MHC Class I Antigen Processing Distinguishes Endogenous Antigens Based on Their Translation from Cellular Vs

MHC class I antigen processing distinguishes endogenous antigens based on their translation from cellular vs. viral mRNA

Brian P. Dolan, Aditi A. Sharma, James S. Gibbs, Tshaka J. Cunningham, Jack R. Bennink, and Jonathan W. Yewdell1

Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892

Edited by Thierry Boon, Ludwig Institute for Cancer Research, Brussels, Belgium, and approved March 23, 2012 (received for review July 29, 2011) To better understand the generation of MHC class I-associated presented at high (15–20%) efficiency in a macrophage-like cell peptides, we used a model antigenic protein whose proteasome- line whether synthesized by VV or intracellular Listeria (8), showing mediated degradation is rapidly and reversibly controlled by Shield- that two very different systems of antigen introduction into the 1, a cell-permeant drug. When expressed from a stably transfected cytosol need not differ widely in processing efficiency. gene, the efficiency of antigen presentation is ∼2%, that is, one cell- Taken together, these findings demonstrate system-dependent- surface MHC class I–peptide complex is generated for every 50 folded wide variation in the efficiencies of generating complexes from source proteins degraded upon Shield-1 withdrawal. By contrast, full-length proteins and oligopeptides. To better understand these when the same protein is expressed by vaccinia virus, its antigen differences, here we measure the context dependence of antigen- presentation efficiency is reduced ∼10-fold to values similar to those processing efficiency using a number of antigens, cells, and ex- reported for other vaccinia virus-encoded model antigens. Virus in- pression vectors. fection per se does not modify the efficiency of antigen processing. Rather, the efficiency difference between cellular and virus-encoded Results antigens is based on whether the antigen is synthesized from trans- Kb–SIINFEKL Is Generated from SCRAP at Remarkably High Efficiency gene- vs. virus-encoded mRNA. Thus, class I antigen-processing ma- in EL4 Cells. Building on the work of the Wandless laboratory (9),

