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Portion of HCMV

Definition
The commonly used cytomegalovirus (CMV) peptides used for therapeutic approaches are pp65 and pp71. pp65 is also known as glycoprotein 64 or UL83 is a virion tegument protein and the main component of the enveloped subviral particle of CMV. The human cytomegalovirus (HCMV) tegument protein pp71 is an upper matrix protein  involved in gene regulation and is encoded by gene UL82.

Discovery
The identification of DNA sequences coding for a virion phosphoprotein of 71 kDa and a viral 65-kDa polypeptide was done by Nowak B et.al, in early in 1980s1.

Structural Characteristics
CMV pp65 belongs to the herpesviridae pp65 family. HCMV contains a phosphorylated matrix protein of 65,000 apparent molecular weight (65K phosphoprotein; pp65) and a related phosphoprotein of 71,000 molecular weight (pp71). The 65K phosphoprotein is usually by far the most abundant structural component found in culture-grown purified virus particles. The 65K phosphoprotein is coded for by the 5'-terminal part of an abundant 4-kilobase (kb) mRNA. The structural protein pp65 forms about 95% of the protein mass in dense bodies. The nucleotide sequence of the entire coding domain for pp65 and pp7l,identifies separate translational reading frames and transcripts for each protein, and analyzes their RNA structures. CMV pp65 contains elements of the prototypic nuclear localization signal (NLS) in which arginine and lysine predominate within a bipartite motif in which short regions of basic amino acids are separated by 10 or more nonconserved amino acids. The nuclear localization signals of CMV pp65 consist of at least two such motifs located in the carboxy-terminal region of the polypeptide.A second NLS of CMV pp65 consists of a basic region of amino acids between aa 537 and 561; this region was termed the C-D motif by Schmolke et al,2, 3. The primary sequence of HCMV is Asn-Leu-Val-Pro-Met-Val-Ala-Thr-Val.

Mode  of Action
CMV pp65 as a nucleotropic protein which enters the nucleus immediately after infection. It binds to polo-like kinase 1 (PLK-1), an enzyme important in mitosis and it is likely that the protein has specific effects on cell cycle events 4. CMV pp65 has been shown to have protein kinase activity. CMV pp65 is an immunodominant target of CD4+ and CD8+ Tcell response in CMV. CMV pp65 specific T cell predominantly produces cytokines like IFN-γ, IL-2, and TNF-α. HCMV pp71 is delivered directly to cells by infecting HCMV virions. At the start of lytic infections, it travels to the nucleus and stimulates viral IE gene expression by displacing the chromatin remodeling protein ATRX from Daxx and by mediating Daxx degradation through a rare ubiquitin-independent, proteasome-dependent process5.

Functions
CMV pp65 has been the prototypic antigen for the demonstration of CMV-specific T-cell immunity, it is likely that other proteins of CMV will be necessary for the development of a vaccine that generates humoral and cellular protection. CMV pp65-specific T-cell responses have been used for the development of other immunotherapeutic approaches to the control of CMV infection.

HCMV protein pp65 is an efficient protein carrier system into human dendritic cells 6. It is also major target for the cellular immune response. HCMV Protein pp71 disrupts major histocompatibility complex class I cell surface expression. HCMV tegument protein pp71 (ppUL82) enhances the infectivity of viral DNA and accelerates the infectious cycle. Human CMV pp65 virion protein inhibits antiviral gene expression in infected cells. HCMV pp65 mediates accumulation of HLA-DR in lysosomes and destruction of the HLA-DR α-chain 7.

References

1.     Nowak B, Gmeiner A, Sarnow P, Levine AJ, Fleckenstein B (1984). Physical mapping of human cytomegalovirus genes: identification of DNA sequences coding for a virion phosphoprotein of 71 kDa and a viral 65-kDa polypeptide. Virology, 134(1):91-102.

2.     Rüger B, Klages S, Walla B, Albrecht J, Fleckenstein B, Tomlinson P, Barrell B (1987). Primary structure and transcription of the genes coding for the two virion phosphoproteins pp65 and pp71 of human cytomegalovirus. J Virol., 61(2):446-453.
3.     Schmolke S, Drescher P, Jahn G, Plachter B (1995). Nuclear targeting of the tegument protein pp65 (UL83) of human cytomegalovirus: an unusual bipartite nuclear localization signal functions with other portions of the protein to mediate its efficient nuclear transport. J Virol., 69(2):1071-1078.
4.     Zaia JA, Li X, Franck AE, Wu X, Thao L, Gallez-Hawkins G (2009). Biologic and immunologic effects of knockout of human cytomegalovirus pp65 nuclear localization signal. Clin Vaccine Immunol., 16(6):935-943.
5.     Hwang J, Kalejta RF (2009). Human cytomegalovirus protein pp71 induces Daxx SUMOylation. J Virol., 83(13):6591-6598.
6.     Scheller N, Furtwängler R, Sester U, Maier R, Breinig T, Meyerhans A (2008). Human cytomegalovirus protein pp65: an efficient protein carrier system into human dendritic cells. Gene Ther.,15(4):318-325.
7.     Browne EP, Shenk T (2003). Human cytomegalovirus UL83-coded pp65 virion protein inhibits antiviral gene expression in infected cells. PNAS., 100(20):11439-11444.

Definition

The CEF control peptides are 8-12 amino acids in length, with sequences derived from the human Cytomegalovirus, Epstein-Barr Virus and Influenza Virus1 These peptides are used in the stimulation of IFNg release from CD8+ T cells in individuals with defined HLA types1.  They are useful as positive control peptides in several cytokine assays such as Elispot.

Discovery

CEF peptides were first selected in 2002 based on their ability to recognize CD8+ T cells1.

Classification

They are derived from epitopes of viruses and hence have antigenic properties1.

Structural Characteristics

CEF peptides are 8-11 amino acids long with sequences: GILGFVFTL (Influenza A, HLA-A2), FMYSDFHFI (Influenza A, HLA-A2), CLGGLLTMV (EBV, HLA-A2), GLCTLVAML (EBV, HLA-A2), NLVPMVATV (HCMV, HLA-A2).

Mode of action

CEF peptides are effective epitopes for CD8+ T cells2.  They bind to these cells and trigger the production of IFNg.

Functions

CEF control peptides are used as positive control in Elispot assay that is used to investigate specific immune responses in various diseases including infections, cancer, allergies and autoimmune diseases2.  In this case the CEF peptides ensure that the cells under study are active and viable2.  Elispot is also useful in the development of vaccines especially for HIV where CEF peptides are used also as controls2.

References

1.     Currier JR, Kuta EG, Turk E, Earhart LB, Loomis-Price L, Janetzki S, Ferrari G, Birx DL, Cox JH (2002). A panel of MHC class I restricted viral peptides for use as a quality control for vaccine trial ELISPOT assays, J Immunol Methods, 260, 157-172.

2.     Gazagne A, Claret E, Wijdenes J, Yssel H, Bousquet F, Levy E, Vielh P, Scotte F, Goupil T, Fridman WH, Tartour E (2003). A Fluorospot assay to detect single T lymphocytes simultaneously producing multiple cytokines, J Immunol Methods, 283(1-2), 91-98.