Adaptor protein complexes AP-1 and AP-3 are required by the HHV-7 Immunoevasin U21 for rerouting of class I MHC molecules to the lysosomal compartment.

The human herpesvirus-7 (HHV-7) U21 gene product binds to class I major histocompatibility complex (MHC) molecules and reroutes them to a lysosomal compartment. Trafficking of integral membrane proteins to lysosomes is mediated through cytoplasmic sorting signals that recruit heterotetrameric clathrin adaptor protein (AP) complexes, which in turn mediate protein sorting in post-Golgi vesicular transport. Since U21 can mediate rerouting of class I molecules to lysosomes even when lacking its cytoplasmic tail, we hypothesize the existence of a cellular protein that contains the lysosomal sorting information required to escort class I molecules to the lysosomal compartment. If such a protein exists, we expect that it might recruit clathrin adaptor protein complexes as a means of lysosomal sorting. Here we describe experiments demonstrating that the μ adaptins from AP-1 and AP-3 are involved in U21-mediated trafficking of class I molecules to lysosomes. These experiments support the idea that a cellular protein(s) is necessary for U21-mediated lysosomal sorting of class I molecules. We also examine the impact of transient versus chronic knockdown of these adaptor protein complexes, and show that the few remaining μ subunits in the cells are eventually able to reroute class I molecules to lysosomes

Tailless U21 is affected by depletion of adaptor complexes.

<p>Immunofluorescence analysis of class I MHC molecules in tailless-U21-expressing cells (U21NT), − (a) and + (b) AP-1μ shRNA expression, as indicated. Arrows in panel b point to the plasma membrane. Scale bar = 10 µm.</p

Rescue of AP-3 depletion.

<p>a) anti-AP-3μ immunoblot analysis of lysates from U373-U21 cells (lanes 1,2) or U373-U21 cells expressing FLAG-AP-3μ^rescue (lanes 3,4), −/+ AP-3μ shRNA, as indicated. The Ponceau S stained nitrocellulose is shown beneath the immunoblots as a loading control. b) Flow cytometric analysis of surface class I MHC molecules on U373-U21 or U373-U21 cells expressing FLAG-AP-3 µ^rescue, −/+ AP-3μ shRNA depletion, as indicated.</p

Physical Association of the K3 Protein of Gamma-2 Herpesvirus 68 with Major Histocompatibility Complex Class I Molecules with Impaired Peptide and β(2)-Microglobulin Assembly

To persist in the presence of an active immune system, viruses encode proteins that decrease expression of major histocompatibility complex class I molecules by using a variety of mechanisms. For example, murine gamma-2 herpesvirus 68 expresses the K3 protein, which causes the rapid turnover of nascent class I molecules. In this report we show that certain mouse class I alleles are more susceptible than others to K3-mediated down regulation. Prior to their rapid degradation, class I molecules in K3-expressing cells exhibit impaired assembly with β(2)-microglobulin. Furthermore, K3 is detected predominantly in association with class I molecules lacking assembly with high-affinity peptides, including class I molecules associated with the peptide loading complex TAP/tapasin/calreticulin. The detection of K3 with class I assembly intermediates raises the possibility that molecular chaperones involved in class I assembly are involved in K3-mediated class I regulation

U21 does not reroute class I MHC molecules in the absence of AP3µ.

<p>a) Schematic representation of the AP-3 complex (redrawn from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099139#pone.0099139-Bonifacino1" target="_blank">[15]</a>). b) AP-3µ immunoblot of lysates from U373-U21 cells before and 5 days after introduction of AP-3µ shRNA. The Ponceau S stained nitrocellulose is shown beneath the immunoblot as a loading control c) Flow cytometric analysis of class I MHC molecules on the cell surface of U373 or U373-U21 cells, 5 days after introduction of AP-3µ shRNA. Cell lines − (red) and + (blue) AP-3µ shRNA are indicated. d,e) U373 and U373-U21 cells or AP-3µ shRNA-expressing U373 and U373-U21 cells were labeled with W6/32, as indicated, 5 days after introduction of AP-3µ shRNA. Arrows in panel e point to the plasma membrane. Scale bar = 10 µm.</p

MCMV gp48-mediated trafficking of class I MHC molecules is affected by depletion of AP-1μ or AP-3μ subunits.

