Reviews in Medical Virology

REVIEW

Rev. Med. Virol. 2015; 25: 273–285. Published online 20 July 2015 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/rmv.1843

Biology and oncogenicity of the Kaposi sarcoma herpesvirus K1 protein Annie Cristhine Moraes Sousa-Squiavinato1, Renata Nacasaki Silvestre1 and Deilson Elgui De Oliveira1,2* 1

Viral Carcinogenesis and Cancer Biology Research Group (ViriCan) at Botucatu Medical School, São Paulo State University (UNESP), Botucatu, SP, Brazil 2 Biotechnology Institute (IBTEC), São Paulo State University (UNESP), Botucatu, SP, Brazil

S U M M A RY The Kaposi sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, is a gammaherpesvirus etiologically linked to the development of Kaposi sarcoma, primary effusion lymphomas, and multicentric Castleman disease in humans. KSHV is unique among other human herpesviruses because of the elevated number of viral products that mimic human cellular proteins, such as a viral cyclin, a viral G protein-coupled receptor, anti-apoptotic proteins (e.g. v-bcl2 and v-FLIP), viral interferon regulatory factors, and CC chemokine viral homologues. Several KSHV products have oncogenic properties, including the transmembrane K1 glycoprotein. KSHV K1 is encoded in the viral ORFK1, which is the most variable portion of the viral genome, commonly used to discriminate among viral genotypes. The extracellular region of K1 has homology with the light chain of lambda immunoglobulin, and its cytoplasmic region contains an immunoreceptor tyrosine-based activation motif (ITAM). KSHV K1 ITAM activates several intracellular signaling pathways, notably PI3K/AKT. Consequently, K1 expression inhibits proapoptotic proteins and increases the life-span of KSHV-infected cells. Another remarkable effect of K1 activity is the production of inflammatory cytokines and proangiogenic factors, such as vascular endothelial growth factor. KSHV K1 immortalizes primary human endothelial cells and transforms rodent fibroblasts in vitro; moreover, K1 induces tumors in vivo in transgenic mice expressing this viral protein. This review aims to consolidate and discuss the current knowledge on this intriguing KSHV protein, focusing on activities of K1 that can contribute to the pathogenesis of KSHV-associated human cancers. Copyright © 2015 John Wiley & Sons, Ltd. Received: 12 January 2015; Revised: 1 June 2015; Accepted: 2 June 2015 *Correspondence author: D. Elgui de Oliveira, Faculdade de Medicina, Universidade Estadual Paulista (UNESP), Rua Bento Lopes, s/nDistrito de Rubião Jr., Botucatu, SP, CEP 18618-970, Brazil. E-mail: [email protected]

INTRODUCTION

Abbreviations used AIDS-KS, AIDS-associated KS; BCR, B-cell receptor; DISC, death-inducing signaling complex; Erdj3, ER-associated DNAJ protein 3; FasL, Fas ligand; GM-CSF, granulocyte-macrophage colony-stimulating factor; GSK3β, glycogen synthase kinase 3β; Hsp90β, heat shock protein 90β; HVS, herpesvirus saimiri; Igλ, lambda immunoglobulin light chain; ITAM, immunoreceptor tyrosine-based activation motif; KS, Kaposi sarcoma; KSHV, Kaposi sarcoma-associated herpesvirus; LANA, latent nuclear antigen; LMP1, latent membrane protein; MCD, macrophagederived chemokine; MCD, multicentric Castleman disease; MCP-1, monocyte chemotactic protein-1; MMPs, matrix metalloproteinases; MSM, men who have sex with other men; NCBI, National Center for Biotechnology Information; PDGF, platelet-derived growth factor; PDK, phosphoinositide-dependent kinases; PEL, primary effusion lymphoma; PKB/Akt, protein kinase B/AKT; PTEN, phosphatase and tensin homolog protein; RREs, Rta-responsive elements; RTA, replication and transcription activator; SH-PTP, SH2-containing tyrosine phosphatases; STP, saimiri transformation protein; TPA, 12-O-tetradecanoyl phorbol-13acetate; v-Cyc, viral cyclin D; VE-cadherin, vascular endothelial cadherin; VEGF, vascular endothelial growth factor; v-FLIP, viral FLICE-inhibitory protein; vGPCR, viral G protein-coupled receptor.

