Murine CMV pp 89 Fragments Products

Definition
Murine CMV (cytomegalovirus) pp 89 Fragments have been derived from the amino acid sequence of murine cytomegalovirus protein pp 89, an immediate-early phase regulatory protein of murine cytomegalovirus.

Discovery
Cytomegalovirus (CMV) is an ubiquitous human pathogen that is responsible for congenital malformations. Within the 235-kilobase-pair MCMV DNA, gene expression during the IE (immediate-early) phase is limited to a region of 12 kbp, which includes the transcription units IEl, IE2, and IE3. Gene IEl contained in transcription unit iel encodes the major MCMV IE protein, the non-structural regulatory phosphoprotein pp89. The murine cytomegalovirus protein pp89 is expressed in the immediate-early phase of the viral replication cycle and located mainly in the nucleus of infected cells ^1,2,3.

Structural Characteristics
The amino acid sequence of the Murine CMV IE1 protein contains a region of homology to histone H2B and a highly acidic carboxy-terminal region. In vitro studies have revealed that pp89 is eluted from chromatin at low salt concentration, but binds strongly to isolated histones. Murine CMV pp89 does not bind to condensed chromatin and that neither the region of homology to histone H2B nor the acidic region are essential for histone binding but interacts with both DNA associated and isolated histones in vitro. Analysis of pp89 deletion mutants and of fragments generated by cleavage at pH 2.5 revealed that the regions responsible for association with histone are located between amino acids 71 and 415, and are not identical with the domain that shows homology to histone H2B or the highly acidic carboxy-terminal region. A potential gene-activating role of pp89 is the high affinity for isolated histones and the low affinity for DNA associated histones ⁴.

Mode of Action
Virus-specific, major histocompatibility complex class I-restricted cytolytic T lymphocytes (CTL) recognize pp89. Vaccination with a -recombinant vaccinia virus, MCMV-IEI-VAC, expressing pp89 as the only MCMV protein, induces protective immunity. A single domain of pp89 is relevant for recognition by CD8+ T lymphocytes in BALB/c mice and led to the identification of an epitope 19 amino acids in length within this domain. The domain is entirely encoded by the fourth exon of gene IEI and contains the sequences essential for both immunogenicity and antigenicity of pp89 ^5,6.

Functions

Activation of E gene promoters- Thorough functional analysis of the homologous gene complex, IE1/IE2, in human CMV has revealed that IE1 products activate the IE1 gene promoter, whereas IE2 products have been found to be essential for the activation of E (early) gene promoters. Histone-binding domains of pp89 support the establishment of stable transcription complexes during nucleosomal organization by direct interaction with histones. In addition, the highly acidic C-terminal sequence of pp89 is free to exert functions ^4,7.

Murine CMV protection- IE protein pp89 is the only MCMV gene product that can protect against a lethal challenge infection with Murine CMV. Protection is mediated by pp89-specific CD8+ T lymphocyte. The epitope comprises amino acids 161 to 179, contains a proposed pattern at residues 170 to 174, and is the first described to be presented by the major histocompatibility complex class I molecule. This peptide contains three prolines, an amino acid seldom found in a-helices and in epitopes recognized by T lymphocytes ⁸.

DHFR stimulation- Murine CMV infection of quiescent NIH 3T3 cells markedly stimulates transcription, expression and activity of the cellular dihydrofolate reductase (DHFR), a key enzyme in the synthesis of DNA precursors. DHFR stimulation by Murine CMV is sensitive to UV irradiation and seems to depend on expression of the viral immediate-early protein pp89 ^9,10.

References

1. Keil GM, Ebeling-Keil A, Koszinowski UH (1984). Temporal regulation of murine cytomegalovirus transcription and mapping of viral RNA synthesized at immediate early times after infection. J. Virol., 50:784-795.
2. Keil GM, Ebeling-Keil A, Koszinowski UH (1987). Immediate-early genes of murine cytomegalovirus: location, transcripts, and translation products. J. Virol., 61:526-533.
3. Keil GM, Ebeling-Keil A, Koszinowski UH (1987). Sequence and structural organization of murine cytomegalovirus immediate-early gene 1. J. Virol., 61: 901-1908.
4. Münch K, Bühler B, Messerle M, Koszinowski UH (1991). The core histone-binding region of the murine cytomegalovirus 89K immediate early protein. Journal of General Virology., 72:1967-1974.
5. Koszinowski UH, Reddehase MJ, Keil GM, Schickedanz J (1987). Host immune response to cytomegalovirus: products of transfected viral immediate-early genes are recognized by cloned cytolytic T lymphocytes. J. Virol., 61:2054-2058.
6. Volkmer H, Bertholet C, Jonjic S, Wittek R, Koszinowski (1987). Cytolytic T lymphocyte recognition of the murine cytomegalovirus nonstructural immediate-early protein pp89 expressed by recombinant vaccinia virus. J. Exp. Med., 166:668-677.
7. Stenberg RM, Fortney J, Barlow SW, Magrane BP, Nelson JA, Ghazal P (1990). Promoter-specific trans activation and repression by human cytomegalovirus immediateearly proteins involves common and unique protein domains. Journal of Virolog., 64: 1556-1565.
8. Jonjic S, Del Val M, Keil GM, Reddehase MJ, Koszinowski UH (1988). A nonstructural viral protein expressed by a recombinant vaccinia virus protects against lethal cytomegalovirus infection. J. Virol., 62:1653-1658.
9. Lembo D, Angeretti A, Gariglio M, Landolfo S (1998). Murine cytomegalovirus induces expression and enzyme activity of cellular dihydrofolate reductase in quiescent cells. Journal of General Virology., 79: 2803–2807.
10. Margolis MJ, Pajovic S, Wong EL, Wade M, Jupp R, Nelson JA,  Azizkhan JC (1995). Interaction of the 72-kilodalton human cytomegalovirus IE1 gene product with E2F1 coincides with E2Fdependent activation of dihydrofolate reductase transcription. Journal of Virology., 69: 7759-7767.