1. Kaposi Sarcoma (KS): The Discovery of Human Herpesvirus 8
Quinta Fernandes/BIOL445/Spring2017

2. Kaposi Sarcoma (KS) presents as vascular tumors and lesions associated with immunosuppression
Classical or sporadic KS (CKS), originally described [1] as a slow growing, indolent tumor mostly developing in the extremities of elderly males of eastern and Mediterra- nean Europe.
b) Endemic KS (EKS), predominant in east- ern and central sub-Saharan Africa before the AIDS epidemic and clinically similar to CKS, but also seen in a more fulminant and fatal form in children. The childhood EKS is often lymphoglandular with or without skin involvement.
c) Acquired immunodeficiency syndrome (AIDS)-associated KS (AKS), the most frequent tumor of human immunodeficiency virus type I (HIV-l) infection and the most aggressive and rapidly growing form of KS in AIDS, with early dissemination in the skin and viscera.
d) Iatrogenic KS (IKS), seen in drug related immunosup- pressed patients, e.g. transplant patients, emphasizing the importance of immune disturbance as a co-factor in the pathogenesis of IKS and AKS, and possibly also EKS.
Figure 1. Human immunodeficiency virus–positive patient showing disseminated AIDS-associated cutaneous Kaposi sarcoma plaques on his body (image courtesy of Bruce J. Dezube, MD).
Figure 2. Kaposi sarcoma tumor showing fascicles of spindled cells with extravasated red blood cells and scattered chronic inflammatory cells (hematoxylin-eosin, original magnification 3200).

3. Representational Difference Analysis (RDA) reveals a new DNA virus in KS tumors, Human Herpesvirus-8 (HHV8/KSHV)
For table
Gel:

4. HIV infection and transplant immunosuppression show increased HHV8 seropositivity and risk of KS
Association between HIV and HHV-8 seropositivity in different population groups. The center of the diamonds is the pooled point estimate of the studies included in each population group, and the width of the diamonds represents the 95% confidence interval (CI) of the pooled odds ratio.
Data collected from a meta-analysis of 93 studies with over 58,000 participants in Africa, North America, South America, and Europe.
Modes of transmission are not yet known. Sexual transmission more common between homosexual men in the West. Does not explain endemic African KS.
Rohner et al. 2016

5. KSHV is an double-stranded DNA virus that encodes a large number of cellular homologues involving multiple signaling pathways
KSHV encodes 87 open reading frames (ORFs) and at least 17 microRNAs (purple boxes), 14 of which are co-expressed as a cluster. A striking feature of KSHV is the number (at least 14) of ORFs that encode cellular orthologues. Identified ORFs and encoded proteins are indicated in the figure. Putative latent transcripts are indicated in green, and cellular orthologues in yellow. Infection occurs when mature virions anchor to specific cellular receptors. After viral glycoprotein binding to the necessary receptors, clathrin-mediated endocytosis facilitates entry into cells163. Following infection, rapid circularization of the viral genome occurs164 and, like other herpesviruses, KSHV exists as an episome (double-stranded circular DNA) within the host nucleus.
Reactivation can occur when the promoter of ORF50 is activated (by demethylation, for example)165, resulting in the expression of replication and transcription activator (RTA), the main regulator for the viral lytic replication programme77,166. Early lytic genes include those encoding viral proteins required for DNA replication or viral gene expression, whereas late lytic genes are those encoding viral structural proteins, such as envelope and capsid proteins, that are required for assembly of viral particles (virions).

6. KSHV enhances the angiogenic properties of infected endothelial cells and benefits from and induces inflammatory signaling
Shulz and Cesarman 2015 (professor provided paper)
Model of KS progression. KSHV infected cells from the circulation (B cells, mesenchymal stem cells or endothelial precursors) or circulating cell free virus infect endothelial cells in the skin. KSHV induces expression of angiogenic and inflammatory cytokines, and can transmit virus to other neighboring cells, leading to vascular lesions with formation of abnormal vascular spaces, and containing a mixture of inflammatory cells. KSHV can induce genomic instability, leading to mutations in cellular genes. Some of these mutations, like those in TP53 can lead to further genomic instability and accumulation of more cellular gene mutations. When these provide a proliferative and/or survival advantage, the cell containing these mutations may expand, with the development of an oligo-clonal or (rarely) mono-clonal expansion of neoplastic cells containing the driving mutation(s).

