13.1 Introduction: Kaposi’s sarcoma associated herpes virus (KSHV) is also referred to as human herpes virus 8 (HHV8) was first isolated from the Kaposi’s sarcoma (KS) lesion of patient with AIDS. It is a ds DNA virus classified as member of gamma 2-herpes virus subfamily. Earlier studies had confirmed that KSHV was the etiologic agent of KS. KS is a neoplasm derived from lymphatic endothelial cells infected with KSHV, made up of spindle-shaped cells and inflammatory mononuclear cells. KS is grouped into 4 epidemiological types: classic, endemic, iatrogenic and AIDS-associated. KSHV is also associated with primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD)
13.1.1 Kaposi’s sarcoma (KS): KS is a vascular tumor which is made of interweaving bands of cells, embedded with reticular and collagen fibers, and inflammatory infiltrates of mononuclear cell and plasma cells. The tumor is highly vascular that contain abnormally dense and irregular blood vessel that leak red blood cells into the surrounding tissue thereby giving the tumor the characteristic dark color.
Skin lesions are divided into three stages: patch, plaque, and nodular. The patch stage shows a proliferation of irregular branch blood vessels that maybe grouped around normal looking blood vessels. The patch stage is characterized by the appearance of spindle cells forming bundles in the vascular spaces. There is evidence of extravasation of red blood cells and macrophage while mitoses and nuclear abnormalities are more profound in both the spindle and endothelial cells. Biomarkers associated with KSHV –associated lesions include tissue-specific markers such as CD34, vascular-endothelial cadherin, endothelial leukocytes adhesion molecule type 1, CD4, CD68, CD14 and PECAM. Others are cytokine activate-ECs.
The classical form of KS is usually found in the lower extremities while AIDS-associated KS is mostly involves other parts of the body. The skin of the face, the extremities, torso, and the mucus membrane of the oral cavity are mostly affected. Studies reported of gastrointestinal tract involvement among 40% of patients with AIDS-associated KS at the time of initial diagnosis and 80% at autopsy. KS may also involve the lung parenchyma, bronchial tree, and pleural surface.
13.1.2 Primary Effusion Lymphoma (PEL): PEL is also called body-cavity-based lymphoma, a rare lymphoma found in HIV-infected patients. It is a unique form of NHL and derived from clonally expanded malignant B cells and present as a lymphomatous effusion tumor containing various body cavities such as the pericardium, peritoneum, and pleurum. Others have also reported PEL as a solid mass in the lymph nodes and other regions. PEL is aggressive and can rapidly progress, and cause high fatality; with mean survival time for patients with PEL about 2-6 months. Biomarkers associated with PEL include CD45, activation-associated antigens, clonal immunoglobulin rearrangement, CD138/ syndecan-1. Other biomarkers are the expression of viral proteins LANA-1, vCyclin, vFLIP, kaposin, and LANA-2. KSHV is detected as either monoclonal or oligoclonal episome in PEL samples. KSHV genome is found in PEL in high copy number; about 50-150 viral genome per infected cells and can be used as diagnostic criteria for this lymphoma.
13.1.3 Multicentric Castleman’s Disease (MCD): MCD is a localized lymphoproliferative condition which is characterized by expansion of germinal centers with B-cell and vascular proliferations. There are different types of MCDs with the plasmablastic variant form more commonly seen in AIDS patients and transplant recipients. The plasmablastic MCD can be aggressive and rapidly progress to high fatality. MCD is frequently but not always associated with KSHV infection. Biomarkers associated with KSHV-associated MCD include dysregulated high levels of IL-6 and VEGF. KSHV is also detectable in almost all HIV-positive MCD cases and about 50% of HIV-negative MCD cases.
KSHV is postulated to be the cause of other diseases due to dysfunction of the immune system; for e.g. a newly characterized KSHV –associated condition abbreviated as KICS (KSHV inflammatory cytokine syndrome) has been reported in patients with HIV and KSHV co-infection, displaying elevated levels of IL-6 production. Additionally, KSHV has been linked to different forms of lymphomas, including Burkitt’s lymphoma, multiple myeloma, germinotropic lymphoproliferative disorder (GLD), malignant skin tumors, angio-sarcomas, angio-immunoblastic lymphoma and primary pulmonary hypertension. Others have also reported of a KSHV/HHV8-associated germinotrophic lymphoproliferative disorder in HIV-seronegative individuals. More studies are needed to analysis causal association between KSHV and other diseases since there are limited evidence to strongly link KSHV to other diseases such as Kikuchi’s diseases, saliva gland tumors, etc.
