow & Schulz, unpublished data). In contrast to the similar expression kinetics of K1 and K15, regulation of K15 gene expression may therefore differ from that of K1.

At the protein level, different TMP isoforms have been demonstrated in transient transfection assays using expression constructs with differentially spliced mRNAs (Glenn et al., 1999; Choi et al., 2000; Sharp et al., 2002; Brinkmann et al., 2003). Recent findings suggest that the 45 kDa K15 protein is expressed in epithelial cell lines harboring a recombinant KSHV genome (M. M. Brinkmann et al., unpublished data). A 21 kDa isoform, much smaller than the expected molecular weight of most TMPs, has been seen in some PEL cell lines and could represent a proteolytic cleavage fragment (Sharp et al., 2002). Immunoreactive K15 protein has been detected in a small number of B cells in MCD biopsies using a monoclonal antibody to K15, in keeping with the predicted lytic expression pattern of K15 (Sharp et al., 2002).

“Kaposin” locus

The region between nt 117,432 and nt 118,758 of the prototype KSHV sequence was originally predicted to contain an open reading frame, ORFK12, defined by a start ATG at position 117 919 and a stop codon at position 118 101 and expected to encode a small hydrophobic protein of 60 aa (Russo et al., 1996; see Fig. 28.8). Independently, an 0.7 kb mRNA (T0.7) was cloned from a pulmonary KS sample and found to be latently expressed in KS biopsies and PEL cell lines by in situ hybridization (Zhong et al., 1996; Staskus et al., 1997; Stürzl et al., 1997). By Northern blot, an mRNA originating in this region was also strongly expressed in uncultured PEL cells (Li et al., 2002). K12/T0.7 has since been regarded as a marker of latently infected cells. However, in PEL cell lines, K12/T0.7 mRNA expression is induced by treatment with TPA or butyrate, with one group classifying it as a class Ⅱ gene in view of its detectable basic expression in the BC-1 cell line in the absence of chemical treatment (Sarid et al., 1998), while another classified it as an early gene because of the cycloheximide-sensitive induction of T0.7 mRNA 13 hours following Na-butyrate treatment of the same PEL cell line (Sun et al., 1999). In more recent gene array experiments K12/T0.7 was classified as a latent/lytic gene (Jenner et al., 2001) or as a lytic gene with relatively late doubling of expression (Paulose-Murphy et al., 2001; Nakamura et al., 2003). The contrast between the in vivo gene expression pattern (constitutive) and that in cultured PEL cell lines (inducible) suggests that the regulation of gene expression in PEL cells may have been affected by in vitro culture.

Although the term T0.7/Kaposin is now often used as a synonym for K12-derived transcripts, recent work has shown that in most PEL cell lines, in a primary PEL sample and in KS biopsies a larger transcript of varying size (approx. 1.4–2.4 kb, depending on the sample studied) predominates (Sadler et al., 1999; Li et al., 2002). The varying size of these transcripts is explained by the variable number of repeat units in two groups of repeats, DR1 and DR2 (Sadler et al., 1999; Li et al., 2002). This genomic arrangement has been seen in tumor samples or PEL lines of subtype A (see Fig. 28.8; Poole et al., 1999). In addition to the variable length of the DR1 and DR2 repeats, additional repeat elements, Ⅰ and Ⅱa-c, have been described in a primary PEL sample of subtype B (Li et al., 2002). However, the assignation of genomic subtypes in the K12 region is based on the sequence of the original K12 open reading frame (Fig. 28.8) and no extensive analysis of the genomic arrangement in the upstream repeat region in different KSHV subtypes has been carried out.

The larger mRNA encoded in this region originates at position 118,758 according to one report (Sadler et al., 1999), or at position 123,842 between ORFs 72 (vCYC) and 73 (LANA1) and involving a splice event between nt 118,779 and 123,595 (Li et al., 2002; see Fig. 28.8). The latter report found evidence for conservation of this splice event in all examined PEL cell lines and showed that the region between ORFs 72 and 73 contains a constitutive promoter element between nt 123,842 and 124,242, i.e., overlapping with ORF73 (LANA1) (Li et al., 2002). The existence of this promoter has recently been confirmed by two other groups, although several start sites for the latent transcript originating at this promoter have been identified around 123751/60 (Pearce et al., 2005; Cai et al., 2006; see Fig. 28.8).

