Processing and MHC class I presentation of human cytomegalovirus pp65-derived peptides persist despite gpUS2–11-mediated immune evasion

Abstract

Immune control of human cytomegalovirus (HCMV) infection can be mediated by CD8 + cytolytic T lymphocytes (CTL). Adoptive transfer of antiviral CTL confers protection against HCMV reactivation and disease. The tegument protein pp65 and the immediate-early 1 protein (IE1) are recognized to be major CTL targets, even though during productive infection the viral immunoevasion proteins gpUS2-11 act to suppress major histocompatibility complex (MHC) class I-restricted antigen presentation. Thus it was not clear how infected cells could be labelled with antigenic peptides in the face of immunoevasion. We show here that the immunodominant peptide pp65 NLV was presented by MHC class I in cells infected with a gpUS2-11-competent virus. Presentation of pp65 NLV was still detectable at 96 h post-infection, although at low levels. Partial suppression of pp65 NLV presentation was dependent on the ability of the infecting strain to express gpUS2-11. MHC class I-restricted antigen presentation in HCMV-infected cells (encoding gpUS2-11) exhibited specificity for pp65-derived peptides, as infected fibroblasts did not present the IE1-derived nonapeptide IE1 TMY . Remarkably, infected cells could restore pp65 NLV peptide presentation after acid removal of MHC class I despite gpUS2-11 expression. This recovery was shown to be dependent on proteasome functionality. In contrast to IE1, pp65 peptides are loaded on MHC class I molecules to be transported to the cell surface at early and late times after infection in the face of gpUS2-11-mediated immunoevasion. pp65 is therefore the first example of an HCMV protein only incompletely subjected to gpUS2-11-mediated immunoevasion. INTRODUCTIONInfection with human cytomegalovirus (HCMV) affects more than 50 % of the human population. Individuals with immature or compromised immune defence functions are more likely to encounter severe clinical disease conditions. In healthy individuals, however, HCMV is normally efficiently controlled by the immune system (Pass, 2001), and in particular by HMCV-specific CD8 + cytoxic T lymphocytes (CTL) (Reddehase et al., 1985;Reusser et al., 1991). The CTL response is primed against short peptides that are proteolytically processed from intracellular viral proteins and presented on the cell surface by major histocompatibility complex (MHC) class I molecules (Kloetzel, 2004). For this, proteins are labelled by ubiquitination to be targeted to proteasomes for degradation. Peptides of appropriate length are released from proteasomes and processed further at their amino terminus by cytosolic aminopeptidases. Ultimately they are translocated to the endoplasmic reticulum (ER) by the transporter associated with antigen presentation (TAP), where they associate with MHC class I. In some instances, ER-resident proteases contribute to the trimming of antigenic peptides (reviewed by Kloetzel, 2004).The HCMV genome encodes over 150 proteins, which can potentially serve as target antigens for CTL (Chee et al., 1990). Recent studies showed that memory CTL isolated from HC...

Exogenous introduction of an immunodominant peptide from the non-structural IE1 protein of human cytomegalovirus into the MHC class I presentation pathway by recombinant dense bodies

“…Human foreskin fibroblasts (HFF) and CTL lines were grown as described previously (Besold et al, 2007). For mutagenesis, the HCMV strain RV-HB5 was used as parental strain (Borst et al, 1999).…”

“…SDS-PAGE, immunoblotting and immunofluorescence analyses were carried out as described previously (Schmolke et al, 1995 Gamma interferon (IFN-c)-based ELISpot assay and cytotoxicity assay. ELISpot assays and cytotoxicity assays were performed as described previously (Besold et al, 2007;Frankenberg et al, 2002). CTL lines specific for the HLA-A0201 (A2)-restricted HCMV-derived peptides pp65 [495][496][497][498][499][500][501][502][503] (pp65 NLV -CTL) (Diamond et al, 1997;Wills et al, 1996) and IE1 297-305 (IE1 TMY -CTL) (Gallez-Hawkins et al, 2003) were used in these analyses.…”

“…Signal intensity was comparable for the two viruses at each time point tested. Cells infected with the pp65 deletion mutant RVHd65 (Besold et al, 2007), used as a negative control, showed no specific staining. These experiments demonstrated that the kinetics of intracellular transport of the pp65-IE1 TMY fusion protein in RV-VM3-infected cells resembled those of wt pp65.…”

“…Previous studies provided evidence that peptides derived from exogenously delivered, particle-associated HCMV proteins are presented by MHC class I molecules without requiring de novo protein synthesis (Besold et al, 2007;Manley et al, 2004;McLaughlin-Taylor et al, 1994;Pepperl et al, 2000;Reddehase et al, 1984;Riddell et al, 1991). It was previously unknown whether peptides not usually included in viral particles would also be introduced into MHC class I presentation.…”

“…CTL lines specific for the HLA-A0201 (A2)-restricted HCMV-derived peptides pp65 [495][496][497][498][499][500][501][502][503] (pp65 NLV -CTL) (Diamond et al, 1997;Wills et al, 1996) and IE1 297-305 (IE1 TMY -CTL) (Gallez-Hawkins et al, 2003) were used in these analyses. The CTL lines had been generated by immunizing HLA-A2/huCD8 double-transgenic (tg) mice (Besold et al, 2007). …”