Functional analysis of the Epstein-Barr virus LMP1 variants isolated from nasopharyngeal carcinoma in Hong Kong

Abstract

Nasopharyngeal carcinoma (NPC) occurs with a striking geographic incidence and is endemic in southern China. In Hong Kong, the annual incidence rate of NPC is about 25 fold higher than most other countries. The closest association of EBV infection is found in the poorly differentiated NPC where infection rate is close to 100%. The latent membrane protein 1(LMP1), encoded by EBV, is commonly expressed in the NPC cells. The LMP1 protein possesses oncogenic potential and activates several intracellular pathways involved in regulation of cell death and survival. Expression of LMP1 in host cells constitutively actives both the NF-κB and JNK pathways, which contribute to the oncogenic effect of LMP1. There is evidence that sequence variations can affect the intracellular signaling capacity of LMP1 and has been implicated to play a role in NPC pathogenesis. In the present study, we have carried out a systematic analysis of phenotype and genotype correlation from LMP1 sequences isolated from NPC to elucidate the relationship between sequence variations and intracellular signaling ability. The full-length sequences of 35 LMP1 variants from 27 NPC subjects, 4 healthy individual and 4 LMP1 variants commonly used in previous study (B95-8 LMP1, Raji-LMP1, Cao-LMP1, and 2117-LMP1) were cloned into expression vectors, sequenced and analyzed for their abilities to activate various intracellular signaling pathways. A comprehensive phylogenic comparison on the sequences of all these 35 LMP1 variants reveals two major groups of LMP1 variants. Interestingly, these two phylogenetic groups of LMP1 variants have distinct capacities to activate NF-kB signaling determined by the sensitive luciferase reporter assay. One group of LMP1 variants (n = 6) has a lower capacity to activate NF-kB and includes the B95.8-LMP1, Raji-LMP1 and 4 NPC-LMP1 variants. These LMP1 variants have similar sequences at their C-terminal cytoplasmic domains. The other group of LMP1 variants has a higher capacity (3 to 10 folds) to activate NF-kB and includes Cao-LMP1, 2117-LMP1and other NPC-LMP1 variants isolated from NPC subjects and 4 cases of healthy donors. This group of LMP1 variants all shared the deletion of 10-amino-acid sequence at residues 343 to 352. None of these LMP1 variants with higher capacities to activate NF-KB shows mutations in the core TRAF/TRADD/RIP binding sequences in CTAR1 and CTAR2. However, numerous mutations were detected in the putative JAK-3-binding Box-1 and Box-2 motifs in CTAR3. This study strongly indicated that LMP1 variants isolated from NPC patients has higher capacity to activate NF-kB than the prototype B95-8 LMP1, which has been used in many laboratories for its ability to immortalize and oncogenic transformation of B cells. Our results suggest that LMP1 variants isolated from NPC patients have distinct biological properties from the prototype B95-8 LMP1 and support their involvement in NPC pathogenesis.