This latent mRNA uses alternative start codons (CUG, GUG) to translate several proteins in different frames whose sequence is derived from the repeats upstream of the original K12 (Sadler et al., 1999; see Fig. 28.8). In frame 1 the original ORFK12 is also translated from a conventional ATG start codon, giving rise to the small (60 aa) hydrophobic membrane associated protein “kaposin A.” Being thus located at the 3′ end of a bicistronic mRNA it is not clear at present whether its translation involves an internal ribosomal entry site, the inefficient use of the upstream alternative CUG start codon, ribosomal scanning, or a separate smaller mRNA. However, evidence for its expression in PEL cell lines has been presented (Kliche et al., 2001).

A CUG start codon (nt 118 679) in frame 2 directs the expression of “kaposin B”, a 48 kDa protein; evidence for its expression comes from transfection studies with an expression vector that contained an epitope tag in frame with, and downstream of, the predicted “kaposin B” sequence (Sadler et al., 1999). “kaposin B” was the predominant protein expressed from the repeat region upstream of K12 (Sadler et al., 1999) and also detected in a PEL cell line (Kliche et al., 2001). However, the CUG start codon used by “kaposin B” was absent from the subtype B PEL sample investigated by Li et al. (2002). Not all KSHV subtypes may therefore express “kaposin B.” A further alternative start codon (CUG) in frame 1 could be used to translate a third protein, “kaposin C”; however, expression of this protein appeared inefficient in the studies by Sadler et al. (1999) and Kliche et al. (2001). It is conceivable that it predominates in other KSHV subtypes (Li et al., 2002), but no direct evidence for this currently exists.

As a consequence of the 23 bp repeat elements that constitute the DR1 and DR2 region the reading frame with regard to each individual element shifts in each consecutive element. The resulting proteins translated from the three different frames therefore share a repetitive 23 aa sequence motif (PGTWCPPPREPGALLPGNLVPSS for DR1; HPRNPARRTPGTRRGAPQEPGAA for DR2) and a monoclonal antibody to the DR1 motif will react with proteins translated in all three reading frames (Sadler et al., 1999). This antibody detects DR1 containing proteins in a subpopulation of latently infected KS spindle cells, underscoring the expression of at least some DR1-derived proteins in vivo (Sadler et al., 1999).

Viral microRNAs

In 2005, several groups independently identified a cluster of microRNAs (miRNAs) in the KSHV genome. Of 11 currently known miRNAs, 10 are encoded between the “Kaposin” locus and ORF71/K13 (miR K1-9,11), while a single miRNA has been identified in ORFK12 (Fig. 28.8). All KSHV miRNAs are derived from latent mRNAs directed by either the latency promoter in the 3′ end of ORF73/LANA1 or the major latency promoter upstream of ORF73/LANA1 (position 127,935–127,968; see Figs. 28.4 and 28.8). While miR K10 is located in an exon present in all spliced forms of these latent mRNAs the miR K1-9, 11 cluster is located in an intron present in the corresponding pre-mRNAs (Fig. 28.8; Cai et al., 2005; Pfeffer et al., 2005; Samols et al., 2005; Grundhoff et al., 2006). This ensures the expression of these miRNAs during latency, suggesting a role in the regulation of latent persistence. Potential viral and cellular mRNAs that could be targeted by these viral miRNAs are currently being explored.

Other spliced genes in the KSHV genome

ORF4 (KSHV complement control protein-KCP)

ORF4 encompasses nucleotides 1,142 through 1,794 and shares homology with cellular genes encoding proteins referred to as regulators of complement activation. Northern blot analysis and RT-PCR studies in PEL-derived cell lines demonstrate at least two alternatively spliced co-terminal transcripts in addition to an unspliced, full length mRNA of 1,679 bp (Fig. 28.9) which are induced by TPA treatment (Spiller et al., 2003). These three transcripts encode proteins which retain C-terminal transmembrane domains and four N-terminal complement control protein (CCP) domains required for membrane attachment and complement regulation respectively. Analysis of complement regulation by soluble and membrane associated KCP demonstrated its ability to inhibit C3b deposition on cell surface and to act as a cofactor for factor I-mediated inactivation of complement proteins C3b and C4b, subunits of classical C3 convertase (Mullick et al., 2003; Spiller et al., 2003).

Fig. 28.9. Splicing patterns of ORF4 transcripts.

Fig. 28.9

Splicing patterns of ORF4 transcripts. Two of several alternatively spliced transcripts co-terminal with an unspliced full length transcript of ORF4, all inducible with TPA.