chinery can distinguish folded proteins based on the precise details of we recently described SCRAP, a chimeric antigen whose stability IMMUNOLOGY their synthesis to modulate antigen presentation efficiency. is precisely controlled by the cell-permeant drug Shield-1 (10). SCRAP consists of a Shield-1 interaction domain NH2-terminally D8+ T lymphocytes play a key role in immunosurveillance by fused to SIINFEKL and GFP (10) (Fig. 1A). In the absence of Ceradicating tumor cells and cells harboring viruses and other Shield-1, nascent SCRAP is degraded with a t1/2 of 16 min. In the intracellular pathogens. Recognition is based on the interaction presence of Shield-1, a stable pool of SCRAP accumulates that is of the clonally restricted T-cell receptor with MHC class I mol- degraded with a t1/2 of 30 min upon removal of Shield-1 (10). ecules bearing oligopeptides. Peptides are predominantly gener- Fig. 1 B and C illustrate the behavior of SCRAP when expressed b ated from defective ribosomal products (DRiPs), nascent as a permanently transfected gene in EL4 cells, an H-2 T-cell translation products that are degraded rapidly either by design lymphoma. Via flow cytometry, properly folded fluorescent b (i.e., dedicated for antigen presentation) or necessity (defective SCRAP and K –SIINFEKL are measured simultaneously in live forms of proteins that can interfere with cell function) (1). The cells based on, respectively, GFP and directly conjugated 25-D1.16 use of DRiPs greatly speeds recognition of virus-infected cells, mAb (11). Because GFP fluorescence requires proper folding and because many peptides derive from otherwise highly stable pro- not all SCRAP is properly folded, only measuring fluorescent teins. For viral infections, the efficiency of recognition afforded SCRAP will underestimate the amount of SCRAP present in cells. by DRiPs is of the essence, because viruses can replicate within Via immunoblotting, however, we can measure all SCRAP forms in hours, and killing must be fast to be effective. Time is not limiting total cell extracts that interact with a mAb specific for GFP (Fig. for tumor cells but efficiency is critical, because T cells often 1D). Using purified GFP of known concentration to generate recognize antigens derived from gene products expressed at ex- a standard curve, we could calculate the average number of im- tremely low levels. Indeed, T cells may be the only means of munoreactive SCRAP molecules expressed per cell (Fig. 1D). We detecting expression of the source antigen (2). simultaneously determined absolute numbers of directly conju- Only a handful of studies have addressed the efficiency of pep- gated 25-D1.16 bound to cells using flow cytometry standardization tide generation, a critical issue with broad implications for the ef- beads as described in Materials and Methods. At the start of the ficiency of protein synthesis and cytosolic compartmentalization of efficiency measurement period, we treated cells with mild acid (pH antigen processing (3). Pamer and colleagues seminally reported 3) to destroy preexisting Kb–SIINFEKL complexes, thereby im- that class I–peptide complexes are generated with an efficiency of proving the signal-to-noise ratio. 3–25% (i.e., 3–25 complexes generated per 100 proteins degraded) The SCRAP system enabled us to determine the efficiency of from Listeria monocytogenes proteins secreted into the cytosol of peptide generation from nascent proteins vs. retirees (12). mouse macrophage-like cells (4, 5). Using recombinant vaccinia Retirees are folded, functional proteins that are selected for viruses (rVVs) to express a rapidly degraded full-length chimeric degradation either stochastically according to the classical view protein, we reported that the efficiency of complex (Kb–SIIN- (13) or, in this case, due to unfolding based on Shield-1 with- FEKL) generation was much lower in a variety of mouse fibroblast drawal. For nascent proteins, we determined the amount of and macrophage/dendritic cell (DC)-like cell lines, 0.25%–0.05% (1/400–1/2,000), with an efficiency of ∼2% for SIINFEKL synthe- sized as the MSIINFEKL minigene product (6). Fruci et al. devised Author contributions: B.P.D., J.R.B., and J.W.Y. designed research; B.P.D. and A.A.S. per- a clever method for quantitating minimal peptides released into the formed research; J.S.G. and T.J.C. contributed new reagents/analytic tools; B.P.D., A.A.S., cytosol based on liberation from chimeric GFP-ubiquitin fusion J.R.B., and J.W.Y. analyzed data; and B.P.D., A.A.S., J.R.B., and J.W.Y. wrote the paper. proteins by ubiquitin hydrolases, reporting an efficiency of 0.2% (1/ The authors declare no conflict of interest. 500) for EBV-transformed human B cells expressing permanently This article is a PNAS Direct Submission. transfected genes (7). Extending this approach, Wolf and Princiotta Freely available online through the PNAS open access option. demonstrated that ubiquitin hydrolase-liberated SIINFEKL is 1To whom correspondence should be addressed. E-mail: [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.1112387109 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 Fig. 1. Model antigens and a schematic of SCRAP presentation and quantification. (A) The various constructs used in this study are depicted. The red “S” box denotes the SIINFEKL peptide, influenza A virus (IAV) nucleoprotein is in orange, and the green “Ub” box represents ubiquitin. (B) EL4/SCRAP cells were washed in a mild citric acid buffer (pH 3.0) to remove existing Kb–SIINFEKL complexes and cultured in the presence or absence of 5 μM Shield-1. At the indicated times, Kb–SIINFEKL complexes and GFP levels were determined by flow cytometry. (C) Same as in A, except Shield-1 was removed following an initial 3.5-h incubation and, following a second acid wash, cells were cultured in the presence of cycloheximide (CHX). (D) An example of a Western blot for GFP using recombinant GFP standards and lysates from EL4/SCRAP cells treated with or without Shield-1 for 3 h.

