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Cell division, which leads to the birth of two daughter cells, is essential for the growth and development of all organisms. The reproduction occurs in a series of events separated in time, designated as the cell cycle. The cell cycle progression is controlled by the activity of cyclin-dependent kinases (CDK). CDKs pair with cyclins to become catalytically active and phosphorylate a broad range of substrates required for cell cycle progression. In addition to cyclins, CDKs are regulated by inhibitory and activating phosphorylation events, binding to CDK-inhibitory proteins (CKI), and also by subcellular localization. The control of the CDK activity is crucial in preventing unscheduled progression of the cell cycle with mistakes having potentially hazardous consequences, such as uncontrolled proliferation of the cells, a hallmark of cancer. The mammalian cell cycle is a target of several DNA tumor viruses that can deregulate the host s cell cycle with their viral oncoproteins. A human herpesvirus called Kaposi s sarcoma herpesvirus (KSHV) is implicated in the cause of Kaposi s sarcoma (KS) and lymphoproliferative diseases such as primary effusion lymphomas (PEL). KSHV has pirated several cell cycle regulatory genes that it uses to manipulate its host cell and to induce proliferation. Among these gene products is a cellular cyclin D homologue, called viral cyclin (v-cyclin) that can activate cellular CDKs leading to the phosphorylation of multiple target proteins. Intriguingly, PELs that are naturally infected with KSHV consistently express high levels of CDK inhibitor protein p27Kip1 and still proliferate actively. The aim of this study was to investigate v-cyclin complexes and their activity in PELs, and search for an explanation why CKIs, such as p27Kip1 and p21Cip1 are unable to inhibit cell proliferation in this type of lymphoma. In this study, we found that v-cyclin binds to p27Kip1 in PELs, and confirmed this novel interaction also in the overexpression models. We observed that p27Kip1 associated with v-cyclin was also phosphorylated by a v-cyclin-associated kinase and identified cellular CDK6 as the major kinase partner of v-cyclin responsible for this phosphorylation. Analysis of the p27Kip1 residues targeted by v-cyclin-CDK6 revealed that serine 10 (S10) is the major phosphorylation site during the latent phase of the KSHV replication cycle. This phosphorylation led to the relocalization of p27Kip1 to the cytoplasm, where it is unable to inhibit nuclear cyclin-CDK complexes. In the lytic phase of the viral replication cycle, the preferred phosphorylation site on p27Kip1 by v-cyclin-CDK6 changed to threonine 187 (T187). T187 phosphorylation has been shown to lead to ubiquitin-mediated degradation of p27Kip1 and downregulation of p27Kip1 was also observed here. v-cyclin was detected also in complex with p21Cip1, both in overexpression models and in PELs. Phosphorylation of p21Cip1 on serine 130 (S130) site by v-cyclin-CDK6 functionally inactivated p21Cip1 and led to the circumvention of G1 arrest induced by p21Cip1. Moreover, p21Cip1 phosphorylated by v-cyclin-associated kinase showed reduced binding to CDK2, which provides a plausible explanation why p21Cip1 is unable to inhibit cell cycle progression upon v-cyclin expression. Our findings clarify the