ORF40/41 (PAF)

ORF40 and ORF41 are both located on a spliced 2.2 kb mRNA that removes the region between these two ORFs and generates a long joint reading frame, ORF40/41, and has been shown to encode a protein of 75 kDa. Based on sequence homology, the 75 kDa protein is likely to be a primase-associated factor (PAF) (AuCoin and Pari, 2002; Wu et al., 2001). The joint ORF40/41 transcript starts at position 60, 226, eliminates the genomic region between nt 61,658 and 61,784 and uses a polyadenylation site at position 62,546 (AuCoin and Pari, 2002; Wu et al., 2001). A region 5′ to the start of this transcript has been found to contain a strong promoter activity when inserted into a luciferase vector (AuCoin and Pari, 2002). In addition, a second transcript of 0.7 kb initiates at nt 61,871 and thus only contains a part of ORF41; the corresponding protein is predicted to be translated from an ATG at position 61,908, within ORF41 (AuCoin and Pari, 2002). A 439 bp fragment upstream of the start site for this mRNA (nt 61,372–61,811) also has strong promoter activity (AuCoin and Pari, 2002). However, the existence of a protein translated from this mRNA has not yet been demonstrated. ORF57: The originally assigned ORF57 is located between nt 82,717 and 83,541 and was predicted to encode a homologue of an early herpesviral gene widely conserved among different herpesviruses, e.g., herpesvirus saimiri (HVS) ORF57, herpes simplex virus (HSV) ICP27 and Epstein–Barr Virus (EBV) BMLF1 (Russo et al., 1996). For many of these homologues a role in RNA processing and nuclear export of unspliced mRNAs has been shown (detailed literature in Bello et al., 1999). Subsequent RT-PCR studies, prompted by the smaller than expected size of KSHV ORF57, showed that the ORF57 mRNA is spliced and thus encodes a larger reading frame, of which the originally assigned ORF57 represents the c-terminal end. This splice removes an intron (nt 82,118–82,225) and with it an in frame stop codon upstream of the original ORF57. The spliced ORF57 mRNA initiates at nt 82,003, contains several in frame translational start codons in its first exon, uses the stop codon of the original ORF57 at 83,544 and a polyadenylation site at nt 83,608 (Bello et al., 1999; Kirshner et al., 2000). ORF57 is an early lytic gene whose expression becomes detectable on northern blots 2–4 hours after TPA induction of BCBL-1 cells, i.e., slightly later than ORF50/RTA and at about the same time as ORFK8/KbZIP, but before other early lytic genes (Lukac et al., 1999). One of the more recent gene array studies classified it as a primary lytic gene (Jenner et al., 2001), while another found a doubling of expression at 8h and a peak of expression at 72 h (Paulose-Murphy et al., 2001). The ORF57 promoter is activated by RTA (Lukac et al., 1999; Wang et al., 2003a, b, c), placing ORF57 expression immediately downstream of the expression of RTA.

The ORF57 protein (SSM or MTA) enhances the expression of the bicistronic ORF59/ORF58 mRNA (as well as that of the ORF59/PFA protein), of the untranslated nuclear T1.1/PAN, RNA, and, in the presence of ORF50, RTA, of luciferase reporters driven by the nut-1 and kaposin promoters (Kirshner et al., 1999). ORF57/MTA does not activate these promoters on its own, but enhances their ORF50/RTA-mediated activation. These findings suggest that ORF57 acts at a post-transcriptional level, but does so in a promoter-specific manner (Kirshner et al., 2000).

ORFK3

The proteins encoded by ORFs K3 and K5 downmodulate major histocompatibility class Ⅰ (MHC-I) proteins, NK receptors and coactivation molecules, thereby allowing KSHV-infected cells to escape both cytotoxic T-cell (CTL) and natural killer (NK) cell responses (Coscoy and Ganem, 2000; Haque et al., 2000; Ishido et al., 2000). Viral transcripts containing ORFK3 include three early (cycloheximide-sensitive) transcripts that also cover the neighboring ORF70 (TS). As shown in Fig. 28.10, one of these, an unspliced bicistronic mRNA, includes the entire ORF70 gene with the ORF70 translational start codon and presumably serves to translate the viral thymidylate synthase (Rimessi et al., 2001). Another unspliced mRNA initiates downstream of the ORF70 start codon and could therefore translate a shortened ORF70 protein, or represent a monocistronic mRNA from which only the ORFK3 protein (MIR1, ZMP-B) could be translated. The third early mRNA splices out most of ORF70 and, although it does retain the ORF70 start codon and therefore represents a bicistronic transcript, could again serve to translate the ORFK3 protein. In addition to these early transcripts, an immediate-early, doubly spliced transcript that lacks most of the ORF70 coding region (but does retain the ORF70 ATG and is therefore bicistronic) could serve to translate the ORFK3 protein (Rimessi et al., 2001).

Fig. 28.10. Splicing patterns in the ORFK3-ORF70 region of the genome.

