To identify subregions responsible for self-inhibition of LANA1 protein degradation, we generated LANA1 deletion constructs of CR domains (LANAΔCR123) and each CR subdomain (LANAΔCR1, LANAΔCR2, LANAΔCR3), in the context of full-length LANA1 (Kwun et al., 2007
). A PEST-sequence destabilized EGFP (d1EGFP) protein (Clontech) was cloned into the C-terminus () or N-terminus (Fig. S1
) and protein turnover rates were assessed by measuring fluorescence. LANA1 full-length and deletion mutant constructs were transfected into 293 cells and allowed to recover for 24 hours. Cells were then treated with 100 μg/ml cycloheximide (CHX) for 12 hours to inhibit new protein synthesis and residual EGFP-tagged protein was determined by fluorimetry.
Fig. 1 The central repeat (CR) 2 domain of LANA1 inhibits LANA1 turnover. (A) LANA1 repeat regions (CR1, CR2, CR3) were individually and in combination deleted from full-length LANA1, and cloned as fusion proteins with a destabilized enhanced green fluorescent (more ...)
As seen in , full-length LANA1 protein markedly stabilizes the turnover of PEST-EGFP sequences: 67.5% of LANA1-d1EGFP protein remains 12 hours after CHX inhibition of new protein synthesis while nearly all unfused vector (d1EGFP) protein has undergone degradation during the same period. To exclude the possible effect of the position of GFP tag artificially affecting the stabilization of the fusion proteins, both C- () and N-terminally (Fig. S1
) fused PEST-EGFP constructs were examined and show similar patterns. When each repeat domain is expressed as a PEST-EGFP expression construct individually or in combination with other repeat domains, only LANA1 CR2-containing peptides have significantly diminished protein turnover (). Taken together, these data suggest that CR2 primarily inhibits cis protein turnover in the context of the larger LANA1 structure and also that the primary structure of CR2 alone is sufficient to retard proteasomal degradation.
To determine whether, like EBNA1, LANA1 domains retarding cis
LANA1 translation also inhibit proteasomal degradation, we examined a CR2-EcoRV-CR3 fragment containing an inserted EcoRV DNA sequence that disrupts the junction between the CR2 and CR3 domains. We previously found that this insertion diminishes LANA1 CR translation retardation mediated by the CR2CR3 junction (Kwun et al., 2007
). Turnover of CR2-EcoRV-CR3, however, was unchanged from the parental fragment having an intact CR2-CR3 junction suggesting that this region is not active in preventing protein turnover. Unlike EBNA1, the translation retardation of KSHV LANA1 (CR2-CR3 junction) can be physically separated from its proteasomal inhibition function (CR2 domain).
We took advantage of an ability to dissociate these two processing functions to determine their importance in MHC class I presentation. LANA1 full-length and deletion constructs were fused with EGFP in the N-terminus and the ovalbumin (OVA) peptide SIINFEKL in the C-terminus (). Surface presentation of SIINFEKL by the murine MHC I allele H2Kb
is detected by flow cytometry using the 25-D1.16 antibody (eBioscience) directed against the peptide bound to the H2Kb
cleft (Porgador et al., 1997
). We expressed each construct in human 293KbC2 cells engineered for stable expression of murine H2Kb
(a kind gift of Jonathan Yewdell) (Tscharke et al., 2005
). Expression of each deletion construct was confirmed by immunoblotting (), in vitro
translation (), as well as fluorescence microscopy ().
Fig. 2 Construction and expression of EGFP-LANAOVA. (A) Schematic diagram of EGFP-LANA1OVA constructs used to measure SIINFEKL antigen presentation. Protein expression was determined by immunoblotting (B), in vitro translation labeled with [35S]-Met (C) and (more ...)
As expected, the constructs with deleted CR2 or CR3 domains had higher translation efficiencies compared to full-length LANA1 since this disrupts the CR2-CR3 junction (). Moreover, expression of the CR1 deletion construct had similar translation efficiency as full-length LANA1, consistent with the site of translation retardation lying between CR2 and CR3. These proteins thus accumulated in the nucleus similar to parental LANA1 due to retention of nuclear localization signals present in the N and C-termini of LANA1 ().
Two color flow cytometry for allophycocyanin (APC)-conjugated 25-D1.16 antibody (eBioscience) and EGFP were next used to measure SIINFEKL surface expression in cells expressing the various LANA1 deletion constructs. EGFP vector and EGFPOVA
constructs were used as negative and positive controls, respectively. Consistent with the results of Zaldumbide et al.