In 1872, Moritz Kaposi reported five cases of men aged 40 to 68 years old presenting with a rare cutaneous malignant tumor on the feet [1], now recognized as Kaposi sarcoma (KS). KS is a mesenchymal cancer of vascular vessels with prominent inflammatory reaction. Its etiology was elusive until 1994, when the KS-associated herpesvirus (KSHV)—formally human herpesvirus 8—was first detected in KS lesions in patients with AIDS [2]. The viral DNA was consistently found in virtually all KS cases evaluated in the following years, even in individuals without HIV [3]. KSHV infection is also associated with the development of multicentric Castleman disease (MCD) and primary effusion lymphoma (PEL), two rare human lymphoproliferative diseases [4,5]. MCD is

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Kaposi sarcoma

274 a B-cell non-neoplastic disorder that compromises the architecture of lymph nodes. PEL, on the other hand, is characterized by neoplastic B-cell proliferation in body cavities (e.g. pleural, pericardial, and peritoneal spaces). Both diseases occur predominantly in HIV-positive patients, and they typically show systemic symptoms and poor prognosis [6]. KS is the most common cancer associated with KSHV infection, and it has four main forms: classic, endemic, iatrogenic, and AIDS-associated KS (AIDS-KS). Classic KS is the disease described by M. Kaposi: it presents as isolated lesions on the lower limbs of mid-aged to elderly men, notably of Jewish or Mediterranean European origin. Involvement of internal organs and lymph nodes by classic KS is rather uncommon. Endemic KS is typically observed in children living in Central and South Africa; it has an aggressive evolution, involving lymph nodes and viscera. Adults show milder clinical course compared with children. Iatrogenic KS arises in patients receiving immunosuppressive therapy to facilitate organ transplantation, and disease remission can be achieved upon suspension of immunosuppressive drugs. Finally, the AIDS-KS arises in individuals with uncontrolled HIV infection, especially among men who have sex with other men. Untreated disease has a very aggressive behavior, with multiple fast-growing lesions and involvement of skin (Figure 1), mucosa, and internal organs, especially in the gastrointestinal tract and lungs [7]. Early cutaneous KS lesions present as patch and plaques and evolve to nodules, the tumoral stage of the disease. Lesions are brownish to bluish red or purple in color, reflecting the high degree of

Figure 1. Clinical aspect of AIDS-associated Kaposi sarcoma. Young male patient, homosexual, HIV positive, showing multiple violaceous to brownish cutaneous lesions in the trunk (courtesy of Helio A. Miot, MD, PhD)

Copyright © 2015 John Wiley & Sons, Ltd.

A. C. M. Sousa-Squiavinato et al. vascularization in the neoplastic tissue [8]. Cellularity of KS lesions is heterogeneous and varies according to the stage of the lesion. Tumors are mostly formed by neoplastic spindle cells (Figure 2), bland and atypical endothelial cells, extravasated erythrocytes, and mononuclear cells. Most of the spindle cells are KSHV positive and express the vascular marker CD34 and markers for lymphatic endothelial cell LYVE-1, VEGFR-3, and D2-40 [9]. Malignant KS spindle cells induce an inflammatory microenvironment due to secretion of a variety of chemical immune modulators, including IFNγ, TNFs, IL-6, and IL-1; granulocyte-macrophage colony-stimulating factor (GM-CSF); proangiogenic molecules, such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor; and chemokines, such as monocyte chemotactic protein1 and IL-8 [10]. These molecules participate in a complex network of cell signaling processes, promoting the development and progression of KS [8].

Kaposi sarcoma-associated herpesvirus KSHV infection is required but insufficient for KS development, and the disease only arises under some degree of immune dysfunction. HIV-1 infection plays a pivotal role for tumorigenesis in AIDS-KS cases. Besides the HIV-1-induced immunosuppresion, it is reported that the retroviral proteins tat and nef induce expression of KSHV products that contribute to the development of KS lesions [11–13]. KSHV belongs to the order Herpesvirales, family Herpesviridae, subfamily Gammaherpesvirinae, and

Figure 2. Histopathology of Kaposi sarcoma tumors: the lesions are predominantly composed by neoplastic spindle cells (S) and anomalous endothelial cells and blood vessels (BV) (ViriCan’s Kaposi sarcoma research collection)

