Date

June 2011

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Molecular Microbiology and Immunology

Institution

Oregon Health & Science University

Abstract

Cytomegalovirus (CMV) efficiently evades host immune defenses, and encodes a number of proteins that prevent antigen presentation by major histocompatibility complex class I molecules (MHC-I) in order to escape recognition and killing of infected cells by cytotoxic CD8+ T cells. We recently identified Rh178 as a Rhesus cytomegalovirus (RhCMV)-specific protein that interferes with MHC-I expression. Here, we demonstrate that Rh178 localizes to the membrane of the endoplasmic reticulum displaying a short luminal and large cytosolic domain, and that the membrane-proximal cytosolic portion is essential for inhibition of MHC-I expression. We further observed that Rh178 does not require synthesis of full-length MHC-I heavy chains but is capable of inhibiting the translation of short, unstable amino-terminal fragments of MHC-I. The cytosolic orientation of Rh178 and its ability to target protein fragments carrying the MHC-I signal peptide are consistent with Rh178 intercepting partially translated MHC-I heavy chains after signal recognition particle-dependent transfer to the endoplasmic reticulum membrane. However, interference with MHC-I translation by Rh178 seems to occur prior to Sec61-dependent protein translocation since inhibition of MHC-I translocation by Eeyarestatin-1 resulted in a full-length degradation intermediate that can be stabilized by proteasome inhibitors. These data are consistent with Rh178 blocking protein translation of MHC-I heavy chains at a step prior to the start of translocation, thereby downregulating MHC-I at a very early stage of translation. We have also investigated the in vivo role of Rh178 along with other RhCMVencoded MHC-I inhibitors in RhCMV superinfection of rhesus macaques. RhCMV, like human CMV (HCMV) can promote secondary infection despite preexisting anti-CMV immunity. We have identified the MHC-I inhibitors encoded by RhCMV as being solely responsible for allowing superinfection to occur. RhCMV that lacks the MHC-I inhibitors encoded within the Rh182-189 region can no longer superinfect. This is true whether or not Rh178 is present, so Rh178 alone is not sufficient for superinfection. The same is true for Rh189, the RhCMV orthologue of HCMV US11. Based on these results, a majority of MHC-I inhibitors must be encoded for RhCMV to be competent for superinfection. We also identified the same region of genes as responsible for altering the immunodominance profile of CD8+ T cells against a recombinant RhCMV carrying SIVgag. With these results, we have gained a greater understanding of immune evasion and immune stimulation by RhCMV, and these are the first in vivo data demonstrating the importance of MHC-I immune evasion genes in a non-human primate system. Information gained from this study will inform rational vaccine design both against CMV and for the use of CMV as a vaccine vector.

Identifier

doi:10.6083/M4Z31WN6

School

School of Medicine

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