(Zaldumbide et al., 2007
), the LANA1-SIINFEKL fusion protein shows marked reduction of SIINFEKL presentation compared to the EGFPOVA
control. We also confirm that LANA1 inhibition of antigen presentation is dependent on the central repeat region since the fusion protein lacking this segment (LANAΔCR123OVA
) is presented to a similar extent as the EGFPOVA
control protein (). Surprisingly, we find that neither the CR2-CR3 junction, which impedes LANA1 translation (), nor the CR2 domain, which inhibits proteasomal processing () are predominantly responsible for SIINFEKL immune evasion (). Instead, all LANA1 constructs lacking CR1, including deletion of CR1 alone (LANAΔCR1OVA
), deletion of the entire CR region (LANAΔCR123OVA
) and deletion of CR1CR2 (LANAΔCR12OVA
), show markedly increased SIINFEKL peptide presentation compared to LANAOVA
. In constrast, LANA1 proteins with the CR2 region deleted (LANAΔCR2OVA
) inhibit MHC I peptide presentation similar to the parental LANA1 molecule. Transfection efficiency for each of the constucts was similar (), as monitored through the expression of EGFP protein by flow cytometry analysis (50 to 70% of 25,000 cells were positive for EGFP expression, ), regardless of translation efficiency (). The amount of total MHC class I level on the surface determined by flow cytometry using anti-mouse MHC class I (H-2Kb) (clone AF6-184.108.40.206, eBioscience) was unchanged despite LANA1 expression (data not shown) indicating that inhibition of SIINFEKL peptide presentation is not due to a general downregulation of MHC I surface expression by LANA1. In addition, cotransfection of CR1 and EGFPOVA
from separate plasmids does not inhibit SIINFEKL presentation (data not shown), indicating that CR1 acts in cis
to prevent MHC presentation of SIINFEKL rather than in trans
. Since surface expression level of MHC I is unchanged by LANA1, it is unlikely that total MHC I is targeted for trans
downregulation as is the case with the KSHV K3 and K5 proteins (Coscoy, 2007
Fig. 3 The CR1 region of LANA1 inhibits MHC I peptide antigen presentation in cis. (A) Representative results from flow cytometry analysis for SIINFEKL presentation on 293KbC2 cells expressing EGFP-LANA1OVA constructs (). Cells were stained with APC anti-mouse (more ...)
We next examined whether LANA1 antigen is processed in a proteasomal-dependent or -independent fashion (e.g., autophagy) by pretreating 293KbC2 cells transfected with various LANA1OVA constructs with MG132 (). As seen in , presentation is largely abolished by MG132 pretreatment, indicating that LANA1 is processed for MHC I presentation through canonical proteasomal processing. Preliminary experiments suggest that neither rapamycin nor chloroquine pretreatment modifies LANA1OVA presentation, consistent with autophagy playing a small role, if any, in LANA1 immune processing (unpublished data). To determine if immunoproteasomal processing is critical to LANA1 presentation, we pretreated cells with interferon gamma (). Relatively higher antigen presentation of LANA1 suggests that immunoproteasomal processing enhances LANA1 presentation.
That LANA1 CR1 inhibits presentation in cis
but does not either retard synthesis of LANA1 or its proteasomal degradation suggests that it might reduce translocation of LANA1 peptides from the cytosol into the ER for loading onto MHC I. We therefore bypassed this step by introducing endoplasmic reticulum (ER) signal peptide (SP) sequences (Persson et al., 1980
) into the N-terminal of EGFP-LANA1OVA
) and then examined expression and antigen presentation. SP construct-expressing cells (green) were also loaded with ER-tracker (red), a marker for the ER, to determine the localization of LANA1 (). In contrast to LANAOVA
without SP, SP-LANAOVA
is efficiently processed for MHC I presentation (). Both LANADΔCR1OVA
proteins, with or without the SP, undergo similar levels of antigen presentation suggesting that CR1 inhibits pre-ER steps in peptide presentation (). Pretreatment of SP-LANAOVA
with MG132 diminished peptide presentation and so we cannot exclude that this protein undergoes retro-translocation and proteasomal degradation.
Fig. 4 The effect of endoplasmic reticulum (ER) targeting signal peptide (SP) on LANA1 antigen presentation. (A) LANA1 expression was determined with EGFP (green) and ER-Tracker (red) fluorescence in living cells. The EGFP signal of SP constructs precisely overlapped (more ...)