Toll-like receptor (TLR)-dependent pathways control the production of IFNalphabeta, a key cytokine in innate immune control of viruses including mouse cytomegalovirus (MCMV). The lymphotoxin (LT) alphabeta-LTbeta receptor signaling pathway is also critical for defense against MCMV and thought to aid in the IFNbeta response. We find that upon MCMV infection, mice deficient for lymphotoxin (LT)alphabeta signaling cannot mount the initial part of a biphasic IFNalphabeta response, but show normal levels of IFNalphabeta during the sustained phase of infection. Significantly, the LTalphabeta-dependent, IFNalphabeta response is independent of TLR signaling. B, but not T, cells expressing LTbeta are essential for promoting the initial IFNalphabeta response. LTbetaR expression is required strictly in splenic stromal cells for initial IFNalphabeta production to MCMV and is dependent upon the NF-kappaB-inducing kinase (NIK). These results reveal a TLR-independent innate host defense strategy directed by B cells in communication with stromal cells via the LTalphabeta cytokine system.

CD4 T lymphocytes regulate the adaptive immune response to most viruses, both by providing help to CD8 T cells and B cells as well as through direct antiviral activity. Currently, no mouse cytomegalovirus (MCMV)-specific CD4 T cell responses are known. In this study, we identify and characterize 15 I-Ab-restricted CD4 T cell responses specific for MCMV epitopes. CD4 T cells accumulate to high levels in the spleen and lungs during acute infection and produce multiple cytokines (IFN-γ, TNF, IL-2, IL-10, and IL-17). Interestingly, IL-17 and IFN-γ production within epitope-specific cells was found to be mutually exclusive. CD4 T cells recognizing a peptide derived from m09 were only detectable at later times of infection and displayed a unique cytokine production profile. In total, this study reveals that the MCMV-specific CD4 T cell response is complex and functionally diverse, highlighting its important role in controlling this persistent pathogen.

The Drosophila BTB domain containing gene mod(mdg4) produces a large number of protein isoforms combining a common Nterminal region of 402 aa with different C termini. We have deduced the genomic structure of this complex locus and found that at least seven of the mod(mdg4) isoforms are encoded on both of its antiparallel DNA strands, suggesting the generation of mature mRNAs by trans-splicing. In transgenic assays, we demonstrate the ability of Drosophila to produce mod(mdg4) mRNAs by trans-splicing of pre-mRNAs generated from transgenes inserted at distant chromosomal positions. Furthermore, evidence is presented for occurring of trans-splicing of mod(mdg4)-specific exons encoded by the parallel DNA strand. The mod(mdg4) locus represents a new type of comlpex gene structure in which genetic complexity is resolved by extensive trans-splicing, giving important implications for genome sequencing projects. Demonstration of naturally occurring trans-splicing in the model organism Drosophila opens new experimental approaches toward an analysis of the underlying mechanisms.T rans-splicing is a process that produces mature transcripts by combining exons of independent pre-mRNA molecules and was first reported in trypanosomes (1-3). The capability of mammalian cells for mRNA trans-splicing was first shown by Eul et al. (4). Recently, additional reports demonstrated transsplicing in higher eukaryotes. Li et al. (5) identified a splice variant of the human acyltransferase ACAT-1 that contains a 5Ј-untranslated exon encoded from chromosome 7 connected with exons 1-16 encoded from chromosome 1. In rat liver cells, carnitine octanoyltransferase mRNA variants with duplications of coding exons 2 and 3 not present in the genomic complement have been identified (6). In this case, different proteins are encoded by the splice variants. However, the physiological significance of these trans-splicing events remains largely unknown.So far no experimental proof for naturally occurring transsplicing in insects has been documented. Recently, Labrador et al. (7) have shown that the mod(mdg4)-67.2 isoform in Drosophila is encoded on both DNA strands and suggested formation of the mature mRNA by trans-splicing of two independent transcripts.Sequence analysis of a large number of mod(mdg4) cDNA clones (8-12) and the available genomic sequence of the mod-(mdg4) locus (13) indicated that usage of both DNA strands as coding strands is a general property of the mod(mdg4) locus. The common exons 1-4, which are found in all identified transcripts, are located at the 5Ј end of the locus whereas the alternatively spliced 3Ј-specific exons are organized in five groups on both DNA strands. In transgenic assays, we provide evidence that trans-splicing is the basic mechanism responsible for production of multiple mod(mdg4) isoforms. (14) in both orientations. The construct was partially sequenced and used for germline mediated transformation. However, by sequencing the transgene specific reverse transcription (RT)-PCR product, we found a frame shift...

The NK cell–activating receptor NKG2D interacts with three different cellular ligands, all of which are regulated by mouse cytomegalovirus (MCMV). We set out to define the viral gene product regulating murine UL16-binding protein-like transcript (MULT)-1, a newly described NKG2D ligand. We show that MCMV infection strongly induces MULT-1 gene expression, but surface expression of this glycoprotein is nevertheless completely abolished by the virus. Screening a panel of MCMV deletion mutants defined the gene m145 as the viral regulator of MULT-1. The MCMV m145-encoded glycoprotein turned out to be necessary and sufficient to regulate MULT-1 by preventing plasma membrane residence of MULT-1. The importance of MULT-1 in NK cell regulation in vivo was confirmed by the attenuating effect of the m145 deletion that was lifted after NK cell depletion. Our findings underline the significance of escaping MULT-1/NKG2D signaling for viral survival and maintenance.

SummaryDeath receptors (DRs) of the TNFR superfamily contribute to antiviral immunity by promoting apoptosis and regulating immune homeostasis during infection, and viral inhibition of DR signaling can alter immune defenses. Here we identify the human cytomegalovirus (HCMV) UL141 glycoprotein as necessary and sufficient to restrict TRAIL DR function. Despite showing no primary sequence homology to TNF family cytokines, UL141 binds the ectodomains of both human TRAIL DRs with affinities comparable to the natural ligand TRAIL. UL141 binding promotes intracellular retention of the DRs, thus protecting virus infected cells from TRAIL and TRAIL-dependent NK cell-mediated killing. The identification of UL141 as a herpesvirus modulator of the TRAIL DRs strongly implicates this pathway as a regulator of host defense to HCMV and highlights UL141 as a pleiotropic inhibitor of NK cell effector function.