13.2 KSHV Genome
Analysis of DNA extracts from purified KSHV showed that the full length genome is 165 to 170 kb. Moore et al performed the primary characterization of the genome. The genome of the virus is similar to that of herpesvirus saimiri in that it has a single contiguous region of 140 to 145 kb which contains all the coding regions. Studies have shown permissive and non-permissive tumor cell lines contain KSHV DNA up to 270 kb in size. The genome has repeats of 803 bp in length of which over 85% are guanidine and cytosine (G+C). The genome is surrounded by icosahedral protein capsid, thick tegument, and a lipid bilayer envelope. A mature KSHV contains at least 24 virus-associated proteins; these include 5 capsid proteins, 8 envelope proteins glycoprotein, 6 tegument proteins, and 5 proteins with unidentified location. KSHV gene seems to have circular conformation; however active DNA found in lytic replication is linear. Close to 100 genes/ORF (Figure 1) encoded by 140 kb long unique region (LUR) with 53.5% G+C content have been described. Many of them are conserved in most herpesvirus. The LUR is about 138 to 140.5 kb long and contains all of KSHV ORFs which are designated K1 to K15 depending on their relative location in the KSHV genome normally from left to right. In addition, KSHV contains many genes which originated from the host genome and are homologous of cellular genes. A number of the genes plays significant role in the pathogenesis of KS. Some such as K1 and K15 are involved in signal transduction, cell cycle regulation by vCyclin, inhibition of apoptosis by vFLIP and immune modulation by viral chemokines receptors.
Figure 1: KSHV genome description (Adopted from Russo et al, 1996)
In addition, a total of 12 microRNAs have been reported in KSHV genome. 10 were found in the non-coding region between K12/kaposin and K13/orf71/vFLIP, and 2 were found within K12 ORF. All of them were expressed during the period of latency, with a sub-set of them upregulated during lytic cycle. The viral genes encoded by KSHV are classified into three groups: 1. Herpesvirus-common genes, 2. KSHV-unique genes, and 3. Cellular-homologues genes which may contain group 1 and 2. There are some gaps in our understanding of herpes viruses, further studies are needed to identify genes associated with KSHV pathogenesis.
13.3 KSHV Life Cycle
To initiate its replication (figure 2), the virion particle binds to the host cell surface receptor and penetrates into the host cell cytoplasm via a complex multistep process. KSHV has two different phases in its life cycle. The latent phase is characterized by a circular episome tightly packed as nucleosome and expression of small subset of latent transcripts in the infected cells. The circular episome is chromatinized as a result of its association with cellular histones in order to ensure: 1. Protection of viral DNA ends to escape the host DNA damage response, 2. Stable maintenance, replication and segregation of the viral genome to daughter cells during mitosis, 3. Successful completion of viral life cycle, and 4 regulation of viral gene expression. There is no production of functional viral particles during this phase. OrfK12/Kaposin, orf71/K13/vFLIP, orf72/vCyclin, and orf73/LANA have been detected in the latent phase of all the KSHV-associated cancers. The second phase is the lytic phase which is characterized by the replication of linear viral genomes, and the expression of more than 80 transcripts in a highly planned temporal order of immediate (IE), early, and late groups.
Figure 2: KSHV life cycle (Adopted from Uppal et al, 2015)
The IE gene does not rely on viral protein synthesis but are important for regulating transcriptional cascade. A KSHV-encoded IE gene, Rta, is required and sufficient for initiating the lytic replication cycle to completion while another IE gene, orf45 is essential for the suppression of IFN induction during lytic viral infection or reactivation. The general function of the early and late genes is to facilitate the replication of viral genome, viral assemble and egress.