SCRAP rescued over a 4-h period by Shield-1 and the concomi- cytometry as cells infected with rVV expressing NP-S-GFP, and tant decrease in Kb–SIINFEKL expression (Fig. 1B). For retirees, generated essentially identical amounts of Kb–SIINFEKL. In the we accumulated a pool of SCRAP by treating for 4 h with Shield- absence of Shield-1, nearly identical amounts of fluorescent GFP 1, and then induced degradation by removing Shield-1 while were detected compared with Ub-R-S-GFP (6), and peptides measuring the increase in Kb–SIINFEKL expression concomitant were generated at half the rate from SCRAP vs. rapidly degraded with SCRAP degradation (Fig. 1C) in the presence of cyclohexi- NP. Therefore, an intrinsic difference between SCRAP and NP-S- mide to prevent newly synthesized and rapidly degraded SCRAP GFP (or Ova, also reported in the original study) does not account from entering the antigen presentation pathway. for the high efficiency of peptide generation from SCRAP in The efficiency of generating Kb–SIINFEKL complexes was EL4 transfectants. statistically indistinguishable between Shield-1–sensitive nascent and retiree pools (Tables 1 and 2), and was calculated to be ∼2%. Remarkably, this is 40-fold more efficient than generation Table 1. Efficient antigen presentation of peptides derived of Kb–SIINFEKL complexes from VV-expressed rapidly de- from rapidly degraded self-proteins graded nucleoprotein (NP)- SIINFEKL (S)-GFP [ubiquitin Kb–SIINFEKL GFP Calculated fi (Ub)-R-S-GFP in Fig. 1A], and 10-fold more ef cient than Nascent Experiment complexes molecules efficiency (%) misfolded NP-S-GFP due to the insertion of a KEKE sequence in the NP domain (6, 14). 1 5,445 2.54 × 105 2.1 These large differences could be related to differences in the cell 2 2,958 5.62 × 105 0.5 type studied (EL4- vs. L-Kb), antigen (SCRAP vs. NP-S-GFP), or 3 3,104 6.83 × 104 4.5 expression system (transfection vs. VV infection). To distinguish Average 2.4 fi between the types of antigens, we measured the ef ciency of anti- EL4/SCRAP cells were treated as in Fig. 1. Peptide–MHC complexes were gen presentation from VVs expressing SCRAP vs. NP-S-GFP (Fig. determined by quantitative flow cytometry. Shield-1–sensitive nascent b 2). L-K cells infected with VV SCRAP in the presence of Shield-1 SCRAP molecules were quantitated via immunoblotting. Efficiencies were expressed nearly identical amounts of GFP as detected by flow calculated at 4 h after addition of Shield-1.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1112387109 Dolan et al. Downloaded by guest on September 29, 2021 Table 2. Retired SCRAP is presented with similar efficiency as Could the specifics of gene expression related to host- vs. virus- nascent, rapidly degraded SCRAP directed expression influence the efficiency of antigen processing? Kb–SIINFEKL GFP- Calculated To explore this possibility, we transiently transfected SCRAP in b fi Retired Experiment complexes degraded efficiency (%) HeLa K cells and compared their antigen-processing ef ciency to HeLa Kb cells infected with rVV SCRAP at a dose titrated to 1 297 15,556 1.9 match the rate of SCRAP synthesis in the transfected cells (Fig. 4 A 2 793 33,106 2.4 and B). Although the cells were synthesizing near-identical 3 1,156 51,454 2.2 amounts of SCRAP from host vs. VV mRNA, transfectants gen- Average 2.2 erated Kb–SIINFEKL complexes at a much higher rate from the Shield-1–sensitive pool (Fig. 4 A and B). In five separate experi- EL4/SCRAP cells were treated as in Fig. 1C. Peptide–MHC complexes were fl ments (Table 3), we determined that the average fold increase in determined by quantitative ow cytometry. Retired SCRAP molecules were fi quantitated via immunoblotting. Efficiencies were calculated 2 h following antigen presentation ef ciency of transfect-encoded protein com- the removal of Shield-1. pared with VV-encoded protein was 8.3 (±1.6 SD units, P < 0.05). Notably, the efficiency of Kb–SIINFEKL generation from transfected SCRAP in HeLa vs. EL4 cells was similar (0.6% vs. Are there cell type-related differences in antigen-processing ef- 2.4%), particularly when considering that a mismatch between the ficiency? Because EL4 cells are highly resistant to VV infection, we mouse class I molecule (Kb) and human antigen-processing ma- compared the antigen-processing efficiencies of EL4 vs. L-Kb cells chinery (TAP, tapasin, other chaperones, and endoplasmic re- using recombinant vesicular stomatitis viruses (rVSVs) expressing ticulum aminopeptidase) likely lowers peptide loading efficiency. Venus fluorescent protein (VFP) fused to ubiquitin followed by the There was a reasonably good match between the efficiency of SIINFEKL peptide (VFP-Ub-S). Following infection with VSV- generating Kb–SIINFEKL from the Shield-1–sensitive pool of VFP-Ub-S, the cell lines expressed nearly identical amounts of Kb– VV-encoded SCRAP (1/1,250) and our previous determination SIINFEKL (Fig. 3A, red trace), although EL4 cells expressed al- of the efficiency of generating Kb–SIINFEKL from rapidly de- most 50% more fluorescent protein signal (Fig. 3A, black trace). As graded NP in various mouse cell types (as high as 1/1,400 in DC peptides are in excess under these conditions (3), this demonstrates 2.4 cells) or slowly degraded, misfolded NP (1/440) (6). that EL4 and L-Kb cells have a similar overall capacity to generate What is the contribution of virus-induced alterations in cell surface Kb–SIINFEKL complexes from a cytosolic SIINFEKL function to modulating antigen presentation efficiency? We pool. This implies similar functional levels of the transporter infected EL4/SCRAP cells with VSV or HeLa Kb/transient IMMUNOLOGY associated with antigen processing (TAP), Kb, and intracellular SCRAP cells with rVV, acid-stripped preexisting Kb–SIINFEKL trafficking machinery in EL4 and L-Kb cells. complexes, and measured the efficiency of Kb–SIINFEKL gener- We next compared the abilities of EL4 and L-Kb cells to ation from nascent, Shield-1–sensitive SCRAP synthesized during generate Kb–SIINFEKL complexes from a full-length stable viral infection (Fig. 4C). Infection with VSV or VV resulted in protein expressed by VSV. Following infection with rVSV-NP-S- a partial decrease in both Kb–SIINFEKL and GFP expression, but GFP, EL4 and L-Kb cells expressed nearly identical levels of had only a minor (less than 20%) inhibitory effect on the overall fluorescent NP-S-GFP between 1 and 3 h postinfection (hpi) (Fig. efficiency of antigen presentation. 3A). Levels of Kb–SIINFEKL were similar, although L-Kb cells Taken with the data in Table 3, these findings point to the demonstrated a more complicated pattern, with increased im- remarkable conclusion that the antigen-processing machinery mediate presentation and a 50% decrease in complex generation has the capacity to distinguish folded proteins based on their between 2 and 3 hpi Overall, however, the efficiency of gener- synthesis from cell- vs. virus-encoded mRNA. The same peptide ating Kb–SIINFEKL complexes from DRiPs derived from this from the same folded protein pool in the same cell line is gen- full-length stable protein was similar between the two cell types. erated approximately eightfold more efficiently when synthesized We also examined the efficiency of Kb–SIINFEKL generation from cell- vs. VV-encoded RNA. in L-Kb cells infected with rVV-NP-S-GFP or rVSV-NP-S-GFP What about DRiP efficiency? The SCRAP system allows us to with virus dose adjusted to yield equivalent levels of NP-S-GFP separate the presentation of peptides from retired vs. DRiP sub- fluorescence. Generation of Kb–SIINFEKL complexes was su- strates, which are the only source of peptides when saturating perimposable (Fig. 3B), demonstrating that antigen presentation amounts of Shield-1 are added to SCRAP-expressing cells (10). can occur at similar efficiency from two very different viral vectors. We compared the amount of GFP to Kb–SIINFEKL synthesized in acid-stripped HeLa cells treated with Shield-1 and expressing Antigen Processing Efficiency is Dependent on the mRNA Source of SCRAP from transfected vs. VV-expressed genes. This revealed the Antigen. The findings demonstrate that the efficiency of gen- that VV infection of HeLa Kb cells per se has little effect on Kb– erating Kb–SIINFEKL complexes is similar between EL4 and L- SIINFEKL processing from DRiPs derived from cell-encoded Kb cells and between rVV and rVSV expressing NP-S-GFP. SCRAP (Fig. 4D, infected), whereas DRiPs derived from

