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* Department of Microbiology and
Immunology Graduate Program, University of Iowa, Iowa City, IA 52242;
The Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton; and
Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria, Australia
Cytotoxic T lymphocyte escape occurs in many human infections, as well as mice infected with the JHM strain of mouse hepatitis virus, which exhibit CTL escape variants with mutations in a single epitope from the spike glycoprotein (S510). In all CTL epitopes prone to escape, only a subset of all potential variants is generally detected, even though many of the changes that are not selected would result in evasion of the T cell response. It is postulated that these unselected mutations significantly impair virus fitness. To define more precisely the basis for this preferential selection, we combine x-ray crystallographic studies of the MHC class I (Db)/S510 complexes with viral reverse genetics to identify a prominent TCR contact residue (tryptophan at position 4) prone to escape mutations. The data show that a mutation that is commonly detected in chronically infected mice (tryptophan to arginine) potently disrupts the topology of the complex, explaining its selection. However, other mutations at this residue, which also abrogate the CTL response, are never selected in vivo even though they do not compromise virus fitness in acutely infected animals or induce a significant de novo CTL response. Thus, while structural analyses of the S510/Db complex provide a strong basis for why some CTL escape variants are selected, our results also show that factors other than effects on virus fitness limit the diversification of CD8 T cell epitopes.
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1 This work was supported by National Institutes of Health Grant R01 NS036592-09 (to S.P. and A.W.P.), and a National Institutes of Health Predoctoral Training Grant on Mechanisms of Parasitism (T32 AI007511 to N.S.B.). A.W.P. is a Senior Research Fellow, M.A.D. is a Doherty Post-doctoral Fellow, and A.I.W. is a C. J. Martin Fellow of the National Health and Medical Research Council of Australia. J.R. is an Australian Research Council Federation Fellow.
2 N.B. and A.T. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Stanley Perlman, Department of Microbiology, Bowen Science Building 3-712, University of Iowa, Iowa City, IA 52242. E-mail address: Stanley-perlman{at}uiowa.edu or Dr. Anthony Purcell, Department of Biochemistry and Molecular Biology, The Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, 3010 Victoria, Australia. E-mail address: apurcell{at}unimelb.edu.au
4 Abbreviations used in this paper: HCV, hepatitis C virus; JHMV, JHM strain of mouse hepatitis virus; S510, the immunodominant H-2Db-restricted epitope from the spike glycoprotein of JHMV (CSLWNGPHL); S598, subdominant H-2Kb-restricted epitope spanning residues 598–605 of the spike glycoprotein of JHMV (RCQIFANI); p.i., postinfection; Aba, L-
aminobutyric acid; W513R, position 4 Arg-substituted S510 epitope (CSLRNGPHL); W513G, position 4 Gly-substituted S510 epitope (CSLGNGPHL); W513S, position 4 Ser-substituted S510 epitope (CSLSNGPHL); W513L, position 4 Leu-substituted S510 epitope (CSLLNGPHL); PEG, polyethylene glycol; rJ, recombinant JHMV; rJ.SW513G, recombinant JHMV bearing the W513G mutation introduced by reverse genetics; rJ.SW513S, recombinant JHMV bearing the W513S mutation introduced by reverse genetics; rJ.SW513L, recombinant JHMV bearing the W513L mutation introduced by reverse genetics; rJ.SW513R, recombinant JHMV bearing the W513R mutation introduced by reverse genetics; MOI, multiplicity of infection; WT, wild type; OVA, chicken ovalbumin; DC, dendritic cell; CD, circular dichroism; Tm, midpoint of thermal denaturation; rmsd, root mean square deviation; LCMV, lymphocytic choriomeningitis virus; HGV, hepatitis G virus.
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