RESUMEN
CD8(+) CTLs detect virus-infected cells through recognition of virus-derived peptides presented at the cell surface by MHC class I molecules. The cowpox virus protein CPXV012 deprives the endoplasmic reticulum (ER) lumen of peptides for loading onto newly synthesized MHC class I molecules by inhibiting the transporter associated with Ag processing (TAP). This evasion strategy allows the virus to avoid detection by the immune system. In this article, we show that CPXV012, a 9-kDa type II transmembrane protein, prevents peptide transport by inhibiting ATP binding to TAP. We identified a segment within the ER-luminal domain of CPXV012 that imposes the block in peptide transport by TAP. Biophysical studies show that this domain has a strong affinity for phospholipids that are also abundant in the ER membrane. We discuss these findings in an evolutionary context and show that a frameshift deletion in the CPXV012 gene in an ancestral cowpox virus created the current form of CPXV012 that is capable of inhibiting TAP. In conclusion, our findings indicate that the ER-luminal domain of CPXV012 inserts into the ER membrane, where it interacts with TAP. CPXV012 presumably induces a conformational arrest that precludes ATP binding to TAP and, thus, activity of TAP, thereby preventing the presentation of viral peptides to CTLs.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Virus de la Viruela Vacuna/inmunología , Evasión Inmune/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas Virales/inmunología , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Presentación de Antígeno/genética , Presentación de Antígeno/inmunología , Línea Celular Tumoral , Membrana Celular/metabolismo , Virus de la Viruela Vacuna/genética , Retículo Endoplásmico/inmunología , Mutación del Sistema de Lectura , Células HEK293 , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Unión Proteica/inmunología , Transporte de Proteínas/inmunología , Proteínas Virales/genéticaRESUMEN
To get insight into the insertion mechanism of small newly synthesized single-spanning membrane proteins, Pf3 coat protein mutants were constructed with potential glycosylation sites in the N-terminus. Some of these proteins, when synthesized in vitro in the presence of microsomes, became efficiently glycosylated, proving that they insert into the membrane and translocate their N-terminus to the lumenal side. Such Pf3 constructs also insert efficiently into Escherichia coli vesicles and even in pure lipid vesicles, suggesting a common mechanism, which might be spontaneous. Glycosylation was sensitive to changes in the amino acid sequence of the N-terminus, suggesting that it depends on the structure of the protein and/or its positioning with respect to the lipid-water interface.