mechanisms on how v-cyclin evades the inhibition of cell cycle inhibitors and suggests an explanation to the uncontrolled proliferation of KSHV-infected cells. Solunjakautuminen, joka johtaa kahden uuden tytärsolun syntymiseen on välttämätäntä kaikkien eliöiden kasvulle ja kehitykselle. Solunjakautumista edeltää solun valmistautuminen jakautumiseen (välivaihe eli interfaasi), jolloin esim. kromosomit kahdentuvat, jotta kummatkin tytärsolut saavat mitoosissa eli tuman jakautumisessa saman geneettisen kopion. Nämä useat vaiheet muodostavat solusyklin, jota säätelee monimutkainen, sykliinistä riippuvien proteiinikinaasien (CDK) toimintaan perustuva koneisto. CDKt muodostavat aktiivisia komplekseja sykliiniproteiinien kanssa ja fosforyloivat monia eri substraattiproteiineja. CDKien toimintaa säätelee sykliiniproteiinien lisäksi myös erilaiset aktivoivat/inaktivoivat fosforylaatiot, CDKta inhiboivat proteiinit (CKI) sekä myös lokalisaatio solun sisällä. Monet DNA syöpävirukset pystyvät häritsemään solusyklin kulkua tuottamalla omia syöpäproteiinejaan. Kaposin sarkooma herpesvirus (KSHV) on liitetty Kaposin sarkooman sekä tiettyjen non-Hodkin lymfoomien kuten primaaristen effuusiolymfoomien (PEL) syntyyn. KSHV:n genomi koodaa useita solusykliä sääteleviä proteiineja, joista moni on luultavasti kaapattu isäntäsolun geeneistä. Yksi näistä on viruksen sykliini eli v-sykliini. KSHV:n v-sykliini sitoutuu solun omaan kinaasiin ja aktivoi sen fosforyloimaan suuren joukon kohdeproteiineja. Mielenkiintomme herätti se, että KSHV-infektoituneet PEL lymfoomasolut tuottavat suuria määriä CDKta inhiboivaa proteiinia, p27Kip1, joka ei kuitenkaan pysty estämään solunjakautumista, mikä on sen tehtävä normaalissa solussa. Tutkimuksen tarkoitus oli selvittää v-sykliinin merkitystä PEL-lymfoomien patogeneesissa ja etsiä vastausta sille miksi solusykliä inhiboivat proteiinit kuten p27Kip1 ja p21Cip1, eivät pysty estämään näiden lymfoomasolujen jakautumista. Tutkimuksemme osoittivat että v-sykliini yhdistyy PEL-lymfoomasoluissa solun omaan CDK6-kinaasiproteiiniin ja aktivoi sen fosforyloimaan useita solunjakautumista edistäviä kohdeproteiineja. Osoitimme että v-sykliini sitoutuu solusykliä säätelevän p27Kip1 proteiinin ja että v-sykliini-CDK6-kompleksi fosforyloi tämän proteiinin. Selvitimme että v-sykliini-CDK6 fosforyloi eri kohtia p27Kip1 proteiinissa riippuen viruksen elinvaiheesta. Viruksen ns. latentin vaiheen aikana, jolloin virus on hiljaisena isäntäsolussa ja uusia viruksia ei tuoteta, v-sykliini-CDK6 kinaasikompleksin aiheuttama fosforylaatio johtaa p27Kip1 lokalisaatioon sytoplasmaan, missä p27Kip1 ei voi inhiboida solun omien sykliini-CDK kompleksien aktiivisuutta. KSHVn lyyttisen vaiheen aikana, jolloin isäntäsolussa tuotetaan uusia viruspartikkeleita, v-sykliini-CDK6-välitteisen fosforylaation seurauksena p27Kip1 proteiini hajoitetaan, minkä havaittiin tapahtuvan myös PEL-lymfoomasoluissa. Osoitimme lisäksi että v-sykliini sitoutuu myös toiseen solusykliä inhiboivaan proteiiniin, nimeltään p21Cip, joka oli myös kohteena v-sykliini-CDK6-välitteiselle fosforylaatiolle sekä inaktivaatiolle. Tutkimuksemme selventävät miksi PEL-lymfoomat pystyvät kasvamaan olosuhteissa jotka normaalisti estäisivät solujen jakautumisen sekä tuovat uutta tietoa siitä miten v-sykliini-CDK- kinaasikompleksi estää solusykliä jarruttavien proteiininen toimintaa.