Although there are some limitation in our understanding of some aspects of viral life cycle, studies indicates that there are key regulatory steps in the life cycle of many viruses that are essential for the establishment and persistence of viral latency. After entering the host cell nucleus, the viral genome must adopt a structure that is similar to the host genome and interact with cellular chromatin. The chromatinzation of viral DNA is influenced by the same epigenetic factors as cellular DNA resulting in the generation of viral epigenome which has essential role in both latency and lytic reactivation of the viral genome. In addition, some studies have shown that when viruses enter and hide in the host nucleus, they co-evolve with numerous cellular chromatin modulation mechanisms which ensure their survival and propagation.
13.4 Viral genes involved in KSHV-associated Transformation and Oncogenesis
A number of genes are involved in lytic and latent cycle replications which are essential for the long term persistence of the virus. These gene products contribute to KSHV-induced pathogenesis. This section will review some of the genes associated with KSHV oncogenesis.
13.4.1 Latency-associated nuclear antigen (LANA): LANA is a 222-232 kDA nuclear protein that tethers the viral episomal DNA to cellular chromosome through histone HI binding. It is the main latent protein of KSHV which is expressed in all types of KSHV-associated tumors and is the key viral protein associated with viral oncogenesis. LANA is a transcriptional regulator that suppresses KSHV Rta expression resulting in the inhibition of viral lytic replication and maintaining latency. LANA is highly promiscuous in its transforming activities, interacts with many other cellular proteins, and is involved in the disruption of a number of cellular proliferation control mechanisms by: binding to glycogen synthese kinase 3β (GSK-3β), a signal protein in the Wut pathway and negatively regulates β-catenin thereby increasing the levels of β-catenin and activity of downstream transcription factor TCF/LEF. Other reported targets include p53, pRB, AP-1, STAT, and p300.
13.4.2 V- Cyclin: It is a homolog of cellular cyclin D2 which is encoded by ORF72. It is known to activate cellular cyclin-dependent kinase 6 (CDK6) to regulate cellular proliferation and viral replication, promotes G1-S transition of the cell cycle, apoptosis, induce DNA damage, has oncogenic potential and autophagic properties and activates NF-kβ. It is expressed both during KSHV latency and lytic replication phase. The v-cyclin-CDK6 complex can phosphorylate pRB protein. The exact role of this protein in KSHV replication is still not fully elucidated but studies indicates that v-cyclin-CDK6 complex mediates the phosphorylation of nucleophosmin (NPM) which facilitates NPM-LANA interaction as well as recruitment of HDAC1 resulting in KSHV latency. Furthermore, studies has shown that v-cyclin share close functional relationship with murine gammaherpesvirus 68 v-cyclin which is known to mediate efficient lytic reactivation from latency. A study also showed that in vivo expression of v-cyclin in B- and T-cell lymphocytes led to a markedly low survival due to frequency of early onset T-cell lymphoma and pancarditis. Finally a study suggested that cyclin can play a role in the initiation of Notch-dependent lymphomagenesis because the Notch pathway is known to have a role in T-cell development and lymphoma initiation.
13.4.3 v-FLICE (fas-associated death domain like il-1 β-covertase enzyme) inhibitory protein (vFLIP): vFLIP, also known as K13 is encoded by ORF17. The FLIP proteins are group of cellular and viral proteins that inhibits death receptor –induced (DR-induced) apoptosis. They are made of two death effector domains (DED) capable of inhibiting DED-DED interaction between FAS-associated protein with death domain (FADD) and procaspases 8 -10 with death signaling complex (DISC). VFLIP is latently expressed through splicing of LANA transcript from messenger RNA, and through the use of IRES in v-cyclin coding sequence. In KSHV, vFLIP prevents recruitment and processing of procaspases 8, thereby inhibiting FAS-induced apoptosis, thus providing a survival advantage for KSHV infected cells. Another function of vFLIP is it binds to IkB kinase (IKK) complex and heat shock protein 90 (hsp90) resulting in both classical and alternative NF-kB survival signaling. Elimination of vFLIP by RNA interference results in appreciable decrease in NF-kB activity and apoptosis. This confirms that vFLIP has a role in the survival of PEL cells. More studies are needed to understand the role of vFLIP in the initiation of KSHV-associated diseases.