Fig. 28.10

Splicing patterns in the ORFK3-ORF70 region of the genome. Four transcripts are expressed during lytic induction of virus while a fifth transcript is detected at low levels during latent infection in PEL cell lines.

Finally, a low abundance 2.5 kb latent transcript was recently identified by Taylor et al. (2005) which is coterminal with and encodes K3 in its entirety. When mapped, this transcript shows a complicated splicing pattern and encodes the entire K3 open reading frame (nt 18 585–19 671) along with 208 base pairs of the amino terminus of ORF70 (nt 20,096–20,304) and 70 base pairs of a region more than 3kb upstream (nt 23,770–23,840 (Fig. 28.10). Although low levels of ORFK3 protein expression has been documented in KS biopsies in vivo (Rimessi et al., 2001), it is not yet clear which of these mRNAs is most efficiently translated to yield the ORFK3 protein and whether the novel 2.5kb latent transcript also plays a role in down-regulation of MHC class Ⅰ during latent infection.

Events leading to the activation of immediate–early and early KSHV genes

In the experimental model commonly used to study the early stages of the lytic cycle, i.e., the induction of PEL cells by TPA or Na-butyrate, the immediate–early ORF50 gene, encoding RTA, is the earliest viral gene to be expressed. The ORF50/RTA promoter contains several transcription factorbinding sites, including AP-1, Sp1, Oct 1, CEBP/alpha (Deng et al., 2000; Sakakibara et al., 2001; Wang et al., 2003a,b,c). RTA activates its own promoter through two of three CEBP/alpha binding sites, most – likely by physically associating with CEBP/alpha (Wang et al., 2003a,b). RTA also increases the expression of CEBP/alpha, thereby generating an amplification loop that leads to the expression of not only the immediate–early ORF50/RTA gene, but also of other early genes (see below).

In persistently infected PEL cell lines the ORF50/RTA promoter appears to be methylated in a region (–315 to –255 of the transcriptional start site) located between the two CEBP/alpha sites that are important for RTA-mediated activation (Chen et al., 2001; Wang et al., 2003a,b). In KSHV-infected B-cells in vivo this regions also appears to be heavily methylated, whereas in PEL biopsies, MCD lesions and in KS tumors in vivo, the methylation pattern is lighter, with an inverse correlation between the expression of lytic viral proteins and the methylation status of the ORF50 promoter in individual samples (Chen et al., 2001). Treatment of persistently infected PEL cell lines with TPA or 5-azacytidine reduces the number of methylated CpG residues in the ORF50 promoter and activates the lytic cycle (Chen et al., 2001). It is therefore, conceivable that methylation of the ORF50 promoter during viral persistence regulates, at least in part, the spontaneous activation of the lytic cycle in persistently infected cells. However, given the presence of transcription factor sites that are targeted by components of signaling pathways known to be induced by TPA or Na-butyrate (e.g., AP-1) these could also contribute to the activation of the lytic cycle.

RTA also activates a number of viral early promoters, including those of ORF, K8 (KbZIP/RAP), nut-1 (T1.1/PAN), ORF57 (MTA), ORFK2 (vIL6), vMIP, ORFK12 (kaposin), ORF74 (vOX2/GPCR). In at least some target promoters, i.e., T1.1/PAN, ORFK12, vIL6, RTA binds to specific DNA sequence elements (type Ⅲ RTA responsive elements; RRE) (Chang et al., 2002; Deng et al., 2002; Song et al., 2002; Wang et al., 2003a,b). In contrast, the promoters for ORF50 (RTA), ORFK8 (KbZIP/RAP), nut-1 (PAN), and ORF57 (MTA) contain CEBP/alpha binding sites and their activation by RTA involves an interaction of RTA with DNA-bound CEBP/alpha (Wang et al., 2003a,b).

In addition to the ability to directly bind DNA of target viral promoters, RTA can also target promoters lacking direct recognition elements by interaction with host DNA-binding factors such a RBP-Jκ. RBP-Jκ belongs to a family of sequence-specific transcriptional repressors which recruits other corepressors to silence gene activation. By binding to RBP-Jκ, it appears that RTA not only displaces associated corepressors, but also allows for ligand independent activation of target genes. Although specific genes including PAN, ORF57, and SSB have been individually shown to be regulated in this manner, the broader implication is that RTA-mediated redirection of RBP-Jκ activity from repression to activation is critical for lytic reactivation (Liang et al., 2002; Liang and Ganem, 2003).