Rev. Med. Virol. 2015; 25: 273–285. DOI: 10.1002/rmv

Biology and oncogenicity of KSHV K1 protein genus Rhadinovirus. Another human gammaherpesvirus, EBV, is well known for establishing latency in lymphocytes and causes proliferative diseases in both natural and experimental hosts, including cancer [14]. The KSHV virion is spherical, and it has linear double-stranded DNA surrounded by a capsid with 162 capsomers immersed in an amorphous tegument. It is enclosed by an envelope containing glycoproteins required for viral attachment to the cell surface. The viral genome has approximately 160 kpb and more than 90 ORFs. Of note, KSHV encodes products with high homology with human proteins, including a viral Dtype cyclin (v-Cyc; ORF-72), a protein similar to the anti-apoptotic bcl-2 (vBcl-2; ORF-16), a viral interleukin 6 (vIL-6; ORF-K2), and several viral interferon regulatory factors (vIRFs, encoded in ORFs K9, K11, K11.1, K10.5, K10.6, and ORF K10) [15]. KSHV life cycle consists of latent and lytic phases. During the latent phase, a limited number of viral genes are expressed, and the viral genome is kept as an episome in chromatin of the infected cell. KSHV LANA protein (encoded in ORF-73) binds the viral genome to cellular histones, allowing the episome to be shared between daughter cells upon cell division [16]. LANA also represses p53 transcriptional activity [17], which confers oncogenic potential to KSHV. Other examples of KSHV latent proteins that foster cell transformation are the viral FLICE-inhibitory protein (v-FLIP; ORF-K7) and v-Cyc. The former has antiapoptotic properties due to its interaction with Fas [18] and v-FLIP-mediated NF-kB activation [19], while vCyc activates the cell cycle owing to functional homology with the cellular D-type cyclins [20], although it is resistant to cellular CDK inhibitors [21]. While only a small set of KSHV genes are expressed in latency, all genes are expressed during the lytic cycle, and the viral DNA is replicated. The KSHV replication and transcription activator (ORF-50) is essential and sufficient to control the entire viral lytic cycle [22]. Among the lytic viral proteins, it is important to mention vIL-6, which induces hematopoiesis and angiogenesis, as well as local VEGF accumulation [23]. The lytic phase of KSHV life cycle is also characterized by the expression of viral proteins relevant for immune evasion, such as viral CC chemokine homologues (vCCL-1, vCCL-2, and vCCL-3 encoded by ORFs K6, K4, and K4, respectively) [15], and vIRFs, for instance vIRF-1 (ORF-K9) [24]. Several surface proteins expressed during the lytic phase of KSHV life cycle are pivotal for KSHV pathogenesis owing to their role in intracellular Copyright © 2015 John Wiley & Sons, Ltd.

275 signaling. For instance, the viral G protein-coupled receptor (vGPCR; ORF-74) resembles a constitutively active IL-8 receptor [25], and it can induce cell transformation and tumorigenesis in nude mice [26]. Likewise, the KSHV K1 protein has important oncogenic properties, which will be outlined and discussed further. KSHV K1 PROTEIN

General features KSHV K1 is a transmembrane glycoprotein of Mr 46 000 containing 289 amino acids encoded by the viral ORF-K1. It is structurally divided into a peptide sequence signal at the N-terminal region, an extracellular domain, a transmembrane domain, and a short cytoplasmic tail at the C-terminal region [27]. Its extracellular portion is similar to the lambda immunoglobulin light chain (Igλ) and has approximately 79 glycosylated amino acids [28]. K1 is structurally and functionally similar to the B-cell receptor (BCR), and it impacts the activation of B lymphocytes initiating several signaling pathways and intracellular calcium mobilization [28–30]. As shown in Figure 3, the cytoplasmic region of K1 contains an immunoreceptor tyrosine-based activation motif (ITAM), similar to immunoglobulins α and β [28,30]. ITAMs contain a peptide sequence of approximately 26 amino acids, in which six conserved residues are precisely spaced, (D/E)X7x(D/ E)X2xYX2xLX7xYX2x(L/I), where X is any amino acid. ITAMs are critical for transducing intracellular signals after receptor–ligand binding events, which may culminate in cell differentiation, proliferation, or even cell death [31]. Intact KSHV K1 ITAM is activated upon protein multimerization, leading to phosphorylation of its tyrosine residues by src kinases. The ITAM activation motifs with phosphorylated tyrosine (Y1Y2SL… Y3TQP) recruit specific syk kinases (e.g. lyn, syk, p85α, PLCγ2, RasGap, vav SH-PTP1/2, and GRB2) by means of their Src homology 2 (SH2) domains. The interaction of K1 with syk and PLCγ2 requires phosphorylation of the ITAM at tyrosine Y1 and Y3; on the other hand, phosphorylation of Y1 or Y3 is sufficient for recruitment of lyn (Y3 mainly), p85 (particularly Y1), and GRB2 SH2-PTP1/2. The Y2 residue does not play a direct role on K1 signal transduction, although it presumably regulates the binding stability between phosphorylated ITAM and cellular SH2 kinases that interact with K1 [32]. Rev. Med. Virol. 2015; 25: 273–285. DOI: 10.1002/rmv