13.4.4 Kaposin (Kpsn): Kpsn are the most abundantly expressed viral transcript during KSHV latency. Three types of Kpsn have been described: Kaposin A, Kaposin B, and Kaposin C. Kpsn A has oncogenic properties and transforms cells in culture. A study reported of morphological change in Rat-3 cells through the interaction with cytohedsin-1. Kpsn B increases the expression of cytokine by blocking the degradation of mRNAs thereby stabilizing cytokine expression such as IL-6 and GM-CSF. mRNA stabilization activity depends on direct repeats (DR1 and DR2) element of Kaposin B. Study has found that Kpsn B is abundant in PEL cell line BCBL-1. The function of Kpsn C is not known.
13.4.5 Replication and Transcription Activator (Rta): Rta is novel E3 ubiquitin ligase which is encoded by ORF50. It targets a number of transcriptional repressor ubiquitin proteasome pathway. A study showed that Rta interacts with the cellular transcriptional repressor protein Hey1 and that Hey1 has a contributory role in the maintenance of KSHV latency although other studies showed that Rta results in the disruption of latency. In addition, Rta is reported to be important in the induction of KSHV lytic replication from latency through the activation of the lytic cascade.
13.4.6 KSHV MicroRNAs (MiRNAs): MiRNAs are non-coding RNAs of 19-23 nucleotides in length with the ability of regulating gene expression post-transcriptionally by targeting 3’ untranslated regions (UTRs) of messenger RNAs. A number of cellular targets of KSHV-encoded miRNAs have been identified. They have highly expressed in latency and KS tumors which shows that they have essential functions in the viral life cycle and development of KS tumors. Their roles include inhibition of apoptosis and transformation, cell cycles regulation, and promotion of angiogenesis. Earlier it was suggested that these viral post-transcriptional regulators might be promoters of latency by targeting lytic genes. Ziegelbauer et al in a study profiling mRNA mimic- and antagomir reported that KSHV miRNAs can modulate the latent/lytic transition through direct targeting of RTA by miR-K12-9. Other also showed that miR-K12-5 and miR-K12-7 can directly target RTA. This data is consistent with in silico prediction of targets within RTA 3’UTR. Targeting of RTA could prevent reactivation. MiRNAs can also contribute to latency by targeting host factors that are involved in viral reactivation. A study showed that KSHV miRNAs play a role in maintaining latency by targeting cellular transcription factors and that miR-k12-11 and miR-k12-3 prevents lytic reactivation through modulating the expression of transcription factors MYB, C/EPPα and Ets-1. Moody et al found in a study that miRNA redundantly targets the NF-kB pathway to regulate cell cycle progression and apoptosis.
13.4.7 K1: K1 is a 46 kDA type 1 membrane glycoprotein encoded by ORFK1, which is the most variable portion of the viral genome. It is also referred to as variable ITAM containing protein (VIP) because it contains an immunoreceptor tyrosine –based activation motif (ITAM). KSHV K1 ITAM activates several intracellular signaling pathways especially P13K/AKT. This means the expression of K1 results in the inhibition of proapoptotic proteins and increases the life-span of KSHV infected cells. Additionally, K1 enhances the production of inflammatory cytokines and proangiogenic factors such as vascular endothelial growth factor. KSVH K1 immortalizes primary human umbilical vein endothelial cells (HUVEC) in culture and transforms rodent fibroblasts as well as inducing tumor in vivo in transgenic mice. K1 has been detected in KS, PEL, and MCD. Available data suggests that K1 is essential in KSHV-associated tumorigenesis and angiogenesis.
13.4.8 Viral interleukin (vIL-6): vIL-6 is encoded by ORFK2 and is homolog to cellular IL-6. Long before the discovery of KSHV, vIL-6 was suspected to be important in the pathogenesis of KS and MCD. Data shows that vIL-6 mimics some of IL-6 activities such as stimulating growth of IL-6 dependent cells and triggering JAK and STAT3, MAPL, and H7-sensitivity pathways. The JAK and STAT3 pathways stimulated by vIL-6 results in increase in VEGF expression. However there are some differences in receptor usage which may give rise to the underlying quantitative and qualitative difference in the utilization of signal pathways. While cellular IL-6 depends on IL-80 and gp 130, vIL-6 signals can be attained through gp130 alone. vIL-6 is capable of inducing transcriptional activation through Type IL-6 response elements (RE) that binds C/EBP. This is indicative of Ras-MAP kinase pathway induction. From available data, it can be suggested that vIL-6 contributes to the progression of KSHV-associated diseases by continued activation of IL-6 stimulated growth and anti-apoptotic pathways; for e.g. a study found that Castleman’s disease involved aberrant IL-6 activity from either endogenous or viral sources.