The ORFK8 encoded protein, KbZIP or RAP, related to the EBV lytic-cycle Z transactivator (ZTA), as discussed above, contains a leucine zipper oligomerization domain and may interact with cellular transcription factors like CBP and CEBP/alpha (Wang et al., 2003b). Although it plays a role in the KSHV lytic replication cycle, as shown by its association with PML domains and recruitment into lytic replication compartments (Wu et al., 2001), it is, unlike EBV ZTA, not sufficient to trigger the activation of the lytic cycle and does not directly bind to viral DNA (Polson et al., 2001; Chiou et al., 2002). There is, however, evidence for its indirect association with the promoters for RTA, ORF57/MTA, as well as its own promoter, most likely as a consequence of its ability to associate with CEBP/alpha (Wang et al., 2003b).

Regulation of the lytic cycle by other viral proteins

In addition to the regulatory role of RTA, KbZIP/RAP and ORF57/MTA during the immediate–early and early phase of the lytic cycle several other KSHV proteins may have the role of modulating lytic replication. Thus the membrane-associated glycoprotein encoded by ORFK1 has been found to inhibit the TPA-induced activation of the lytic cycle in the BCBL-1 PEL cell line (Lee et al., 2002). A detailed analysis of the viral gene expression pattern in this cell line following the overexpression of K1 and treatment with TPA showed that the majority of viral genes appears to be downregulated as the result of K1 overexpression; however, a small number, including ORF72/vCYC, K15/TMP, ORF48 and K1 itself appeared to be upregulated. In contrast, K1 does not appear to affect the ORF50/RTA mediated activation of the lytic cycle, suggesting that TPA-induced events upstream of the ORF50/RTA promoter are modulated by K1. Among these the TPA-mediated activation of AP-1, NF-κB and Oct-1 in this PEL cell line appears to be inhibited by overexpression of K1 (Lee et al., 2002). Lagunoff et al. (2001) also reported that K1 may moderately augment the activation of the lytic replication cycle in a PEL cell line but did observe a dominant negative effect of K1 signaling defective mutants on the ORF50/RTA-mediated activation of the lytic replication cycle in the same PEL cell line (BCBL-1).

Other factors that increase lytic viral replication

Several clinical observations suggest that reactivation of KSHV could be mediated by environmental factors or injury. Thus the frequent localization of classic KS lesions on the feet has been linked to an exposure to volcanic soil (Ziegler, 1993) or to reduced blood flow and poor oxygenation of the lower extremities in elderly individuals. Experimentally, hypoxia has been shown to activate KSHV lytic replication in PEL cell lines (Davis et al., 2001). Haque et al. (2003) reported the presence of functional hypoxia response elements in the ORF50/RTA and ORF34 promoter. These elements are activated by either HIF-2 alpha (ORF50/RTA promoter) or by both HIF-1alpha and HIF-2alpha (ORF34 promoter) and hypoxia induces the transcription of ORF34 and ORF50/RTA mRNAs (Haque et al., 2001). Chang et al. (2000) and Zoeteweij et al. (2001) reported that calcium ionophores, such as ionomycin and thapsigargin, could activate KSHV lytic replication cycle in PEL cells and further synergized with the effects of phorbol esters (Chang et al., 2000). That KS lesions can arise in scar tissue or regions of traumatized skin (Köbner phenomenon) is another well-established clinical observation (Sachsenberg-Struder et al., 1999).

In addition, an extensive body of experimental work has suggested some inflammatory cytokines may accelerate the development of KS lesions in AIDS patients (for a review see Ensoli et al., 2001). Thus Monini et al. (1999) showed that inflammatory cytokines, and in particular interferon gamma, can increase the viral load in cultured PBMC of KSHV infected individuals. In a similar experiment Mercader et al. (2000) identified oncostatin M, hepatocyte growth factor/scatter factor and interferon gamma as cytokines that are released from HIV-1 infected T cells and can induce the expression of ORFK12 and ORF26 mRNA, as well as ORF59 and K8.1 proteins, in the BCBL-1 cell line. In vitro examination of KSHV infected cell lines demonstrates that inflammatory cytokines had diverse effects on KSHV induction. While interferon gamma consistently induced lytic activation, Chang et al. (2000) found other cytokines including tumor necrosis factor, IL-1, IL-2, IL-6, granulocyte-macrophage colony stimulating factor, and basic fibroblast growth factor did not. Further, interferon alpha inhibited KSHV induction (Monini et al., 1999; Chang et al., 2000).

Recently, it was shown that KSHV-infected keratinocytes could activate the lytic replication cycle upon epithelial differentiation in raft cultures (Johnson et al., 2005), suggesting that, similar to human papillomavirus, KSHV infected epithelial cells could be programmed to allow the production of new virions at the epithelial surface.

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