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Figure 3. Overview on KSHV K1 protein structure and intercellular signaling. KSHV K1 is a transmembrane protein that has a cytoplasmic ITAM domain in this C-terminal region and has the potential to multimerize. After activation by src kinases, K1 ITAM recruits syk kinases 2+ such as lyn, syk, and PLCγ2, to cause Ca mobilization, activation of the NF-AT transcription factor and several intracellular cell signaling pathways, AKT/PI3K, but also NFκB, Wnt, MAPKs, and mTOR. The net effect of these signaling events is cell activation, inhibition of apoptosis, cell proliferation, and secretion of inflammatory cytokines and angiogenesis stimulation (for instance, due to VEGF expression mediated by lyn kinase and NF-κB activation)

While tyrosine kinase proteins have a central role at the beginning of signal transduction, to some extent, the negative regulation of some key receptors relies on tyrosine phosphatase proteins. For instance, the negative regulation of BCR activation in B lymphocytes requires SH2-containing tyrosine phosphatases (SH-PTP), enzymes that catalyze tyrosine dephosphorylation, halting the intracellular signal [33]. The interaction between K1 and SHPTP1/2 suggests that either KSHV K1 impairs the SH-PTP1/2 activity or the SH-PTP1/2 modifies the signaling events once initiated by K1; consequently, intercellular signaling may become either continuous or unstable, respectively [32]. Multimerization of cysteine residues in the extracellular domain of K1 possibly turns its ITAM constitutionally active [30]. This is in contrast with the ITAMs within surface receptors of T and B Copyright © 2015 John Wiley & Sons, Ltd.

lymphocytes, which require ligand–receptor interaction for activation [31]. Hence, depending on its expression context (e.g. ectopic expression versus KSHV infection), K1 is prone to trigger uninterrupted intracellular signaling. In BJAB cells (KSHV-negative) expressing the CD8/K1 chimera, intracellular calcium mobilization is observed upon stimulation with anti-CD8 antibody [28], and K1 expression in BJAB cells was also reported to induce NF-AT [30]. Nevertheless, these effects were not significant in BCBL-1 cells (PEL-derived B-cell line constitutionally infected with KSHV) [29].

KSHV K1 variability The ORF-K1 is located within a GC-rich region on the left side of the KSHV genome. Its genomic site is equivalent to the position of the latent membrane protein in the EBV genome [34], the saimiri Rev. Med. Virol. 2015; 25: 273–285. DOI: 10.1002/rmv

Biology and oncogenicity of KSHV K1 protein transformation protein (STP) position in the genome of herpesvirus saimiri [35], and the position of the R1 gene in the genome of rhesus monkey Rhadinovirus [36]. The KSHV ORF-K1 and its encoded K1 protein differ among viral genotypes by approximately 87% and 62% in nucleotide and amino acid sequences, respectively [37]. Variations are concentrated in the extracellular portion of K1, within two hypervariable regions called VR1 and VR2. Nonetheless, 10–12 cysteine residues are well conserved, of which seven are located predominantly in the activation motifs [37,38]. Genetic analysis of the ORFK1 VR1 and VR2 regions allows the discrimination of several viral genotypes, designated A through E (Figure 4). The VR1 and VR2 regions of prototype sequences of viral A and C genotypes extend between codons 54–92 and 199–227, respectively, and between 1–92 and 191–228 for the B genotype. Genotype A differs from B by 39 amino acids (15%), and from C by 85 amino acids (29%) [37]. The high variability of K1 can be noted in the alignment of amino acid sequences of protein prototypes available at the USA’s National Center for Biotechnology Information (NCBI), as shown in Figure 5. The distribution of KSHV genotypes in humans varies according to geographic region and ethnicity. Overall, isolates of A genotype are found mainly in North America; genotype B in Africa; genotype C in Europe, Asia, and the Mediterranean; and genotype D in the Pacific Islands [37,39]. There are also a few more restricted viral genotypes, such as genotype E, identified in South American Amerindians [40], and genotype F, detected in biological samples from individuals in Bantu tribes in Uganda [41]. Figure 4 shows the phylogenetic topology based on K1 sequences from different KSHV genotypes. The transmembrane domain of K1 is well conserved among genotypes A, B, and C, extending between ORF-K1 nucleotides 229–261 [37]. The cytoplasmic domain is conserved in genotypes A and C (3% change) but differs 22–30% in the amino acid sequence between viral genotypes A and B [37,38]. Although the extracellular portion of K1 is highly variable, the ITAM SH2 binding site is usually preserved. The patterns of amino acid substitution in the K1 ITAM diverge among KSHV isolates of genotypes A or C compared with genotype B. In fact, it was suggested that viral K1 from A and C genotypes evolved distinctly from the B Copyright © 2015 John Wiley & Sons, Ltd.

