UL49.5 is responsible for TAP-inhibition in virus-infected natural host cells.

<p>Bovine cells (MDBK), porcine cells (PK15), and equine cells (E.derm) were infected with wild type BHV-1, PRV, or EHV-1, respectively, or with the corresponding UL49.5-negative recombinant viruses. In all experiments, mock-treated (uninfected) cells from the relevant host species were used as a control. Peptide transport was assessed at 5 hrs post-infection in the presence and absence of ATP (black and open bars, respectively). The data are expressed as percentage of translocation, relative to the translocation observed in control cells (defined as 100%).</p

UL49.5 homologs arrest TAP in a translocation-incompetent state.

<p>The lateral mobility of the TAP complex was analyzed in MJS TAP1-GFP cells using confocal microscopy and FRAP. A circular spot in the ER was bleached and recovery of fluorescence was monitored. The half-time for recovery was determined and used to calculate the diffusion coefficient D. Where indicated, ATP was depleted (-ATP) or saturating amounts of substrate peptides (long side chain peptides, l.s.c.p.) were micro-injected into the cells.</p

UL49.5 of PRV and EHV-1 do not interfere with peptide binding to TAP.

<p>To evaluate peptide binding to the TAP complex, microsomal membranes from MJS TAP1-GFP cells (control; ▪), or MJS TAP1-GFP cells expressing UL49.5 of PRV (▴) or EHV-1 (▾) were incubated with increasing concentrations of the radiolabeled peptide (RR[¹²⁵I]YQKSTEL). Unspecific binding was determined in the presence of 200-fold excess of ICP47 (data not shown). The amount of specifically bound peptide per amount of microsomal protein is plotted against the peptide concentration. K_d values for control MJS: 277±58 nM, for MJS UL49.5^PRV: 351±42 nM and for MJS UL49.5^EHV-1: 236±71 nM.</p

UL49.5 of EHV-1 and EHV-4 block ATP binding to human and equine TAP.

<p>(A, B, C) MJS cells expressing the UL49.5 proteins or the HCMV-derived TAP-inhibitor US6 and (D) E.derm cells expressing EHV-4 UL49.5 were lysed using digitonin or NP40 as indicated. Post nuclear lysates were incubated with ATP-agarose. The pellet (P) contains the ATP-binding proteins. The supernatant (S) contains proteins incapable of binding ATP. ATP-bound (ATP-agarose beads; pellet) and unbound (soluble; supernatant) fractions were separated by centrifugation and analyzed using SDS-PAGE and immunoblotting (IB) with antibodies against the proteins indicated.</p

PCR primers used

<p>PCR primers used</p

VZV UL49.5 interacts with the peptide-loading complex, but does not inhibit MHC class I-restricted T cell recognition.

<p>(A) TAP1 was immunoprecipitated (IP) from MJS cells expressing EHV-1 or VZV UL49.5 proteins. Co-precipitating UL49.5 proteins were analyzed by immunoblotting (IB) using antibodies against EHV-1 or VZV UL49.5. Left panel: cell lysates were loaded on SDS-PAGE directly and stained by immunoblotting. (B) BHV-1 or VZV UL49.5-expressing PHA-treated T-cell blasts were labeled with ⁵¹Cr and used as target cells for CTL clones specific for the minor histocompatibility antigens HY and HA-2, recognized in the context of HLA-A1 and HLA-A2, respectively. Specific lysis was determined by measuring ⁵¹Cr release from the target cells after 4 hrs. Effector/target (E/T) ratios are indicated. (C) PHA-treated T-cell blasts or a melanoma cell line (Mel518) carrying the HY antigen were incubated with the HY-specific CTL clone HY HLA-A1. IFN-γ levels released by the CTL clones were determined from the supernatants of the co-cultures after 24 hrs.</p

Phylogenetic tree of alphaherpesvirus UL49.5 proteins constructed on the basis of all presently known UL49.5 amino acid sequences of <i>Alphaherpesvirinae</i> using ClustalV.

<p>The ClustalV (PAM250) method was used under the default settings of the alignment program MegAlignTM 500 of the sequence analysis software DNA* of DNASTAR Inc. The UL49.5 proteins for which TAP inhibition has been tested are shown in bold. *The HHV-3 (VZV) UL49.5 protein binds TAP, but does not block peptide transport. The NCBI accession numbers are provided in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000080#s4" target="_blank">Materials and Methods</a>.</p

Degradation of bovine TAP is mediated by BHV-1UL49.5 through its cytoplasmic tail.

<p>MDBK cells were mock-infected or infected with wild type BHV-1 or BHV-1 expressing UL49.5Δtail for 12 hours. The levels of bovine TAP1 and, as a control, α-tubulin were assessed in lysates of infected cells by immunoblotting (IB). BHV-1 UL49.5 was immunoprecipitated from lysates of metabolically labeled cells (IP).</p

Alignment of the amino acid sequences of UL49.5 homologs of a selection of alphaherpesviruses (NCBI accession numbers are summarized in Materials and Methods).

<p>Hydrophobic residues indicative of N-terminal signal sequences (dashed line) and membrane anchor regions (bold line) are indicated. The conserved cysteine residue involved in disulfide bond formation with the viral glycoprotein M is indicated. The amino acid sequence alignment of UL49.5 homologs was performed using Vector<i>NTI</i> software (Invitrogen).</p

UL49.5 of BHV-1 but not of PRV or EHV-1 reduces TAP1 and TAP2 steady state levels in human cells.

<p>(A) TAP-dependent peptide transport is inhibited in human melanoma cells (MJS) stably expressing the UL49.5 homologs of BHV-1, PRV and EHV-1, but not CHV. Peptide transport is depicted as percentage of translocation, relative to the translocation observed in control cells (defined as 100%). (B) Steady state protein levels of TAP1, TAP2, MHC class I heavy chains (MHC I HC), and UL49.5 in control cells and cell lines expressing UL49.5 of BHV-1, PRV and EHV-1. Proteins present in post-nuclear supernatants were separated using SDS-PAGE and detected by immunoblotting (IB) using antibodies specific for TAP1, TAP2, MHC class I heavy chains, and the UL49.5 proteins of BHV-1, PRV and EHV-1. *The doublet of PRV UL49.5 is probably related to differential glycosylation.</p