13.4.9 Viral interferon regulatory factors (vIRFs): vIRFs are family of genes with homology to cellular interferon regulatory factors (IRFs) .There are four types of vIRFs: 1-4; which inhibits the activity of their cellular counterparts. vIRF-1 downregulates interaction with cellular p53 via its central DNA domain. This interaction inhibits transcriptional activation of p53. A study by Shin et al showed that KSHV vIRF-1 downregulates the total p53 protein level by facilitating its proteasome-mediated degradation. vIRF-1 interacts with cellular ATM (ataxia telangiectasis-mutated) kinase via its carboxyl-terminal transactivation domain. This interaction blocked the activation of ATM kinase activity induced by DNA damage stress. As a result, vIRF-1 expression greatly reduced the level of serine phosphorylation of p53. It leads to increase of p53 ubiquitination and decrease of its protein stability. The study indicated that KSHV vIRF-1 greatly compromised the ATM/p53-mediated DNA damage response checkpoint as it target both upstream ATM and downstream p53 tumor suppressor. This assists it to evade the host growth surveillance and facilitate viral replication in infected cells. vIRF-1 and 2 also directly interact with cellular IRFs. In addition, vIRFs have other functions such as modulation of Myc, transforming growth factor-β, and NF-kβ signaling. These activities of vIRFs have been implicated in KSHV tumorigenesis.
13.4.10 Viral G protein-coupled receptor (vGPCR): vGPCR is encoded by ORF74. It is a lytic cycle associated protein, highly angiogenic, and homologue of IL-8 receptor that signals in part via the cytoplasmic protein tyrosine phosphatase Shp2. It induces several signaling pathways leading to the activation of various transcription factors and ultimately leading to the expression of cellular and viral genes involved in survival, proliferation, and angiogenesis. The expression of vGPCR has been reported in small fraction of KS, PEL, and MCD. The role of this protein in KSHV tumorigenesis has been well documented. A vGPCR transgenic mice developed KS-like angioproliferative lesions with surface markers and cytokine profile resembling those of KS. It has been shown that in KSHV-associated malignancies, the expression of vGPCR was found in some of cells in transgenic tumors and few other tissues which suggest that vGPCR-mediated tumor formation was driven by spontaneous lytic reactivation in the background of latently infected cells. VEGR was reported to be increased in vGPCR-induced tumor. vGPCR can transform NIH3T3 fibroblasts in vitro and vGPCR- expressed 3t3 cells forms tumor in mice. vGPCR also activate mitogen-activated protein kinase (MAPKs), p13K, and Akt in endothelial cells and all these signaling pathways leads to the activation of key cellular transcription factors, such as activating protein-1 (AP-1), NF-kB, nuclear factor activator of T cells, cyclic AMP response element binding protein (CREP), and hypoxia-inducible factor-1 (HIF-1). These transcription factors in turn regulate several viral genes such as vIL-6. Just like K1, autocrine/panacrine signaling of vGPCR might have a role in KSHV-associated oncogenes and angiogenesis.