277 genotype regarding their interference in intracellular signaling pathways [42]. The pattern of nucleotide and amino acid substitutions in different forms of K1 suggests a positive Darwinian selection on VR1 and VR2 [42,43]. The positive selective pressure in VR1 was reported to be as high as in immunogenic regions of wellstudied viral genes, such as HIV-1 env. The analysis of K1 autologous peptides (unique to each individual viral isolate) showed epitopes that induce T cytotoxic response exclusively derived from the VR1 region of K1, with no response to peptides from other individuals. Of note, the analyzed epitopes were derived only from conserved sequences between isolates of the same viral genotype. The maintenance of antigenicity in isolates of the same KSHV genotype may partially explain their specific ethnic and geographical distribution. To some extent, the selection of K1 epitopes is mediated by antigen presentation in the MHC class I context. Conversely, to date, there are no data on putative factors for positive selection in other portions of the K1 protein [43]. Recently, the analysis of K1 nucleotide variation in tissue and PBMC samples of KSHV-positive patients with and without KS from Italy revealed clustering of viral isolates in two major clades, corresponding to the A and C genotypes. Nonsynonymous nucleotide mutations in the K1 protein-coding sequence indicated 12 positively selected sites. One similar amino acid sequence was identified in 4/12 (33.3%) KSHV-positive patients without KS and none of the samples from patients with KS [44]. These data strengthen the idea that the K1 protein might play a role in KS pathogenesis, as detailed further in this review.

Regulation of K1 in cells infected with KSHV The expression of KSHV K1 has been reported in cells derived from PEL [45], MCD [45], and KS [34,46]. This viral protein is significantly expressed in the lytic phase of the viral cycle and can be induced in vitro in PEL cells with treatment with 12-O-tetradecanoyl phorbol-13-acetate (TPA) [46]. In contrast, low levels of K1 are detected in cells latently infected by KSHV [47,48]. In HEK293 cells, the expression of K1 peaks at 24 h after infection with BCBL-1-derived virions, declining sharply after 72 h, when the majority of infected cells are in the latent phase of the viral cycle [49]. Rev. Med. Virol. 2015; 25: 273–285. DOI: 10.1002/rmv

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Figure 4. Phylogenetic analysis based on nucleic acid sequences from prototype KSHV genotypes A, B, C, D, and E. Mega 5.2 Software [79] was used, with the maximum likelihood algorithm and bootstrap validation of the phylogenetic tree (500 replicates). In parentheses, the NCBI Genbank accession number for the sequence is provided. Viral genotypes are indicated on the right

The Rta protein, major viral regulator for the transition between lytic and latent phases, modifies the expression of KSHV genes either by direct binding to Rta-responsive elements (RREs) or indirectly [50,51]. Rta-mediated induction of K1 relies on three binding sites within the ORF-K1 promoter, but only two seem to play a role in protein expression [52]. Rta activates the K1 promoter in B lymphocytes and epithelial cells [49,53]; the effect is less significant in endothelial cells, probably owing to unique features of the transcriptional machinery for this cell type [53]. Expression of K1 in B lymphocytes may modestly increase Rta-mediated lytic reactivation. BCBL-1 cells transfected with vectors encoding KSHV Rta along with a dominant negative version of K1 showed a small reduction of lytic reactivation (75% to 80%) compared with cells expressing Rta only. Furthermore, the suppressive effect of the K1 dominant negative in viral lytic reactivation is abolished when Rta is replaced by TPA treatment [54]. In contrast, BCBL-1 cells expressing the CD8 surface receptor fused to the cytoplasmic portion of K1 (CD8-K1) efficiently suppress TPA-mediated viral reactivation, thus suppressing expression of the K8.1 lytic protein and decreasing transcriptional activation mediated by AP-1, NF-kB, and Oct-1 [29]. These studies are based on the same cell model, so that the discrepancy in results possibly is due to methodological issues. Copyright © 2015 John Wiley & Sons, Ltd.

Treatment with TPA exerts various effects on intracellular signaling, activating several transcriptional pathways that culminate in KSHV lytic reactivation; this does not seem to be the case for the cytoplasmic region K1, which affect a more restricted network of intracellular pathways. In KSHV latently infected cells, K1 expression is downregulated by binding of the C-terminal region LANA to the terminal repeat promoter of ORF-K1 [49]. This is in agreement with the effect of K1 as a mild inducer of lytic cycle, as LANA activity maintains latency partially by reducing K1-induced signaling. Not only does K1 regulate and is itself regulated by viral products, but endogenous proteins of the infected cell also regulate K1. For instance, K1 is inhibited by the heat shock protein 90β (Hsp90β) and ER-associated DNAJ protein 3 (Erdj3), which interact with the Nterminal domain of the protein expressed in BJAB, BCBL-1, and HEK293 cells. It is suggested that Hsp90β chaperone associates with K1 during its synthesis de novo, when the peptide moves through the cytoplasm and the endoplasmic reticulum, while Erdj3 possibly acts in the assembly and folding of newly synthesized proteins into the endoplasmic reticulum [55].