Fig. 2. SCRAP protein expressed by rVV is similar to other model antigens. rVV expressing either SCRAP, NP-S-GFP, or Ub-R-NP-S-GFP was used to infect L-Kb cells. Cells were cultured with or without Shield-1 (for SCRAP infection) and analyzed at the indicated times for GFP (Left)orKb–SIINFEKL (Right)byFACS analysis.

Dolan et al. PNAS Early Edition | 3of6 Downloaded by guest on September 29, 2021 Fig. 3. Similar antigen presentation kinetics between different cell types and viruses. (A) L-Kb and EL4 cells were infected with rVSV expressing either Venus- Ub-SIINFEKL (Left) or NP-S-GFP proteins (Right), and antigen presentation as well as fluorescent protein expression were monitored by FACS. (B)L-Kb cells were infected with rVV and rVSV viruses expressing NP-S-GFP and monitored as in A.

VV-encoded SCRAP have approximately twofold reduced effi- remarkably high efficiency, at 2% (1 Kb–SIINFEKL complex ciency. Thus, viral infection is either associated with fewer generated per 50 precursors degraded). This value is ∼40 times SCRAP DRiPs synthesized or diminished efficiency of DRiP higher than our previous determination of Kb–SIINFEKL gen- conversion to Kb–SIINFEKL complexes. eration from a rapidly degraded VV-encoded protein (6). All things being equal, a 2% antigen-processing efficiency is ex- Discussion tremely difficult to square with cellular protein economy (6, 12, We have made two findings, each startling, that point to major gaps 15). Even in the absence of DRiPs and other rapidly degraded in our understanding of the class I antigen-processing pathway. polypeptides (16), normal cellular protein turnover amounts to − − First, we show that cellular gene products in the form of the ∼109 d 1,or7× 105 proteins min 1, potentially spawning − model antigen SCRAP can be processed from a folded state at peptides that bind a given class I allomorph at 3.5 × 106 min 1

Fig. 4. Virus-expressed antigens are presented at lower efficiencies than self-antigens. (A) HeLa Kb cells were either infected with rVV SCRAP or transfected with SCRAP DNA constructs, and antigen presentation as well as GFP expression were determined 4 hpi or post-acid wash. (B) Representative histograms after 4 h of Shield-1 treatment of SCRAP-expressing cells (blue trace) compared with non-SCRAP-expressing cells (red trace). Kb–SIINFEKL staining with 25-D1.16 mAb (Right) is restricted to GFP-positive cells. (C) EL4/SCRAP or HeLa Kb cells transfected with SCRAP DNA were infected with VSV or rVV, respectively, and both Kb– SIINFEKL and GFP levels were determined 4 hpi. The efficiency of presentation was determined by dividing the MFI of Kb–SIINFEKL staining by the MFI of GFP. Values were compared with uninfected cells and are plotted as a percentage. (D) Levels of Kb–SIINFEKL derived from DRiP substrates (in the presence of Shield- 1) from SCRAP expressed as transfected DNA or by rVV in HeLa Kb cells were normalized to GFP expression. Cells (both transfected and nontransfected) were briefly washed in mild acid and cultured for 2 h in complete media. Cells were then harvested and were either left uninfected or infected with control rVV at an MOI of 10, followed by an additional 4 h in culture before FACS analysis. Background levels of Kb–SIINFEKL were determined immediately following rVV infection. These data are representative of three independent experiments.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1112387109 Dolan et al. Downloaded by guest on September 29, 2021 Table 3. Transfected SCRAP is presented more efficiently than SCRAP expressed by rVV infection Transfected SCRAP rVV-infected SCRAP

