Noncanonical Expression of a Murine Cytomegalovirus Early Protein CD8 T-Cell Epitope as an Immediate Early Epitope Based on Transcription from an Upstream Gene

Abstract

Viral CD8 T-cell epitopes, represented by viral peptides bound to major histocompatibility complex class-I (MHC-I) glycoproteins, are often identified by “reverse immunology”, a strategy not requiring biochemical and structural knowledge of the actual viral protein from which they are derived by antigen processing. Instead, bioinformatic algorithms predicting the probability of C-terminal cleavage in the proteasome, as well as binding affinity to the presenting MHC-I molecules, are applied to amino acid sequences deduced from predicted open reading frames (ORFs) based on the genomic sequence. If the protein corresponding to an antigenic ORF is known, it is usually inferred that the kinetic class of the protein also defines the phase in the viral replicative cycle during which the respective antigenic peptide is presented for recognition by CD8 T cells. We have previously identified a nonapeptide from the predicted ORFm164 of murine cytomegalovirus that is presented by the MHC-I allomorph H-2 Dd and that is immunodominant in BALB/c (H-2d haplotype) mice. Surprisingly, although the ORFm164 protein gp36.5 is expressed as an Early (E) phase protein, the m164 epitope is presented already during the Immediate Early (IE) phase, based on the expression of an upstream mRNA starting within ORFm167 and encompassing ORFm164.

Reconstitution of CD8 T Cells Protective against Cytomegalovirus in a Mouse Model of Hematopoietic Cell Transplantation: Dynamics and Inessentiality of Epitope Immunodominance

“…In essence, for an observation period of 1 year, epitope-specific responses were highly variable between the three HCTs and far from continuity within any of the three HCTs. For the response to IDEs m123/IE1 and m164, both of which are expressed in the IE phase of the viral gene expression program ( 53 , 89 ), the time of the peak acute response was variable and contraction was not pronounced. One may discuss contraction at 10 and 6 weeks in HCT #1 and HCT #3, respectively, but not in HCT #2.…”

“…To account for different kinetic classes of viral protein expression, stimulator cells in the assay were metabolically arrested in the “IE” phase, or in the “E” phase, or were allowed to proceed to the “late” (L) phase. As a remark, IE phase expression of the antigenic peptide assigned to the E Phase protein m164 has recently been shown to result from a transcript starting within an upstream IE gene ( 89 ). With the exception of a minor residual recognition of the Ala-variant L459A of the m145 E-phase peptide, which is paralleled by a still high constitutive proteasome combined processing score (Figure 4 A, upper panel), the recognition of IDEs was abolished by the C-terminal replacements with Ala.…”

Cytomegalovirus-Associated Inhibition of Hematopoiesis Is Preventable by Cytoimmunotherapy With Antiviral CD8 T Cells

“…A virus-specific cytolytic T lymphocyte (CTL) line specific for the D d -presented immunodominant viral epitope m164 Fink et al, 2014) was generated by four rounds of restimulation of CD8 + memory T cells, which were derived from the spleen of latently mCMV-infected female (sry − ) BALB/c mice, with the respective synthetic m164 peptide at a concentration of 10 −9 M Lemmermann et al, 2010). For cytoimmunotherapy of the infection, 1 × 10 6 CTL were infused intravenously together with the HC.…”

“…Virus replication was quantitated on day 14, because group A recipients would die of multiple organ CMV disease and BM aplasia soon afterwards (Mutter et al, 1988). CTL specific for the m164 epitope of mCMV were chosen because the epitope is very special in that it is encoded by transcripts of the viral Early (E) phase gene ORFm164 as well as by a transcript from an upstream Immediate-Early (IE) phase gene (Fink et al, 2014;Renzaho et al, 2019). Accordingly, m164 epitope-specific CTL have the chance to recognize the corresponding D d -presented antigenic peptide on infected cells in the IE phase as well in the E phase of the viral replicative cycle, and this likely enhances the antiviral activity.…”

Recent advances in CMV tropism, latency, and diagnosis during aging

“…We tried to address these points in our group over the past years and provided evidence strongly supporting the hypothesis that MI is driven by viral epitopes presented by latently infected host-tissue cells of non-hematopoietic origin (Seckert et al 2011;Torti et al 2011). We are currently investigating if mCMV gene m164, a second driver of MI in the H2 d haplotype (Holtappels et al 2002;Fink et al 2014), is also expressed in latency as a TEL.…”