Biological effects of K1 Cell membrane effects. Although KSHV K1 is a surface transmembrane protein, it can also be found in early Rev. Med. Virol. 2015; 25: 273–285. DOI: 10.1002/rmv

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Figure 5. Alignment of amino acid sequences for KSHV K1 from viral genotypes A, B, C, D, and E based on sequences deposited in the NCBI Genbank database. In parentheses, the accession number for each sequence is given, followed by the identification of the viral genotype. Regions corresponding to VR1/VR2 and ITAM are indicated

and late endosomes owing to clathrin-mediated K1 internalization. Within endosomes, K1 triggers signal transduction continuously and activates the PKB/Akt pathway. K1 recruits and activates PI3K, causing its own internalization. Both inhibitions of PI3K activation or K1 internalization can suppress signals triggered by this viral protein and its endocytosis. Of note, mutations in K1 ITAM decrease the rates of protein internalization; thus, the signaling events triggered by ITAM play a role in K1 endocytosis [56]. K1 may indirectly contribute to disruption of endothelial cell junctions, allowing an increase in vascular permeability and leakage of blood components in KS lesions. The increased vascular permeability associated to KSHV lytic infection may also be linked to degradation of the vascular endothelial cadherin (VE-cadherin), a putative effect of either Copyright © 2015 John Wiley & Sons, Ltd.

KSHV vGPCR [57] or K15 [58]. Disruption of cell junctions can also be observed in latently infected endothelial cells: KSHV-infected HEK293 cells showed upregulation in the activity of Rac1 GTPase, which is involved in regulation of junctional integrity due to phosphorylation of tyrosine residues of the VEcadherin and β-catenin. K1 expressing cells activate Rac1 more efficiently than non-expressing cells; thus, K1 can also contribute indirectly to the increase in vascular permeability [59]. Increased cell survival. Some KSHV products clearly affect apoptosis regulatory proteins. For instance, LANA represses the transcriptional activity of p53 [17], whereas v-FLIP suppresses apoptosis by blocking the assembly of the death-inducing signaling complex (DISC) [60], as well as stimulating NFRev. Med. Virol. 2015; 25: 273–285. DOI: 10.1002/rmv

280 kB activation [61]. K1 may increase the life-span of KSHV-infected cells at least by interaction with receptors involved in apoptosis (Fas and/or BCR) [62–64], or via ITAM-mediated intracellular signaling [48,55,65]. K1 expression suppresses apoptosis of BJAB, THP-1, U937, and SLK cells treated with a Fas agonist antibody or Fas ligand (FasL) [62]. In addition, mice treated with a lethal dose of an agonist antibody to Fas (JO2, which induces widespread apoptosis in hepatocytes) survive the challenge when transfected with K1 [62]. K1 physically interacts with Fas through its Ig-like domain [64], preventing the action of FasL and Fas agonistic antibodies [62]. On the other hand, K1 partially inhibits the formation of DISC and significantly blocks the activity of caspase 8 [62]. K1 promotes survival of B cells hindering BCR signaling, which may be due to internalization of K1/BCR complexes [56]. Another mechanism involves the accumulation of BCR in the endoplasmic reticulum, due to its interaction with the μ-chain of the N-terminal portion of K1 protein, inhibiting the intracellular transport and cell surface expression of BCR [64]. In BJAB cells, K1 is found mainly in the endoplasmic reticulum, as assessed by confocal microscopy [64]. Owing to interference in the activities of BCR, K1 promotes cell survival and fosters the expansion of the repertoire of cells latently infected by KSHV. The PI3K/Akt pathway is recognized for its important role in the negative regulation of apoptosis [66]. Several components of this pathway are deregulated in cancers, including KS [67]. KSHV proteins vGPCR, vIL-6, ORF45, and K1 compromise proper activity of the PI3K/AKT pathway [68]. Signals triggered by K1 ITAM activate PI3K/Akt in both epithelial cells and B lymphocytes [48,56,62]. K1 expression in endothelial cells and B lymphocytes increases the activation of the p85 subunit of PI3K, Akt, and PDK1 and inactivates the negative regulator PTEN, causing PI3K-Akt hyperactivation [32,48,62]. Additionally, cells expressing K1 typically show phosphorylation and consequent inhibition of proteins involved in apoptosis. For instance, the FOXO proapoptotic transcriptional factor FKHR is phosphorylated in BJAB cells, preventing the transcription of FasL; while HUVECs exhibited phosphorylation of bad, FKHR, FKHRL1, GSK3β, and mTOR, negatively impacting their proapoptotic properties due to cytoplasmic retention [48,62]. Copyright © 2015 John Wiley & Sons, Ltd.

