Major histocompatibility complex class I (MHC I) molecules present antigenic peptides for CD8؉ T-cell recognition. Prior to cell surface expression, proper MHC I loading is conducted by the peptide-loading complex (PLC), composed of the MHC I heavy chain (HC) and ␤ 2 -microglobulin (␤ 2 m), the peptide transporter TAP, and several chaperones, including tapasin. Tapasin connects peptide-receptive MHC I molecules to the PLC, thereby facilitating loading of high-affinity peptides onto MHC I. To cope with CD8 ؉ T-cell responses, human cytomegalovirus (HCMV) encodes several posttranslational strategies inhibiting peptide transport and MHC I biogenesis which have been studied extensively in transfected cells. Here we analyzed assembly of the PLC in naturally HCMVinfected fibroblasts throughout the protracted replication cycle. MHC I incorporation into the PLC was absent early in HCMV infection. Subsequently, tapasin neosynthesis became strongly reduced, while tapasin steady-state levels diminished only slowly in infected cells, revealing a blocked synthesis rather than degradation. Tapasin mRNA levels were continuously downregulated during infection, while tapasin transcripts remained stable and long-lived. Taking advantage of a novel method by which de novo transcribed RNA is selectively labeled and analyzed, an immediate decline of tapasin transcription was seen, followed by downregulation of TAP2 and TAP1 gene expression. However, upon forced expression of tapasin in HCMV-infected cells, repair of MHC I incorporation into the PLC was relatively inefficient, suggesting an additional level of HCMV interference. The data presented here document a two-pronged coordinated attack on tapasin function by HCMV.

Despite a rigorous blockade of interferon-c (IFN-c) signalling in infected fibroblasts as a mechanism of immune evasion by human cytomegalovirus (HCMV), IFN-c induced indoleamine-2,3-dioxygenase (IDO) has been proposed to represent the major antiviral restriction factor limiting HCMV replication in epithelial cells. Here we show that HCMV efficiently blocks transcription of IFN-c-induced IDO mRNA both in infected fibroblasts and epithelial cells even in the presence of a preexisting IFN-induced antiviral state. This interference results in severe suppression of IDO bioactivity in HCMV-infected cells and restoration of vigorous HCMV replication. Depletion of IDO expression nonetheless substantially alleviated the antiviral impact of IFN-c treatment in both cell types. These findings highlight the effectiveness of this IFN-c induced effector gene in restricting HCMV productivity, but also the impact of viral counter-measures.

SummaryCells in our body can induce hundreds of antiviral genes following virus sensing, many of which remain largely uncharacterized. CEACAM1 has been previously shown to be induced by various innate systems; however, the reason for such tight integration to innate sensing systems was not apparent. Here, we show that CEACAM1 is induced following detection of HCMV and influenza viruses by their respective DNA and RNA innate sensors, IFI16 and RIG-I. This induction is mediated by IRF3, which bound to an ISRE element present in the human, but not mouse, CEACAM1 promoter. Furthermore, we demonstrate that, upon induction, CEACAM1 suppresses both HCMV and influenza viruses in an SHP2-dependent process and achieves this broad antiviral efficacy by suppressing mTOR-mediated protein biosynthesis. Finally, we show that CEACAM1 also inhibits viral spread in ex vivo human decidua organ culture.

To assure efficient MHC class I (MHC-I) peptide loading, the peptide loading complex (PLC) recruits the peptide-receptive form of MHC-I, and in this process, tapasin (tpn) connects MHC-I with the peptide transporter TAP and forms a stable disulfide bond with ERp57. Here, we describe an alternatively spliced tpn transcript lacking exon 3, observed in cells infected with human cytomegalovirus. Recognition of exon 3 was regulated via G-runs, suggesting that members of the hnRNP (heterogeneous nuclear ribonucleoprotein)-family regulate expression of the Exon3 variant of tpn. Exon 3 includes Cys-95, which is responsible for the disulfide bond formation with ERp57 and, consequently, interaction of the Exon3 variant with ERp57 was strongly impaired. Although the Exon3 variant specifically stabilized TAP expression but not MHC-I in tpn-deficient cells, in tpn-proficient cells, the Exon3 tpn reduced cell surface expression of the tpn-dependent HLA-B*44:02 allele; the stability of the tpn-independent HLA-B*44:05 was not affected. Most importantly, detailed analysis of the PLC revealed a simultaneous binding of the Exon3 variant and tpn to TAP, suggesting modification of PLC functions. Indeed, an altered MHC-I ligandome was observed in HeLa cells overexpressing the Exon3 variant, highlighting the potential of the alternatively spliced tpn variant to impact CD8 + T-cell responses.Keywords: MHC class I peptide loading r PLC r TAP r Tapasin Additional supporting information may be found in the online version of this article at the publisher's web-site Correspondence: Dr. Anne Halenius e-mail: anne.halenius@uniklink-freiburg.de * These authors contributed equally to this work. Eur. J. Immunol. 2013Immunol. . 43: 1459Immunol. -1469 Introduction Presentation of endogenous peptides by MHC class I (MHC-I) to CD8 + T cells is critical to controlling viral infections and tumor growth. Peptides originating from cytosolic proteasomal degradation are translocated into the ER lumen by the heterodimeric peptide transporter TAP1/2 (transporter associated with Ag processing). Optimally TAP transports peptides with a length of 8-16 amino acids [1,2] and these can be further trimmed in the ER to fit the peptide binding groove of MHC class I (MHC-I) molecules [3], yielding a stable peptide-MHC-I complex that is transported through the secretory pathway for cell surface expression. Proper peptide loading of MHC-I is controlled by several chaperones that form the peptide loading complex (PLC) in the ER [4]. In the PLC the MHC-I heterodimer associates with tapasin (tpn), calreticulin, ERp57, and indirectly with TAP; tpn and TAP interact via their transmembrane segments (TMSs), thereby stabilizing the structure and function of TAP [5][6][7][8][9]. It has been shown that both TAP1 and TAP2 subunits possess binding sites for tpn at their N-terminal TMSs [10][11][12]. The exact stoichiometry of the PLC has been a matter of debate [13,14]; however, recent findings demonstrate a TAP-tpn ratio of 1:2 [15,16] as proposed previously also by Rufer et al...

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