In this study, we hypothesized that the physical interaction between the extracellular domains of the viral protein m152 and the host stress-induced proteins RAE-1 contribute to the regulation of surface expression of RAE-1. Previous efforts to demonstrate the interaction directly by coprecipitation have not been fruitful. Using recombinant RAE-1 isoforms and mutants expressed in E. coli
and m152 expressed in insect cells, we have demonstrated by several different methods the direct physical interaction of the viral and host-encoded molecules. We have shown that m152 binds RAE-1 directly and at a 1:1 ratio. The binding affinities differ detectably among RAE-1 isoforms. RAE-1β and γ, which are expressed on MCMV-susceptible mouse strains such as BALB/c, bind m152 tightly (Kd
=1-3 μM), a level that can be compared to the affinity of the NKG2D/RAE-1 interaction (Kd
=350-730 nM (28
)). In contrast, RAE-1δ, which is expressed on MCMV-resistant mouse strains such as C57BL/6, CBA and C3H, binds m152 weakly (Kd
= 34 μM), which is about 50-fold lower than its affinity for NKG2D (Kd
=726 nM). The difference in affinity of RAE-1β or RAE-1γ for m152 as compared with RAE-1δ suggests that the less effective downregulation of RAE-1δ by m152 as reported recently by Arapovic et al (24
) may be a result of this lower affinity. It is interesting to note that the SV analysis of the m152/RAE-1β interaction revealed a concentration-dependent increase in sfast,
indicative of relatively rapid reversible interaction (). This result may explain the experimental difficulties in detecting m152/RAE-1 interaction by coimmunoprecipitation “pull-down” experiments. Similarly, it was observed that the luminal domain of m152 is involved in the retention of MHC class I molecules in the ERGIC region in the cells, but without biochemical evidence for their direct interaction (23
). This suggests the interactions of m152/RAE-1 and m152/MHC-I may share similar intracellular regulatory pathways.
The differing affinities of RAE-1 isoforms for the MCMV m152 immunoevasin suggest a scenario for the ongoing evolution of MCMV immunoevasins. Genetic resistance of different mouse strains to MCMV infection is determined to some degree by loci that control NK activating receptors such as Ly49H (expressed in the MCMV resistant strain C57BL/6) (39
). In MCMV sensitive strains such as BALB/c, NKG2D, as an ITAM-associated activating receptor, plays an important role in control of viral titre following infection, and NKG2D ligands, particularly the stress-induced MULT-1, H60, and RAE-1 molecules, serve as the stimulus for NK cell activation. The virus gains a counter advantage by evolving evasins that antagonize the expression or function of the NKG2D ligands, particularly important in the setting of Ly49H negative mouse strains. These “sensitive” mouse strains may then gain advantage over the virus by the expression of more potent NKG2D ligands such as RAE-1α, β, and γ, while the resistant strains, because they have an additional NK activating receptor, Ly49H, have less need for the expression of better NKG2D ligands. In response to the expression of “good” NKG2D ligands, the virus refines the function of molecules to downregulate not only MULT-1 and H60, but also RAE-1α, β, and γ, accomplishing this with m152/gp40. The strong direct interaction of the m152 lumenal domain with RAE-1β and γ as we have measured here results in the effective downregulation of these NKG2D ligands, and the quantitative difference between RAE-1β and γ interaction with m152 as compared with RAE-1δ reflects the greater need for effective control of RAE-1β and γ (and presumably RAE-1α as well) to counter the NKG2D susceptibility of MCMV infected cells by NK “sensitive” strains.
This discussion is based on our knowledge of the genetics and relative resistance of several well-characterized inbred mouse strains, and a more complete analysis would require an extensive understanding of the expression patterns and RAE-1 isoform differences among wild outbred mice. To our knowledge such data are not yet available. However, the role of the MCMV genes is beginning to be assessed by the analysis of DNA of a number of independent isolates of virus from free-living mice (40
) Remarkably, the m152
gene has been highly conserved, suggesting that its evolutionary advantage is either to encode a protein that interacts with a molecule with conserved structure, or to serve multiple functions, such as the dual function of m152 interaction both with RAE-1 and host MHC-I. Needless to say, these molecules as well as others play a complex cooperative role in the mutual adaptation of virus and host, and our studies of one set of these interactions offer only a hint of the greater complexity. It should be emphasized, however, that the preservation of a gene that contributes to the fitness of the virus may result from a rather small advantage that may elude detection by laboratory evaluation of viral titre resulting from controlled doses and routes of infection.