A. C. M. Sousa-Squiavinato et al. In summary, K1 induces apoptosis resistance mediated by signaling triggered by the cytoplasmic portion of the protein, which activates Akt, with consequent inhibition of several proapoptotic proteins. Activation of PI3K/Akt and the simultaneous inactivation of PTEN enhances the survival and proliferation of KSHV-infected endothelial cells, promoting viral dissemination, and presumably contributing to the pathogenesis of KSHV-associated diseases. Furthermore, the physical interaction of K1 with the Fas receptor prevents Fas–FasL binding and hinders the apoptotic process. Proangiogenic and proinflammatory properties. The expression of KSHV K1 in B lymphocytes induces cell activation via upregulation of transcription factors such as NF-AT and AP-1, as well as production of proinflammatory cytokines, including macrophage-derived chemokine, IL-1α, IL-1β, IL-8, IL-10, VEGF, and RANTES [30,32]. In KS lesions, cell migration and angiogenesis are stimulated by KSHV-infected cells via expression of IL-6, IL-8, VEGF, and matrix metalloproteinases (MMPs) [69,70]. K1 also has a role in this setting, as it has been reported to increase expression and secretion of MMP-9 and VEGF in epithelial (HEK293) and endothelial (HUVEC) cells transfected with K1 that lacks mutations in ITAM (in the SH2 binding region) [71]. In line with these data, K1-expressing HUVEC and BJAB cells exhibited overactive VEGF/VEGFR signaling and increased secretion of VEGF, respectively [48,72]. Thus, owing to increased expression and secretion of MMP-9 and VEGF, K1 conceivably contributes to angiogenesis and has a role in the growth and proliferation of infected cells in KS lesions in a paracrine fashion [71]. The NF-kB pathway is responsible for a variety of phenomena in inflammation, and its activation is essential for the survival of lymphocytes infected with KSHV. Indeed, it is suggested that NF-kB is hijacked by KSHV to promote viral persistence in vivo in the setting of hostile immune responses [73]. Although there is some debate on the role of K1 in NF-kB activation, some studies report that BC-3, BJAB, and Raji cells transfected with K1 exhibit increased luciferase activity driven by a NFkB-responsive promoter [46,72,74]. This activation seems to be regulated by ITAM and by the lyn kinase, as it is blocked by the specific inhibitor PP2, and NF-kB inhibition is also achieved with ITAM mutation [72]. Splenic B lymphocytes from Rev. Med. Virol. 2015; 25: 273–285. DOI: 10.1002/rmv

Biology and oncogenicity of KSHV K1 protein transgenic mice expressing KSHV K1 showed increased nuclear activity of NF-kB and Oct-2, compared with lymphocytes from non-transgenic mice [74]. Likewise, high NF-κB-driven luciferase activity was shown in KVL-1 cells, from a lymphoma in KSHV K1 transgenic mice [72]. As a result of NF-kB activation, K1-expressing COS-1 cells showed increased levels of IL- 6, IL-8, and IL-12 secretion, and co-expression of KSHV K1 and HIV-1 tat in these cells provided an additive effect on luciferase activity [46]. Conversely, T and B lymphocytes from transgenic mice expressing K1 showed no change in expression of IL-2, IL-4, IL-6, IL-10, GM-CSF, TNF-α, and TNF-β compared with cells from control animals [72]. Interestingly, the expression of K1 in transfected HEK293 cells inhibited, in a dose-dependent fashion, the effect of v-FLIP and ORF-75 protein in the luciferase activity regulated by NF-kB-responsive elements [75]. Hence, further studies are needed to reconcile these results regarding the effects of KSHV K1 on NF-kB signaling.

Role of K1 in carcinogenesis The oncogenic properties of K1 are probably associated with its ability to induce multiple signaling pathways, some pivotal for carcinogenesis. PI3K/Akt activation by K1 conceivably contributes to the immortalization of endothelial and epithelial cells. HUVECs expressing K1 have an increase in the number of culture passages compared with control cells (55 and 32 passages, respectively). After the 32nd passage, control cells stopped growing and exhibited morphological features typical of senescence, while cells expressing K1 continued to grow and showed no morphological changes. In this study, the KSHV K1-expressing HUVECs did not induce tumors when injected into nude mice, and their assumed cell immortalization was not associated with changes in telomerase expression [48]. K1 induces cell transformation in vitro, as rodent fibroblasts expressing K1 displayed both morphologic changes and focus formation compared with negative controls [27]. K1 may also contribute to tumorigenesis in vivo, as marmosets developed lymphomas when inoculated with a plasmid where the HSV STP oncogene was replaced with the KSHV ORF-K1. In addition, immortalized primary T cells can be obtained in vitro from these animals [27]. Copyright © 2015 John Wiley & Sons, Ltd.