7. HHV8 lytic genes like K1 subvert host signaling pathways leading to expression of angiogenic, inflammatory, proliferative factors
Proposed mechanism of KSHV-induced sarcoma. a | In lytic or abortive lytic-infected cells, expression of Kaposi’s sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus 8 (HHV8)) early lytic genes (such as viral G protein-coupled receptor (vGPCR), K1, viral interleukin-6 (vIL-6) and K15; shown in red) subvert host signalling pathways, leading to the expression and secretion of angiogenic, inflammatory and proliferative factors (including, vascular endothelial growth factor (VEGF), platelet-derived growth factor-β (PDGFB), angiopoietin 2 (ANGPT2), IL-6 and IL-8). This can occur together with intracrine activity and the secretion of vIL-6.

8. HHV8 ORF1, K1 expression activates PI3K/Akt signaling pathways and increases phosphorylation of its targets
Because we observed that K1 can activate VEGFR-2, we were interested in determining whether the PI3K/Akt signaling pathway was also activated in endothelial cells. The HUVEC-GFP and HUVEC-K1 cell lines were serum starved for 48 hours. Equivalent amounts of lysate were loaded on SDS-PAGE and subjected to Western blot analysis with antibodies directed against components of the PI3K/Akt pathways. K1 expression resulted in the phosphorylation of the p85 subunit of PI3K (Fig. 2C), indicative of its activation.
Although the cytoplasmic tail of K1 is a highly variable domain among different KSHV viral isolates throughout the world, the SH2 binding motifs present in the cytoplasmic tail are always conserved (39). We mutated the two SH2 binding motifs in K1 by replacing the tyrosine residues with phenylalanine residues and tested whether this mutation (K1SH2 ) abolished PI3K/Akt signaling pathway. We constructed a second set of wild-type (WT) K1, GFP, and K1SH2 mutant expressing endothelial cell lines and analyzed the components of the PI3K/Akt signaling pathway. As shown in Fig. 3B, expression of the mutant K1SH2 protein could not induce phosphorylation and activation of Akt kinase compared with WT K1. Consequently, downstream targets of Akt, including GSK3h, FKHR, Bad, and mTOR, did not display increased phosphorylation in the K1SH2-expressing cells compared with WT K1 (Fig. 3C). These data indicate that the SH2 binding motifs of K1 are necessary for activation of the PI3K/Akt pathway and phosphorylation of its downstream targets in endothelial cells.

9. K1 increases tumor size and tumor angiogenesis in vivo
To determine whether epithelial cells expressing K1 exhibit increased tumorigenic potential in mice, we used the human C33A cervical carcinoma cell line, which is human papillomavirus–negative. C33A cells by themselves are tumorigenic in nude mice (40), and we wanted to determine whether the introduction of K1 in these cells could enhance the tumorigenicity and/or angiogenic nature of these tumors.
K1 and GFP-expressing stable C33A cell lines were made by retroviral infection. Cells were selected with neomycin over several weeks, and three different clones each of the C33A-K1 and C33A- GFP stable cell lines were established. Expression of GFP was analyzed by microscopy, and expression of K1 in each clone was determined by Western blot (Fig. 4A ).
At this dose, V50% of mice injected with C33A-GFP cells developed tumors by 21 days, whereas 100% of mice injected with C33A-K1 cells developed palpable tumors. The cumulative mean tumor-free survival time was shorter in mice injected with the C33A-K1 cells compared with mice injected with C33A-GFP (Fig. 4B).
Taken together, our results show that K1 expression enhances tumor vasculature, tumor size, and tumor weight in vivo.

10. Lack of HAART availability/compliance reveal potential new drugs targeting angiogenesis and lymphangiogenesis
Lack of HAART availability beyond the western world
Novel agents used to treat KS include angiogeneisis inhibitors, tyrosine kinase inhibitors, and matrix metalloproteinase inhibitors (see Figure 3). Thalidomide, which has significant antiangiogenic
One major drawback of these studies is that the transforming function of individual KSHV genes is usually analyzed in over-expression systems, in the absence of other viral genes. Similarly, the majority of the animal assays were conducted in rodent models, which are not natural hosts for KSHV. Another important caveat is that when expressed alone, lytic genes can activate many cellular genes, but during a normal viral infection, a host shutoff function mediated by the lytic SOX (shut off and exonuclease) gene (ORF37) prevents the expression of most cellular genes [43]. Taken as a whole, these data indicate that KSHV encodes many proteins that individually have the ability to immortalize/transform cells in vitro and in vivo, but how they all work together to perform this function in the context of a normal viral infection remains obscure.

11. Works Cited
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