Fold increase Experiment Kb–SIINFEKL GFP-degraded Efficiency (%) Kb–SIINFEKL GFP-degraded Efficiency (%) (transfectant/infected)

1 2,550 2.9 × 105 0.88 857 8.9 × 105 0.10 8.8 2 597 1.1 × 105 0.54 419 7.1 × 105 0.06 9.0 3 879 3.5 × 105 0.25 289 9.4 × 105 0.03 8.3 4 1,543 2.0 × 105 0.79 830 5.9 × 105 0.14 5.6 5 1,680 2.5 × 105 0.69 440 6.0 × 105 0.07 9.9 Average 8.3 ± 1.6

HeLa Kb cells were transfected with DNA constructs encoding SCRAP or infected with rVV expressing SCRAP and treated with or without Shield-1 for 4 h. The number of Kb–SIINFEKL complexes derived from a defined number of SCRAP molecules, determined by quantitative immunoblotting for GFP, is shown. The difference in efficiencies between transfected and infected cells is statistically significant (Student’s t test, P < 0.05).

[a 500-residue protein possesses 500 potential n-mer peptides, of SCRAP with altered primary sequences that enhance antigen which 5 (1%) bind with immunogenic affinity (17)]. A 2% effi- processing. Still, there is nothing known about antigen processing ciency would mean that greater than 7 × 104 peptides are loaded to explain why one form of a folded gene product would be a − onto class I molecules min 1, which would competitively preclude superior source of a given peptide, pointing to a significant lacuna b 2 K –SIINFEKL generation, because there are only ∼10 class I in our understanding. −1 molecules exported min per cell. One or more of these num- One explanation is that SCRAP synthesized from cellular bers, therefore, must either be in error or unrepresentative. mRNA is better-partitioned into a local antigen-processing com- Two factors potentially contribute to the remarkably high ef- partment(s) defined by lack of competition (26) or localized pre- fi ciency of SCRAP retiree processing. First, SIINFEKL may be sentation (27). Whereas it is hard to imagine compartmentalizing fi a far above average peptide. Many other de ned antigenic pep- folded proteins in a manner kinetically dissociated from their

