Functional analysis of the mutated Epstein-Barr virus oncoprotein LMP1(69del): implications for a new role of naturally occurring LMP1 variants.
OBJECTIVE
The role of carboxyterminal deletions of the latent membrane protein-1 (LMP1) in Epstein-Barr virus (EBV) infection and oncogenesis is unclear. Here we describe functional properties of a rare 69-bp LMP1 deletion mutant (LMP1(69del)) isolated from a patient with polyclonal B-cell lymphocytosis.


METHODS
Colony focus assay was used to evaluate the transforming capacity of LMP1(69del) in comparison to that of wild-type LMP1 from EBV strain B95/8. Transient transfectants of B-, T-, epithelial and 3T3 cells, and stable transfectants with ecdysone-inducible LMP1 expression were produced. The signaling capacity of both LMP1s on nuclear transcription factors NFkappaB and AP-1 were studied. Secretion of matrix metalloproteinase MMP-9, apoptosis, and EBV lytic and latent gene expression were also investigated.


RESULTS
LMP(69del) showed transforming properties comparable to those of the wild-type oncoprotein. Induction of NFkappaB but a markedly reduced influence on AP-1 were observed. Both oncoproteins induced secretion of MMP-9, and enhanced pre-apoptotic effects in Jurkat-T cells leading to increased Fas/Apo-1 and doxorubicin-mediated apoptosis. Furthermore, LMP1(69del) showed a more effective down-regulation of the EBV lytic cycle master gene BZLF1(Zebra) than did wild-type LMP1.


CONCLUSIONS
(i) LMP1(69del) possesses oncogenic properties, (ii) the observed impaired activity on AP-1 does not interfere with MMP-9 induction, (iii) the enhanced inhibition of BZLF1 could compensate for previously described mutations of our isolate leading to a more lytic phenotype and may be responsible for counteracting permanent virus replication in the chronic active EBV syndrome observed in this patient.
Larcher
Clara
C
Institute of Hygiene and Social Medicine, University of Innsbruck, Austria. clara.larcher@uibk.ac.at
Bernhard
David
D
Schaadt
Eveline
E
Adler
Barbara
B
Ausserlechner
Michael J
MJ
Mitterer
Manfred
M
Huemer
Hartwig P
HP
D000818:Animals; D017209:Apoptosis; D001402:B-Lymphocytes / Q000473:pathology / Q000821:virology; D002460:Cell Line; D045744:Cell Line, Tumor; D002472:Cell Transformation, Viral / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008218:Lymphocytosis / Q000235:genetics* / Q000473:pathology; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000502:physiology; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014162:Transfection; D014763:Viral Matrix Proteins / Q000235:genetics* / Q000502:physiology; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics; D017735:Virus Latency
C063676:BZLF1 protein, Herpesvirus 4, Human; D004268:DNA-Binding Proteins; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D016328:NF-kappa B; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D018808:Transcription Factor AP-1; D014763:Viral Matrix Proteins; D014764:Viral Proteins; D020780:Matrix Metalloproteinase 9