To understand the molecular mechanisms that determine the differential m152/RAE-1 interaction, we explored whether the PLWY motif, which is present in RAE-1α, β, γ whereas absent from RAE-1δ and mutated to LPWC in RAE-1ε, may play a role in the m152/RAE-1 interaction. Our results confirmed that deletion of PLWY in RAE-1β indeed caused a 10-fold decrease in its binding affinity to m152, suggesting that the PLWY motif has a key role in the m152/RAE-1β interaction. However, the binding affinity of RAE-1γ or δ for m152 was not influenced by the presence or absence of PLWY, indicating that other differences among RAE-1 isoforms contribute to the interaction and to the resulting downregulation of m152. We further found that aside from the PLWY motif, RAE-1 isoforms differ at a number of positions in charged amino acid residues (). To gain insight into the possible effects of these amino acid differences, we have examined the surface electrostatic distribution of the RAE-1β X-ray structure, and have generated molecular models of RAE-1γ and RAE-1δ as well ().
Our first consideration was whether the predicted N-linked glycosylation sites of RAE-1 (Asn38, 70, 83, 143, and 156 --- all conserved, with the exception of Asn156 which is absent from RAE-1δ) might impinge on the m152 interaction. All of these are distant from the NKG2D/RAE-1 interface (26
) and are located either beneath the platform domain (Asn38 and 143) or at various peripheral positions (Asn83 at the N-terminus of the α1 helix, and Asn70 and 156 in loops connecting β strands). Thus, we focused on the “top” of the RAE-1 molecule, which serves as the NKG2D binding interface (26
Looking at a top view of the RAE-1β structure in comparison with the models, there are three major regions that seem to be influenced by the substitutions that distinguish the three isoforms. The first and most obvious region is that where PLWY is present in RAE-1β and γ and where PLWY is deleted in RAE-1δ. This region flanks the α1 helix, focused on the loop connecting the β1 and β2 strands. The second major area involves the third β-strand of the α1 domain as well as the left hand side of the α1 helix (see left box in and highlighted residues of the α1 helix in ). This region is basic in RAE-1β, acidic in RAE-1γ, and partly acidic and partly basic in RAE-1δ. The third region spans the right hand side of both the α1 and α2 helices, is basic in RAE-1β, acidic in RAE-1γ, and has subregions of both acidic and basic character in RAE-1δ. A more detailed understanding clarifying the complexities of the interaction of m152 with the various RAE-1 isoforms must await structure determination of a complex of RAE-1 with m152. Whether the downregulation of host MHC-I molecules by m152 expression is due to a direct interaction of m152 with MHC-I, and whether such a direct interaction is mediated by a surface of MHC-I structurally homologous to that of RAE-1 are questions that demand further detailed investigation.
MCMV infection provides a valid model system for understanding not only available mechanisms for NK and T cell mediated immunity to infection and virus resistance to host immunity, but also for gaining insight into the ongoing coevolution of virus and host. Our studies of the relative differences in the interaction of the viral immunoevasin m152 with host NKG2D ligands provide a glimpse of the ongoing adaptations of virus and host. The recently identified H60 isoforms (H60a, b, c) exhibit some features similar to the RAE-1 isoforms (43
). The relative interactions of m155 for the different H60 isoforms have yet to be addressed. Thus, differential expression and regulation of host ligands for NKG2D, as influenced by the virus, seem to be common features of the ongoing interplay and evolution of host response and viral immunoevasion. The likelihood that mouse and human viruses may share similar immune evasion strategies raises the prospect for identifying targets for novel anti-viral therapeutics based on disrupting the interaction between immunoevasins and their specific ligands in the host.