The importance of cellular immune surveillance in controlling Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8)-related malignancies is reflected by the high rate of disease among immunosuppressed (AIDS and transplant) patients who are KSHV/HHV8 infected. In KS and primary effusion lymphoma (PEL) tumors, the virus is largely latent allowing most viral proteins to escape immune effector surveillance. The latency-associated nuclear antigen 1 (LANA1), however, must be expressed for viral survival during latency and therefore represents an ideal vaccine target to eliminate KSHV reservoir infection. LANA1, like the EBNA1 protein of Epstein Barr virus (EBV), has molecular characteristics that inhibit generation LANA1-specific cytotoxic lymphocyte (CTL) responses. Viral proteins are processed into CD8+ cell-recognized epitopes through proteasomal degradation during protein synthesis or turnover. Degradation at synthesis occurs through cellular recognition of protein misfolding (defective ribosomal products (DRiPs)). We show here that LANA1 protein has specific sequence features that decrease rates of both mature protein turnover and nascent protein translation. LANA1 has markedly enhanced protein stability compared to other cellular and viral proteins and pretreatment with proteasome inhibitors does not lead to its accumulation. Deletion of LANA1 internal repeat domains causes a pronounced increase in LANA1 synthesis rate. Thus, primary LANA1 structural features may limit MHC I presentation of LANA1 peptides by diminishing mature protein turnover and ensuring minimal DRiP presentation through retarded polypeptide translation. Our proposal seeks to define these processes and to determine their significance to LANA1 specific CTL generation.
Specific Aims I and II will define, through fine-mapping, the LANA1 domains involved in mature protein turnover and synthesis retardation. To examine degradation-inhibition (Dl), we will measure ubiquitinylation and turnover of LANA1 derivatives. The putative Dl domain will be cloned into heterologous proteins having defined degradation kinetics in order to assay specific steps of the degradation process. In addition, we will engineer strong proteasome-degradation signals into LANA1 to determine if the Dl function can be overcome. Similarly, to examine synthesis-retardation (SR), quantitation of DRiP formation for LANA1 and derivatives will be measured. Specific mechanisms for SR, such as rare codon usage, will be assessed.
In Specific Aim III, we will directly assess the impact of LANA1 degradation-inhibition and synthesis-retardation on CDS immune responses in mice using LANA1 and a well-established chicken ovalbumin model. These results will lay the groundwork for an effective anti-latency KSHV vaccine.
|Moore, Patrick S; Chang, Yuan (2011) KSHV: forgotten but not gone. Blood 117:6973-4|
|Kwun, Hyun Jin; da Silva, Suzane Ramos; Qin, Huilian et al. (2011) The central repeat domain 1 of Kaposi's sarcoma-associated herpesvirus (KSHV) latency associated-nuclear antigen 1 (LANA1) prevents cis MHC class I peptide presentation. Virology 412:357-65|
|Moore, Patrick S; Chang, Yuan (2010) Why do viruses cause cancer? Highlights of the first century of human tumour virology. Nat Rev Cancer 10:878-89|
|Tolstov, Yanis L; Pastrana, Diana V; Feng, Huichen et al. (2009) Human Merkel cell polyomavirus infection II. MCV is a common human infection that can be detected by conformational capsid epitope immunoassays. Int J Cancer 125:1250-6|
|Kwun, Hyun Jin; da Silva, Suzane Ramos; Shah, Ishita M et al. (2007) Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 1 mimics Epstein-Barr virus EBNA1 immune evasion through central repeat domain effects on protein processing. J Virol 81:8225-35|