Biologic and immunologic effects of knockout of human cytomegalovirus pp65 nuclear localization signal.
The human cytomegalovirus (CMV) pp65 protein contains two bipartite nuclear localization signals (NLSs) at amino acids (aa) 415 to 438 and aa 537 to 561 near the carboxy terminus of CMV pp65 and a phosphate binding site related to kinase activity at lysine-436. A mutation of pp65 with K436N (CMV pp65mII) and further deletion of aa 537 to 561 resulted in a novel protein (pp65mIINLSKO, where NLSKO indicate NLS knockout) that is kinaseless and that has markedly reduced nuclear localization. The purpose of this study was to biologically characterize this protein and its immunogenicity compared to that of native pp65. Unlike the native CMV pp65, following either DNA- or recombinant adeno-associated virus-based transduction of CMV pp65mIINLSKO into cells in vitro, the first observation of pp65mIINLSKO expression was in the cytoplasm and pp65mIINLSKO was expressed at higher levels than the native protein. The CMV pp65mIINLSKO mRNA was more abundant earlier than CMV pp65 mRNA (at 4 h and 8 h, respectively), but the half-lives of the proteins were the same. This modification altered the antigenic processing of CMV pp65 in vitro, as measured by the improved efficiency of cytotoxic killing in a pp65mIINLSKO-transduced human HLA A*0201 target cell line. In HHDII mice expressing HLA A*0201, pp65mIINLSKO was as immunogenic as CMV pp65. By RNA microarray analysis, expression of the CMV pp65mIINLSKO had less of an effect on cell cycle pathways than the native CMV pp65 did and a greater effect on cell surface signaling pathways involving immune activity. It is concluded that the removal of the primary NLS motif from pp65 does not impair its immunogenicity and should be considered in the design of a vaccine.
Beckman Research Institute of the City of Hope, Department of Virology, 1500 E. Duarte Rd., Duarte, CA 91010, USA. jzaia@BRICOH.edu
John A
JA
Zaia
Xiuli
X
Li
Anne E
AE
Franck
Xiwei
X
Wu
Lia
L
Thao
Ghislaine
G
Gallez-Hawkins
D000818:Animals; D000956:Antigens, Viral / Q000235:genetics* / Q000276:immunology* / Q000633:toxicity; D002460:Cell Line; D020869:Gene Expression Profiling; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D019913:Nuclear Localization Signals; D020411:Oligonucleotide Array Sequence Analysis; D010750:Phosphoproteins / Q000235:genetics* / Q000276:immunology* / Q000633:toxicity; D017384:Sequence Deletion; D014763:Viral Matrix Proteins / Q000235:genetics* / Q000276:immunology* / Q000633:toxicity
D000956:Antigens, Viral; D019913:Nuclear Localization Signals; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa