Sequence variations between two Epstein-Barr virus LMP 1 variants have no effect on the activation of NF-kappaB activity.
Previously, we reported that the LMP 1 gene of Epstein-Barr virus (EBV) derived from nasopharyngeal carcinoma (NPC) tissues (i.e., NLMP 1 gene) was able to transform BALB/c3T3 cells. On the other hand, LMP 1 gene of B95-8 strain (i.e., BLMP 1 gene) was not able to transform these cells (Chen et aL, 1992). Further studies indicated that a 10-amino-acid deletion in the carboxyl terminus of NLMP 1 played an important role in transformation (Li et al., 1996). In this study, we tested if this 10-amino-acid deletion affected the induction of NF-kappaB activity by LMP 1. The long terminal repeat of the human immunodeficiency virus type 1 (HIV-1 LTR) contained two copies of NF-kappaB sites and was used to construct the Luc gene-based reporter plasmid, p kappaB-Luc. Plasmid p kappaB-Luc was co-transfected with plasmids containing the NLMP 1 gene, BLMP 1 gene, and their chimeric or deletion constructs, respectively, into C-33A and BALB/c3T3 cells. The activation was then measured by the luciferase activity. Results showed that the full-length proteins induced a similar level of NF-kappaB activity, the two 3' mutants (R15delta and D4delta) still induced a relatively high level of activity, and the two 5' deletion mutants (delta3058 and delta3243) of NLMP 1 gene did not show any significant activation in C-33A cells. However, none of these LMP 1 proteins induced NF-kappaB activity in BALB/c3T3 cells. Using subcellular fractionation analysis and an immunocytostaining method, the truncated proteins of delta3058 and delta3243 were detected in the cytoplasm of the cells whereas the full-length NLMP 1 protein was located at the cytoplasmic membrane. Stable BALB/c3T3 cell clones that expressed both truncated proteins were established and then their ability to induce tumors in nude mice was examined. Data showed that both truncated NLMP 1 proteins still maintained partial transformation activity. Our results suggested that there was no direct correlation between NF-kappaB activation and transformation activity of LMP 1 in BALB/c3T3 cell transformation and that the amino-terminal membrane-spanning domain was important for maintaining both functions of LMP 1.
Yeh
T S
TS
Graduate Institute of Microbiology and Immunology, National Yang-Ming University, Shih-pai, ROC.
Li
S N
SN
Wu
C J
CJ
Liu
S T
ST
Meng
C L
CL
Chang
Y S
YS
D016475:3T3 Cells; D000818:Animals; D000956:Antigens, Viral / Q000737:chemistry / Q000235:genetics*; D002213:Capsid / Q000737:chemistry / Q000235:genetics*; D002472:Cell Transformation, Viral / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism; D004854:Herpesvirus 4, Human / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism*; D009856:Oncogene Proteins, Viral / Q000737:chemistry / Q000235:genetics*; D017433:Protein Structure, Secondary; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000235:genetics*
D000956:Antigens, Viral; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D016328:NF-kappa B; D009856:Oncogene Proteins, Viral; D014763:Viral Matrix Proteins