13.4.11 mRNA Transcript Accumulation (MTA): Mta is encoded by ORF57 protein. It is made of nuclear protein composed of 455 amino acids (aa) residues. Motif analysis of KSHV ORF57 aa sequence showed several sequence motifs which remotely resembles those found in cellular RNA binding proteins. These includes two simple RGG motifs of which RGG1 composed of 138-140 aa and RGG2 made of 372-374 aa have been described. The RGG motifs are similar to RGG-box of RNA-binding proteins; serine/arginine or arginine/serine dipeptides made of 77-95 aa, a nonconsensus putative adenine-thymine (AT) hook made of 119-130 aa, a putative leucine-rich region of 343-364 aa and a γ-herpesvirus glycine/phenylalanine/phenylalanine (GLFF) motif made of 448-451 aa whose function is unknown. Furthermore, the N-terminal half of KSHV ORF57 is enriched with polar residues to form short acidic regions made of approximately 7-52 aa followed with a basic region made of high content of arginine residues that harbours all functionally redundant nuclear localization signals (NLSs) of which three forms have been described: NLS1 made of 101-107 aa, NLS2 made of 121-130 aa, and NLS3 of 143-152 aa. KSHV ORF57 is essential for efficient expression of KSHV lytic genes and replicative replication. Deletion of this protein from the virus genome resulted in inefficient expression of viral lytic genes and abortive viral replication. It possesses a number of activities that are essential for the expression of viral genes, including the three major functions of enhancement of RNA stability, promotion of RNA splicing, and stimulation of protein translation. The ability of Mta to interact with a number of cellular cofactors results in its multifunctional characteristic. These interactions are essential for the formation of MTA-containing ribonucleoprotein complexes are specific binding sites in the target transcripts [referred to as Mta-responsive elements (MREs)]. Two structurally distinct domains have been identified within ORF57 polypeptide: a structure α-helix rich c-terminal and an unstructured intrinsic disordered N-terminal domain. These distinctive structures allow for their unique binding affinities of which N-terminal domain mediates the interaction with cellular cofactors and target RNAs, and the C-terminal contribute to the stability of ORF57 protein in infected cells by counteracting caspase- and proteasome mediation degradation pathway. Although our knowledge of KSHV ORF57 has improved over the past years, there are still some gaps in the data on the biochemistry and biophysics properties of individual domains of this protein; therefore future studies should target these gaps.
13.4.12 Viral Processivity Factor (vPF): vPF is encoded by ORF59 and is one of the factors to be recruited by RTA to oriLyt, where it acts as an accessory factor or sliding clamp, stabilizing the binding. In a study, Rossetto et al showed that binding ORF59 to the C/EBPα binding motif within oriLyt is essential for its function and is dependent on the presence of RTA. This means the function of viral polymerase is also dependent upon its interaction. ORF 59 forms a homodimer, which translocates viral polymerase which is encoded by ORF9 into the nucleus for efficient synthesis of DNA fragment. This is the Processivity function. ORF59 is a phosphoprotein that is phosphorylated by KSHV viral Ser/Thr kinase (ORF36). In a study, McDowell et al showed that ORF36 phosphorylates ORF59 at Ser 378, which is essential for ORF59’s ability to bind RTA and oriLyt. In addition, replacing the phosphorylated serine of these sites within alanines critically reduces viral products. However the precise mechanism by which ORF59 interacts with RTA has not been elucidated. Elucidating this mechanism will result in developing strategy to regulate viral lytic DNA replication at oriLyt.
13.4.13 KSHV bZIP (K-bZIP): Also referred to as K8, it is an immediate early protein which overlaps with ORF50 (Rta), and requires splicing. It can also activate 21 KSHV promoters. K-bZIP gene locus is made of and controls 2 promoters: one early controlling, K-bZIP, and one late controlling, K8.1. K-ZIP can be homodimerized consisting of 237 amino acids. It contains several functional domains: a transcription activation domain at the N terminus, a SUMO interaction motif, a leucine zipper domain at its C terminus, a nuclear localization signal, a DNA binding domain, and a basic region. K-bZIP can be SUMOylated at leucine and this process can affects its interaction with many cellular and viral proteins. A number of proteins are known to interact with K-bZIP, including p53, cAMP-response element-binding protein (CREB)-binding protein (CBP), CCAAT/enhancer-binding protein α, and others. The effect of such interaction on gene regulation is either positive or negative for viral growth. K-bZIP represses the ORF activities of transduction which suggests that it has repressing effect on viral gene expression and viral replication. On the other hand, knockdown of K-bZIP either abolishes the reactivation of KSHV, implying that it is essential for KSHV lytic infection, or lowers viral DNA copies at the latent phase of viral infection, suggesting that K-bZIP might have a role in abortive lytic DNA replication of de novo infection or the maintenance of latent viral genome. In a study, Martinez and Tang found that K-bZIP interacts and colocalizes with histone deacetylase (HDAC) 1 / 2 in the DNA replication domain. This means K-bZIP might function through either recruiting HDAC to have a negative effect on some genes regulation or through segregation of HDAC and inhibiting its activity thereby having positive effect on gene regulation. They also discovered that leucine zipper domain is required by K-bZIP to interact with HDAC 1 /2 and some other KSHV lytic gene promoters. These interactions are essential for KSHV to replicate is HER 293T cells.