fi IMMUNOLOGY tides demonstrate a similar af nity for class I molecules and copy synthesis by hours, it is possible that nascent proteins are imme- number (18), but these may all be far above average (the Mas- diately sequestered in small amounts in regions that favor antigen sachusetts Institute of Technology student-body effect). Second, processing when the protein is eventually degraded by protea- there may be something peculiar about SCRAP that enables it to somes. These concepts are admittedly vague, the real point being that access the class I pathway at high efficiency. As a chimeric protein we still have a lot to learn about MHC class I antigen processing. derived from several sources originating in different organisms, Regardless of the mechanism(s), the present findings point to SCRAP did not experience the honing forces of evolution that features of antigen processing that could be handy in discrimi- assure its integration into the cellular landscape. In some manner, cells may be able to detect SCRAP as a foreign entity and shunt it nating peptides encoded by innocuous vs. danger-associated fi mRNAs. This would be of obvious utility in immunosurveillance for high-ef ciency antigen presentation. This may also contribute fi to the similarly high efficiency of antigen processing from Listeria of tumors. Here the immune system often succeeds in nding the proteins secreted into the cytosol reported in the seminal work of needle in the haystack, by recognizing peptides generated at high fi Pamer and colleagues (4, 5), the high efficiency of SIINFEKL ef ciency from genes whose translation is minimal (2, 28). presentation when liberated from Listeria- or VV-synthesized Ub Materials and Methods fusion proteins (8), and the remarkably high class I occupancy b b exhibited by a some peptides (19–21). Cells, Antibodies, and Viruses. L-K , EL4, EL4/SCRAP, and HeLa K cell culture Our second startling finding is the approximately eightfold has been previously described (10, 29). Recombinant vaccinia virus expressing influenza nucleoprotein fused to the SIINFEKL peptide and GFP (NP-S-GFP) or difference in efficiency of Kb–SIINFEKL generation from retired its rapidly degraded counterpart (Ub-R-NP-S-GFP), SCRAP, or recombinant vi- SCRAP synthesized from cellular vs. VV mRNA. Ironically, from fi rus expressing no protein were previously described (10, 3). Recombinant ve- the perspective of immunosurveillance, viral SCRAP is less ef - sicular stomatitis virus expressing NP-S-GFP has been previously described (30). ciently processed. As discussed above, however, this may in part Generation of rVSV expressing Venus-Ub-SIINFEKL (VFP-Ub-S) was as follows. fi relate to the arti cial nature of SCRAP, and not to an intrinsic Plasmid pVSV-XN2 was kindly provided by J. Rose (Yale University, New Haven, lower efficiency of viral antigen processing. Indeed, Reits et al. CT). Venus-Ub-SIINFEKL was polymerase chain-amplified from recombinant (22) reported that influenza A virus infection rapidly increases the vaccinia virus (3) with the primers Venus 5′ XhoIXmaIMluI (5′-CTCGAGCCC- overall supply of TAP-transported peptides, a critical finding for GGGACGCGTCCATGGTGAGCAAGGGCGAGGAGC-3′) and Ub-SIINFEKL 3′ NheI immunosurveillance that still begs an explanation. (5′-GGCTAGCTTATCATAGCTTTTCGAAGTTGATGATCGAACCACCTCTTAGTCTT- However artificial, it is intriguing and potentially important AAG-3′) using Platinum Taq High Fidelity DNA polymerase (Life Technologies). that cells can distinguish cellular from viral SCRAP. We cannot PCR product was then cloned into pCR4-topo (Life Technologies) according to attribute this to VV-induced changes in cellular physiology, be- the manufacturer’s instructions. Insert DNA containing Venus-Ub-SIINFEKL cause VV infection does not greatly modulate the efficiency of was excised with XhoI and NheI restriction enzymes (Roche) and ligated with cellular Shield-1–sensitive SCRAP presentation. How can the similarly digested pVSV-XN2. Final plasmid pVSV-Venus-Ub-SIINFEKL was fi antigen-processing machinery distinguish between these osten- veri ed by restriction digestion patterns and DNA sequencing of the inserted sibly identical antigen sources? gene. Baby hamster kidney cells (BHK-21; American Type Culture Collection) were maintained in DMEM supplemented with 10% (vol/vol) FBS. Cells were VV SCRAP is likely synthesized in regions of the cytosol or- fi infected with vTF7-3 vaccinia virus expressing T7 polymerase at a multiplicity of ganized speci cally for viral translation that serve as the precursors infection (MOI) of 10 for 1 h, followed by transfection with plasmids pBS-N, for viral factories (23, 24), whereas cellular SCRAP is synthe- “ ” pBS-P, pBS-L (31, 32), and pVSV-VFP-Ub-S using Lipofectamine 2000 (Life sized on normal ribosomes. It is therefore possible that there Technologies) according to the manufacturer’s instructions. Supernatant me- are subtle (or even not so subtle) differences between the two dia containing recombinant virus were recovered at 48 hpi and used to make in their folding/posttranslational modifications. Indeed, because viral stocks. Viral titers were determined by plaque assays on BHK cells. The tRNA aminoacyl synthetases are recruited to locally translating mAb 25D-1.16 was previously described (11). Anti-GFP antibodies were from ribosomes (24), alterations in their specificity (25) could generate Roche. Infrared secondary antibodies were from LI-COR.