<p>Immunofluorescence analysis of class I MHC molecules in cells expressing gp48HA, − (a) and + (b) AP-1μ siRNA, and - (d) and + (e) AP-3μ siRNA, as indicated. Arrows in panels b and e point to the plasma membrane. Scale bar = 10 µm. c and f) Immunoblot analysis of AP-1μ or AP-3μ from lysates of U373-gp48HA cells −/+ treatment with AP-1μ (c) or AP-3μ (f) siRNA, as indicated. The Ponceau S stained nitrocellulose is shown beneath the immunoblots as a loading control.</p

U21 can reroute class I MHC molecules in the absence of AP2µ.

<p>a) Schematic representation of the AP-2 complex (redrawn from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099139#pone.0099139-Bonifacino1" target="_blank">[15]</a>). b) AP2µ immunoblot of lysates from U373-U21 cells before and 5 days after introduction of AP-2µ shRNA. The Ponceau S stained nitrocellulose is shown beneath the immunoblot as a loading control. c) Flow cytometric analysis of class I MHC molecules on the cell surface of U373 or U373-U21 cells, 5 days after introduction of AP-2µ shRNA. Cell lines − (red) and + (blue) AP-2µ shRNA are indicated. d,e) U373 and U373-U21 cells or AP2µ shRNA-expressing U373 and U373-U21 cells were labeled with W6/32, directed against properly-folded class I MHC molecules, as indicated. Arrows in panel d point to the plasma membrane. f,g) U373-U21 cells were double-labeled with W6/32 and anti-lamp2 Arrows point to specific puncta that overlap. h). The images are shown overlayed in (h), with class I molecules in red, and lamp2 in green, as indicated. Cells are shown at the same magnification. Scale bar = 10 µm.</p

siRNA-mediated depletion of AP-1 and AP-3.

<p>a) anti-AP-1μ or anti-AP-3μ immunoblot analysis of lysates from U373-U21 cells −/+ siRNA mediated depletion, as indicated. The Ponceau S stained nitrocellulose is shown beneath the immunoblots as a loading control. b,c) Flow cytometric analysis of class I MHC molecules on the cell surface of U373-U21 cells, −/+ AP-1μ or AP-3µ siRNA. Cell lines − (red traces) and + (blue traces) AP-1μ (b) or AP-3µ (c) siRNA are indicated. d) Cell surface levels of the transferrin receptor (TfR) in U373-U21 cells −/+ siRNA-mediated depletion of AP1μ, as indicated.</p

A Novel MHC-I Surface Targeted for Binding by the MCMV m06 Immunoevasin Revealed by Solution NMR

As part of its strategy to evade detection by the host immune system, murine cytomegalovirus (MCMV) encodes three proteins that modulate cell surface expression of major histocompatibility complex class I (MHC-I) molecules: the MHC-I homolog m152/gp40 as well as the m02-m16 family members m04/gp34 and m06/gp48. Previous studies of the m04 protein revealed a divergent Ig-like fold that is unique to immunoevasins of the m02-m16 family. Here, we engineer and characterize recombinant m06 and investigate its interactions with full-length and truncated forms of the MHC-I molecule H2-L(d) by several techniques. Furthermore, we employ solution NMR to map the interaction footprint of the m06 protein on MHC-I, taking advantage of a truncated H2-L(d), “mini-H2-L(d),” consisting of only the α1α2 platform domain. Mini-H2-L(d) refolded in vitro with a high affinity peptide yields a molecule that shows outstanding NMR spectral features, permitting complete backbone assignments. These NMR-based studies reveal that m06 binds tightly to a discrete site located under the peptide-binding platform that partially overlaps with the β(2)-microglobulin interface on the MHC-I heavy chain, consistent with in vitro binding experiments showing significantly reduced complex formation between m06 and β(2)-microglobulin-associated MHC-I. Moreover, we carry out NMR relaxation experiments to characterize the picosecond-nanosecond dynamics of the free mini-H2-L(d) MHC-I molecule, revealing that the site of interaction is highly ordered. This study provides insight into the mechanism of the interaction of m06 with MHC-I, suggesting a structural manipulation of the target MHC-I molecule at an early stage of the peptide-loading pathway