13.5 KSHV Oncogenesis: As explained earlier, the life cycle of KSHV consists of latent and lytic replication phases which are important for the development of KS tumors. Therefore understanding the mechanisms that result in latency and reactivation holds the key to elucidating KSHV-associated pathogenesis and devising better therapeutic strategies. This section will review recent advances in mechanisms essential in KSHV life cycle.
13.5.1 Mechanism of KSHV Latency and pseudo-latency: A common feature of all human oncoviruses is that they are persistent latent or pseudo-latent infections that do not generally replicate to form infectious particles in tumors. The virus particle is a naked nucleic acid, often as plasmid or episome that relies on host cell machinery to replicate. About 90 KSHV genes are expressed during KSHV-associated latent infection. LANA-1, vFLIP, and vCyclin are expressed in latent infections and are found adjacent to one another in KSHV genome, belonging to the multicistronic transcriptional unit referred to as latent transcript (LT) cluster. It has been suggested that LANA-1 might be the principal translation product of longer miRNAs, while vFLIP and vCyclin are produced from shorter transcript. The three genes are separated from K12 genes, which are usually expressed in low level during latency, by an approximate 4.5 kb KSHV sequence that lack significant ORFs. This represents the largest coding gap in the unique region of KSHV genome. 10 of the KSHV miRNAs are found in this coding gap while miR-K10 is found within K12 and miR-K12 is found within 3’-UTR. KSHV miRNAs are all expressed in latently infected cells and largely uninfected after induction of lytic replication. KSHV K15 is detected in latently infected cells but the expression levels increases following induction of lytic replication. K15 is found adjacent to the terminal repeats, and is transcribed in a leftward orientation from the terminal repeats region. LANA-2 is abundantly expressed in the nuclei of cultured KSHV-infected PEL cells.
13.5.2 Mechanism of KSHV Persistence: For KSHV to persist, the viral genome must replicate and segregate to progeny nuclei with each cell division. LANA-1 is one of the few genes associated with episomal persistence in the absence of other viral genes. It has been reported that both the N-terminal (N-LANA) and C-terminal (C-LANA) are essential for this function with the C-terminal suggested to play a supportive role in binding KSHV episomes to host chromosome. The C-terminal is important for LANA oligomerization and data shows that oligomerization is essential for efficient tethering of KSHV episome. The N-LANA interacts with mitotic chromosome by binding histones H2A/H2B while C-LANA binds to KSHV terminal repeats DNA simultaneously. With this, LANA tethers the viral genome to the host chromosome and distributes viral DNA to daughter cell during mitosis. A study by Sun et al found that LANA recruits replication factor C( RFC), proliferating cell nuclear antigen (PCNA) loader, a DNA polymerase damp to drive DNA replication efficiently. They therefore suggested that PCNA loading is a rate limiting step in DNA replication that is incompatible with viral survival and that LANA enhancement of PCNA loading allows for efficient viral replication and persistence.