Dolan et al. PNAS Early Edition | 5of6 Downloaded by guest on September 29, 2021 Cloning and Transfections. To enhance SCRAP expression in transient trans- transfected HeLa Kb cells were to be infected with rVV, cells were first acid- fectants, a SCRAP construct was generated by PCR amplifying the original washed as described above and recultured for 2 h before infection. In this SCRAP cassette with the primers upstream SacI 5′-TCTAGAGAGCTCCCACC- scenario, the background levels of Kb–SIINFEKL were determined immedi- ATGGGAGTGCAGGTGGAAACCA-3′ and downstream XhoI 5′-AGATCTCTC- ately following rVV infection. In transiently transfected cells, Kb–SIINFEKL GAGTTACTTGTACAGCTCGTCCATGCCCAG-3′ to introduce an upstream SacI levels were determined on GFP+ cells rather than on the entire population. and downstream XhoI restriction site. The PCR product was cut with both enzymes and ligated with similarly digested pCAGGS (a gift from Ronald Quantitative Western Blot and Antigen Presentation Efficiency. Quantitative Harty, University of Pennsylvania, Philadelphia, PA). Plasmid DNA was puri- Western blots were performed based on previous experiments (6). Briefly, b fied (HiSpeed Midi Kit; Qiagen) and used for transfections. HeLa K cells (4 × total cell lysates were prepared from cells at various times after the indicated 5 10 ) were resuspended in 20 μL of Amaxa Solution SF (Lonza) and mixed treatments by boiling cells in SDS sample buffer (Quality Biological) at 107 with 200 ng DNA. Cells were transfected using the Amaxa 96-well Shuttle cells/mL for 20 min, and then an equal volume of 0.05 M DTT (in water) was System using the program CN-114. Following transfection, cells were cul- added to the lysates and boiled for an additional 10 min. Recombinant GFP tured overnight and used the following day. (Clontech) was added to similarly prepared EL4 cell lysates (ranging in con- centration from 1 to 10 nM), and both experimental samples as well as Antigen Presentation Assays and in Vitro Viral Infections. MHC class I antigen recombinant GFP were resolved by SDS/PAGE, blotted onto nitrocellulose, presentation in EL4/SCRAP cells in the presence, absence, or following re- and analyzed by Western blot using the Odyssey Imaging System (LI-COR). moval of Shield-1 was examined as previously reported (10). All flow Odyssey software was used to quantitate GFP signal in samples and stand- cytometry experiments were conducted using a BD LSR II flow cytomter. To ards. A standard curve was generated to determine the concentration of quantify the number of peptide–MHC complexes on the cell surface, acid- GFP in each experimental sample. This concentration was then divided by washed EL4/SCRAP cells were stained with FITC-coupled 25D-1.16 at differ- the number of cell equivalents in the sample (generally 100,000 cells) and ent times postwash, and the mean fluorescence intensity (MFI) of the FITC multiplied by Avogadro’s number to determine the average number of GFP signal was converted to molecular equivalents using FITC-coupled beads molecules present in each cell. The difference between Shield-1–treated and (Spherotech) as previously described (6). Cells were also stained in parallel –untreated samples is reported. For transiently transfected cells, the cell with Alexa 647-coupled 25D-1.16, and the number of peptide–MHC com- equivalent was adjusted based on the transfection efficiency, the number of plexes calculated from FITC-labeled cells was used to generate a standard GFP+ cells as determined by flow cytometry. The efficiency of antigen pre- curve for Alexa 647-labeled cells for comparison of Shield-1–treated and – sentation was determined by dividing the number of peptide–MHC com- untreated cells. For all other antigen presentation experiments, cells were plexes calculated above by the number of GFP molecules per cell. A two- stained with Alexa 647-coupled 25D-1.16 monoclonal antibody and analyzed tailed Student’s t test was used to statistically analyze the datasets using by flow cytometry. For rVV and rVSV infections, cells were resuspended at GraphPad Prism software. a concentration of 2 × 106 in the appropriate solution (saline solution with 0.1% BSA for rVV, serum-free MEM for rVSV) and virus was added at an MOI ACKNOWLEDGMENTS. Glennys Reynoso provided outstanding technical of 10. Cells were incubated at 37 °C for 30 min with occasional agitation, support. This work was generously supported by the Division of Intramural washed, and cultured at 106 cells/mL in complete media. When transiently Research, National Institute of Allergy and Infectious Diseases.

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