281 Another study reported that one out of four BALB/c mice that received intranasal inoculation of a recombinant version of murine herpesvirus 4 encoding KSHV K1 (MHV76-K1) developed salivary gland adenocarcinomas after 120 days [76]. Yet, two out of 13 (15.4%) transgenic mice (C57BL/6J) expressing K1 developed large tumor masses at approximately 14 months of age [74]. One animal developed a submaxilary tumor, supposedly a plasmablastic lymphoma; a second tumor was located in the omentum, and it had suggestive features of sarcomatoid spindle cell neoplasm. The neoplastic cells of plasmablastic lymphoma exhibited higher lyn kinase activity compared with activity in B lymphocytes expressing K1, suggesting that lyn activation was related to a putative role of K1 in lymphomagenesis [74]. The phenotype of transgenic mice expressing K1 has findings similar to KSHV-associated lymphomas in humans, including splenomegaly and abnormal lymphocyte proliferation in response to antigens. Furthermore, high levels of VEGF are found in the KVL-1 cell line, derived from murine tumors expressing K1 [72]. When a lymphoma expressing K1 was subcutaneously injected into nude BALB/c mice, the tumor growth was associated with constitutive activity of NF-kB and VEGF secretion. Additionally, 90% of 12-month-old mice expressing K1 showed lymphoid hyperplasia, and four times more VEGF was produced compared with controls [72]. These results suggest that KSHV K1 may have an impact in the carcinogenic potential of viral infection, notably for lymphomagenesis. However, it must be taken into account that rodent cells are more susceptible to spontaneous transformation and cancer compared with human cells [77]. In time, the acquisition of migratory and invasive traits is the major event for endothelial cells infected by KSHV [78]. K1 increases the angiogenesis in established tumors, and inoculation of C33A epithelial cells expressing K1 into nude mice produced tumors that grew significantly faster than controls. These tumors showed increased Akt phosphorylation, and they have increased expression of the cell proliferation marker Ki67. K1-expressing tumors are highly vascularized probably owing to the activation of VEGF/VEGFR and PI3K pathways [48]. Figure 6 summarizes the data evaluated in the present review regarding the biological and carcinogenic properties of KSHV K1 protein. Rev. Med. Virol. 2015; 25: 273–285. DOI: 10.1002/rmv

282

A. C. M. Sousa-Squiavinato et al.

Figure 6. Concept map produced with the CMapTools Software (Institute for Human and Machine Cognition, Ocala, FL, USA) summarizing data presented in this review. The numbers indicate the studies cited. See text for details

Concluding Remarks

The K1 protein was among the first KSHV products described two decades ago. The available evidence indicates that K1 has a striking effect in intracellular signaling, notably by activation of PI3K/AKT (Figure 3), with effects on cell survival, cell proliferation, and synthesis of inflammatory molecules. This is mainly mediated by the ITAM domain in the intracellular portion of the protein. It should be noted that most of the studies on KSHV K1 activities relies on the expression of this viral protein alone; therefore, the properties of the K1 protein in the actual context of KSHV infection can be substantially modified. Nonetheless, most of the current evidence indicates that the KSHV K1 is capable of fostering a microenvironment favorable to viral dissemination owing to paracrine recruitment of KSHV-permissive inflammatory cells. Furthermore, microenvironmental conditioning by K1 may be pivotal for KSHV-associated cancers, and the small proportion of KSHV-infected cells undergoing spontaneous viral lytic reactivation (neoplastic and non-neoplastic) may play a vital role for Copyright © 2015 John Wiley & Sons, Ltd.

tumor development owing to K1-driven expression of molecules that sustain survival and proliferation of cancer cells. Other possible effects of K1 expression in KSHV latently infected cells are largely unexplored, as it is the eventual biological consequences of the high variability of this protein among viral genotypes. Thus, more substantial effort on KSHV K1 research will be required in the next years to unveil the mysteries of this intriguing viral protein.

CONFLICTS OF INTEREST The authors have no competing interest. ACKNOWLEDGEMENTS The authors are indebted to Hélio Amant Miot, MD, PhD (Dermatology Department, Botucatu School of Medicine at Sao Paulo State University), for providing clinical images of KS lesions for this review. Studies on K1 at the ViriCan laboratories were funded by grants received from DEO from the Sao Paulo Research Foundation (FAPESP Proc. AP 2009/17708-0). Rev. Med. Virol. 2015; 25: 273–285. DOI: 10.1002/rmv

Biology and oncogenicity of KSHV K1 protein

283

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