Herpesviruses belong to the largest viruses known and encode between approximately 80 and 170 viral proteins. Although a multitude of viral genes is homologous to cellular genes and share structural similarities, they possess distinct functions. In most herpesviruses the function of the majority of viral proteins is unknown. Previous work indicated that a large number of herpesviral proteins interfere with the host immune system to establish and maintain latency. Herpesviral functions counteracting the adaptive immune system are well-characterized and include the downregulation of MHC and costimulatory molecules as well as the inhibition of humoral immunity and cytokine signalling (Alcami & Koszinowski, 2000). For CMV, it has recently become clear that the innate immune system also plays an important role in the control of the infection (Scalzo, 2002). CMV inhibits the function of NK cells by multiple mechanisms, for example by expressing viral proteins that bind to inhibitory NK cell receptors, that block ligands for activating NK cell receptors and that stabilize cellular ligands for inhibitory NK receptors (Krmpotic, Hasan et al., 2005;French, Pingel et al., 2004). The role of NK cells in the immune surveillance against #-herpesviruses is less well-characterized. Recently, however, it was found that the immunodeficiency in Epstein-Barr Virus (EBV) associated X-linked proliferative disease is caused by a failure of the 2B4 molecule to activate NK-cell-mediated cytotoxicity against EBV-infected cells, indicating the signifigance of the innate immune control for #-herpesviruses as well (Parolini, Bottino et al., 2000).
In our previous work we received evidence by microarray analysis that the persistent infection of EBV positive B-cells with KSHV induces a phenotype similar to tumor cells derived from primary effusion lymphomas (PEL), which are caused by KSHV. In KSHV+ B-cells basically all B-cell surface molecules including the B-cell receptor (BCR), CD79, CD19 and B220 are downregulated, as well as costimulatory molecules including CD80 (B7-1), CD86 (B7-2) and ICAM (Iotzova, Robertson et al., 2003). A variety of different cytokines and secreted factors including interleukin 6 (IL-6) and IL-10 were found to be increased in supernatants of KSHV+ B-cells. As most of these factors can also be detected in significant amounts in effusion liquids of patients with PEL, KSHV infection of B-cells causes a strikingly similar phenotype as in PEL tumor cells. The downregulation of surface markers (null phenotype) is probably responsible for the fact that these KSHV+ cell lines can not be recognized by allogenetic cytotoxic T-cells (CTL) any more and thus represents a mechanism of immune evasion. It is conceivable that the null phenotype also influences the recognition by NK cells. In order to get hints on the function of previously uncharacterized KSHV proteins, we systematically analysed protein-protein interactions on a genome-wide scale by yeast-two-hybrid (Y2H) matrix analysis similar to previous work done in S. cerevisiae (Uetz, Giot et al., 2000). We identified 123 protein-protein interactions and were able to confirm approximately 50% by co-immunoprecipitation (Uetz, Dong et al., 2005). The KSHV Orfeome was cloned by recombinatorial cloning into the entry vector pDONR207, and subsequently into Y2H bait and prey vectors. Due to viral protein interaction partners with known function, putative functions could be assigned to a variety of previously uncharacterized KSHV proteins (Uetz, Rajagopala et al., 2004).
The goals of this project are to identify #-herpesviral genes modulating the innate immune system by a set of stably transduced B-cell lines expressing all individual viral genes. Subsequently, it shall be investigated whether the b-herpesvirus CMV uses similar mechanisms and whether the in vitro effect can be confirmed in vivo.
Results/Project Status
Generation of a KSHV array in a lentiviral vector
Initial experiments indicated that it is difficult to generate a large number of stably transfected B-cell lines by episomally replicating vectors due to low transfection efficiency. We thus decided to change our strategy and use a lentiviral gene transfer system. The lentiviral vector pLenti6-V5DEST contains a GATEWAY conversion cassette allowing recombinatorial cloning and a blasticidin resistance gene and is based on HIV-1. After cotransfection with three other vectors expressing Gag/Pol, VSV Env and Rev into 293, supernatants containing recombinant transducing particles were used to transduce B-cell lines. Using a lentiviral vector in which we inserted eGFP, we could show that after selection with blasticidin for 7 days almost 100% of the B-cells expressed the transgene. Thus, this system is much more suitable to generate large sets of stable transfectants as aimed for here. We are currently swapping the entire KSHV ORFeome by recombinatorial cloning (RC) from the entry vector pDONR207 into the lentiviral vector and will subsequently generate lentiviral supernatants to generate stably transduced B-cells.
Effect of KSHV on NK cell recognition
We used the two cell lines NKL and NK92 to test whether KSHV+ B-cell lines are recognized and killed by NK cells. We used three pairs of B-cell lines derived from different donors which were either KSHV or EBV positive as target cells in a 51Cr release assay. Whereas neither KSHV nor EBV positive cells were recognized by NKL cells, NK92 killed EBV+ but not KSHV+ B-cells in 2/3 donors. These results have yet to be confirmed, but may in fact hint towards an immune evasion phenomenon. The stably transduced B-cell lines generated as mentioned above will be used to screen for viral proteins which are responsible for NK immune evasion.
Cloning of CMV genes in an eukaryotic expression vector
To compare immunmodulatory mechanisms in KSHV and CMV, we cloned all Orfs of murine CMV into the GATEWAY entry vector pDONR207. Since this array was also used to test for protein interactions, we cloned extra- and intracellular domains of transmembrane proteins separately as well. In total, approximately 280 constructs were generated. This set of mCMV Orfs cloned in pDONR207 will be used to subclone a smaller subset of Orfs into the GATEWAY compatible eukaryotic expression vector pCR3-DEST and the lentiviral vector pLenti6-V5DEST mentioned before.
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