13.5.3 Mechanism of KSHV Reactivation: Switching from latent to lytic infection by KSHV is mediated by a number of stimuli that induces the expression of RTA. This expression of RTA is essential and sufficient to trigger the process of lytic infection which results in the orderly expression of viral proteins, release of viral progeny, and host cell death. The expression of RTA precedes the expression of all other cycloheximide- resistant IE genes and cycloheximide-sensitive early genes. It has also been reported that RTA activation results in complete replication of nascent. After primary infection, KSHV established latent infection in which few genes are transcribed and expression of RTA tightly repressed. However, studies showed that cloned promoter region of RTA is has high basal activity which indicates epigenetic change and chromatin modeling of KSHV genes which may be involved in this repression process. Epigenetic changes like DNA methylation plays a role in regulating gene expression in normal mammalian development and cancer by transcriptionally silencing key growth regulator. A study by Chen et al suggested that mechanism of hypermethylation in RTA protein may regulate its expression and subsequent KSHV reactivation from latency. In addition, other chromatin modification such as histone deacetylation affects the local chromatin structure which coupled with DNA methylation may regulate RTA gene transcription. It has been suggested that methylation of RTA promoter region during latency promotes the association of transcriptional repressors and HDAC. Lytic replication can be induced by chemical such as butyrate, an inhibitor of HDAC and 12-0-tetradecanoylphorbol-13-acetate (TPA), an inducer of histone acetylases (HAT). Both inducers have the ability of affecting the acetylation state of RTA promoter that is dependent on methylation. It can be argued from such data that the control of latency of lytic switching is a function of chromosome architecture which will involve interplay between viral RTA and the host factors that regulates chromatin methylation and acetylation. There has been the suggestion that apart from RTA regulation in KSHV reaction, other cofactors are involved; for example a study suggested that hypoxia is a possible cofactor for KSHV reaction based on clinical observation which showed that KS tumors often appear on body parts such as feet and arms, where blood and oxygen supply are low compared to other parts. In support of this suggestion, a study reported that hypoxia could induce KSHV lytic replication in PEL cells. Hypoxia induces the accumulation of hypoxia-inducible factors (HIF1 /2). In the KSHV genome, promoters of RTA and ORF34 are reported to contain functional hypoxia response elements (HREs). In addition, a number of studies suggested that specific cell cycle phase and cell differentiation could regulate KSHV reactivation.
13.6 Treatment of KSHV-associated Malignancies: A wide range of treatment for KS is available but the treatment options to be used for KS are based on the severity of the disease, KS subtype, and the immune status of the affected. All patients with AIDS-associated KS should receive highly active antiretroviral therapy (HAART). Studies has shown that effective treatment regimen using antiretroviral agents results in both reduction in incidence of AIDS-associated KS, regression in size and numbers of existing lesions, as well as histological regression of existing KS lesions. Several antiviral agents such as ganciclovir, foscarnet, and cidofovir inhibits KSHV replication in vitro but some data showed that antiviral therapy with for e.g. cidofovir aimed at KSHV did not have any effect by itself for treatment of KS. This may be attributed to small amount of lytic KSHV in KS tumor. Antiviral may therefore be effective as adjunct to more conventional chemotherapy with agents such as liposomal anthracyclines such as doxorubicin and daunorubicin as first-line regimen and paxlitaxel as second-line regimen. Other chemotherapeutic agents include vinorelbine, IFN-α, and IL-12. In addition the tyrosine inhibitor or imatinib, and IL-12 demonstrated some activities against AIDS-associated KS.
For PEL, there is no clear established standard of care, and due to its low incidence, randomized clinical studies are not feasible presently. PEL patients have poor prognosis with a median survival of only 2-3 months after diagnosis. As in KS, patient co-infected with AIDS would benefit from HAART as spontaneous regression has been reported. Conventional CHOP-like regimen (cyclophosphomide, doxorubicin, vincristine, and prednisolone) did not improve median survival in comparison to other HIV-associated NHL. For HIV-negative cases of PEL, liposomal anthracycline with or without bortezomib and prednisolone can be giving. Bortezomib, a proteosome inhibitor used for multiple myeloma has been shown to enhance the in vitro cytotoxic effects of doxorubicin and paclitaxel, and has been used successfully in combination with anthracycline-based cytotoxic chemotherapeutic combinations. Rapamycin has also shown some promise in treating PEL cells in culture or xenograft model. Radiation therapy can be initiated for patients who cannot tolerate the above treatments options.
Treatment of MCD with HIV infection, HAART is essential but care should be taken as life threatening flares of MCD have been reported due to manifestations of immune reconstitution. Systemic therapy is the backbone of treating patients with MCD ranging from cytoreduction chemotherapy such as CHOP and ABV (doxorubicine, bleomycin, and vincristine); single agent maintenance chemotherapy, immunomodulating agents, and monoclonal antibodies against IL-6 and CD20 surface markers and inhibitors of KSHV viral replications.
In conclusion, the current treatment strategies employed for KS, PEL, and MCD are sub optimal with their devastating side effects. Targeting signal pathways of these tumors will be ideal strategies resulting in beneficial and efficacious treatment options than the conventional chemotherapy. Therefore more case and randomized clinical studies are needed to advance treatment options for KSHV-associated diseases.
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