Kaposi's sarcoma herpesvirus (KSHV)-encoded v-cyclin, a homolog of cellular cyclin D2, activates cellular CDK6, promotes G1-S transition of the cell cycle, induces DNA damage, apoptosis, autophagy and is reported to have oncogenic potential. Here we show that in vivo expression of v-cyclin in the B- and T-cell lymphocyte compartments results in a markedly low survival due to high penetrance of early-onset T-cell lymphoma and pancarditis. The v-cyclin transgenic mice have smaller pre-tumorigenic lymphoid organs, showing decreased cellularity, and increased proliferation and apoptosis. Furthermore, v-cyclin expression resulted in decreased amounts of CD3-expressing mature T-cells in the secondary lymphoid organs concurrent with alterations in the T-cell subpopulations of the thymus. This suggests that v-cyclin interferes with normal T-cell development. As the Notch pathway is recognized for its role in both T-cell development and lymphoma initiation, we addressed the role of Notch in the v-cyclin-induced alterations. Fittingly, we demonstrate induction of Notch3 and Hes1 in the pre-tumorigenic thymi and lymphomas of v-cyclin expressing mice, and show that lymphoma growth and viability are dependent on activated Notch signaling. Notch3 transcription and growth of the lymphomas was dependent on CDK6, as determined by silencing of CDK6 expression or chemical inhibition, respectively. Our work here reveals a viral cyclin-CDK6 complex as an upstream regulator of Notch receptor, suggesting that cyclins can play a role in the initiation of Notch-dependent lymphomagenesis.

Kaposi's sarcoma herpesvirus (KSHV)-encoded v-cyclin, a homolog of cellular cyclin D2, activates cellular CDK6, promotes G1-S transition of the cell cycle, induces DNA damage, apoptosis, autophagy and is reported to have oncogenic potential. Here we show that in vivo expression of v-cyclin in the B- and T-cell lymphocyte compartments results in a markedly low survival due to high penetrance of early-onset T-cell lymphoma and pancarditis. The v-cyclin transgenic mice have smaller pre-tumorigenic lymphoid organs, showing decreased cellularity, and increased proliferation and apoptosis. Furthermore, v-cyclin expression resulted in decreased amounts of CD3-expressing mature T-cells in the secondary lymphoid organs concurrent with alterations in the T-cell subpopulations of the thymus. This suggests that v-cyclin interferes with normal T-cell development. As the Notch pathway is recognized for its role in both T-cell development and lymphoma initiation, we addressed the role of Notch in the v-cyclin-induced alterations. Fittingly, we demonstrate induction of Notch3 and Hes1 in the pre-tumorigenic thymi and lymphomas of v-cyclin expressing mice, and show that lymphoma growth and viability are dependent on activated Notch signaling. Notch3 transcription and growth of the lymphomas was dependent on CDK6, as determined by silencing of CDK6 expression or chemical inhibition, respectively. Our work here reveals a viral cyclin-CDK6 complex as an upstream regulator of Notch receptor, suggesting that cyclins can play a role in the initiation of Notch-dependent lymphomagenesis.

Kaposi's sarcoma herpesvirus (KSHV)-encoded v-cyclin, a homolog of cellular cyclin D2, activates cellular CDK6, promotes G1-S transition of the cell cycle, induces DNA damage, apoptosis, autophagy and is reported to have oncogenic potential. Here we show that in vivo expression of v-cyclin in the B- and T-cell lymphocyte compartments results in a markedly low survival due to high penetrance of early-onset T-cell lymphoma and pancarditis. The v-cyclin transgenic mice have smaller pre-tumorigenic lymphoid organs, showing decreased cellularity, and increased proliferation and apoptosis. Furthermore, v-cyclin expression resulted in decreased amounts of CD3-expressing mature T-cells in the secondary lymphoid organs concurrent with alterations in the T-cell subpopulations of the thymus. This suggests that v-cyclin interferes with normal T-cell development. As the Notch pathway is recognized for its role in both T-cell development and lymphoma initiation, we addressed the role of Notch in the v-cyclin-induced alterations. Fittingly, we demonstrate induction of Notch3 and Hes1 in the pre-tumorigenic thymi and lymphomas of v-cyclin expressing mice, and show that lymphoma growth and viability are dependent on activated Notch signaling. Notch3 transcription and growth of the lymphomas was dependent on CDK6, as determined by silencing of CDK6 expression or chemical inhibition, respectively. Our work here reveals a viral cyclin-CDK6 complex as an upstream regulator of Notch receptor, suggesting that cyclins can play a role in the initiation of Notch-dependent lymphomagenesis.