Conserved molecular patterns derived from pathogenic microorganisms prime antigen-presenting dendritic cells (DC) to induce adaptive T cell responses. In contrast, virus-infected or tumor cells that express low levels of major histocompatibility complex (MHC) class I activate natural killer (NK) cells for direct killing. It is unknown whether NK cell recognition of MHC class I(low) targets can also induce adaptive T cell responses. Here, we show that MHC class I(low) targets initiate a cascade of immune responses, starting with the immediate activation of NK cells. The activated NK cells then prime DC to produce IL-12 and to induce highly protective CD8 T cell memory responses. Therefore, sensing of MHC class I(low) targets by NK cells can link innate and adaptive immunity to induce protective T cell responses and may alarm the immune system during early infection with noncytopathic viruses.

Essential viral proteins perform vital functions during morphogenesis via a complex interaction with other viral and cellular gene products. Here, we present a novel approach to comprehensive mutagenesis of essential cytomegalovirus genes and biological analysis in the 230-kbp-genome context. A random Tn7-based mutagenesis procedure at the single-gene level was combined with site-specific recombination via the FLP/FLP recognition target site system for viral genome reconstitution. We show the function of more than 100 mutants from a larger library of M50/p35, a protein involved in capsid egress from the nucleus. This protein recruits other viral proteins and cellular enzymes to the inner nuclear membrane. Our approach enabled us to rapidly discriminate between essential and nonessential regions within the coding sequence. Based on the prediction of the screen, we were able to map a site essential for viral protein-protein interaction at the amino acid level.Cytomegaloviruses define the beta subgroup of the Herpesviridae, which are important animal and human pathogens. Human cytomegalovirus (HCMV) is detected with a high prevalence in the human population, and it causes severe and fatal disease in immunocompromised individuals. Murine cytomegalovirus (MCMV) infection serves as an animal model system. CMVs harbor large double-stranded DNA genomes of about 230 kbp (7, 32). The recent cloning of the HCMV and MCMV genomes as infectious bacterial artificial chromosomes (BACs) (4,15,24,27,45,50) rendered these genomes accessible to advanced genetic manipulation (for a review, see reference 46).Infectious herpesvirus particles are formed during lytic virus replication as a result of a complex maturation and egress process (for a review, see reference 28). This multistep process starts in the nucleus with the packaging of viral genomes into capsids. The final envelopment takes place in the trans-Golgi network. Each maturation step is controlled by particular multiprotein assemblies, which are formed by complex interactions between viral and cellular proteins. Efforts to reconstitute the first morphogenesis stage, the capsid assembly, have been successful for alphaherpesviruses (43). In the mature virion, some higher-order protein complexes were studied by crystallography and electron microscopy (for a review, see reference 9). Yet, the logistics of the dynamic transitory stages (e.g., capsid egress from the nucleus) are not accessible for high-resolution analysis using static physical methods. Artificial reconstitution of these intermediate stages would require regulated coexpression of a number of viral proteins in the context of an appropriate host cell environment. Thus, detailed structure-function analyses of protein machines, which are associated with nuclear egress of virus capsids, need the context of virus replication. Gene products involved in herpesvirus morphogenesis are often essential for viral growth. The genetic analysis of these genes is restricted to either the isolated expression of the protein in cell ...

The proteins encoded by the UL34 and UL31 genes of herpes simplex virus are conserved among herpesviruses. They form a complex that is essential for the egress of the herpesvirus nucleocapsids from the nucleus. In previous work on the homologous protein complex in murine cytomegalovirus (MCMV), we defined their mutual binding domains. Here, we started to map binding domains within the UL34/UL31 proteins of alpha-, beta-, and gammaherpesviruses and to locate other functional properties. A protein complementation assay (PCA) using the TEM-1 ␤-lactamase fragments fused to UL31 and UL34 protein homologues was used to study protein-protein interactions in cells. Wild-type MCMV M50 and M53 provided a strong reaction in the PCA, whereas mutants unable to form a complex did not. The homologous pairs of herpes simplex virus type 1, pseudorabies virus, human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and murine herpes virus 68 proteins also reacted, with the exception of the EBV proteins. Cross-complementation was found to be positive only within the same herpesvirus subfamily. Moreover, the HCMV homologues rescued replication-defective MCMV genomes lacking one or the other gene. We identified the binding site of M53 for M50 in the first conserved region (CR1) (M. Loetzerich, Z. Ruzsics, and U. H. Koszinowski, J. Virol. 80:73-84). Here we show that the CR1 of all tested UL31 proteins contains the UL34 binding site, and chimeric proteins carrying the subfamily-specific CR1 rescued the ability to cross-complement in the PCA.Human herpesviruses cause highly prevalent infections associated with usually mild symptoms resulting in lifelong latency. However, they can provoke fatal disease in immunecompromised and immune-immature patients (12,23,41). In spite of their medical importance, at this time herpesvirus infections can be controlled only by antiviral therapy targeting viral DNA replication (7,15). Within the last few years, promising new agents that affect protein-protein interactions involved in herpesvirus replication have been identified by highthroughput screening (14,36). Such screening requires the identification of proteins that interact in an important or essential fashion.Recently the interaction map, the so-called interactome, of the two herpesviruses Kaposi's sarcoma-associated herpesvirus and varicella-zoster virus was annotated (38). The protein networks predicted by yeast two-hybrid screening suggest a common core set of herpesvirus protein interactions. Predicted interactions need to be validated in the natural context. Here we have tested a protein complementation assay (PCA) (10, 37, 40), which can be used to characterize viral protein-protein interactions in cells. In principle, the same assay is applicable to the screening (26) and validation of compounds interfering with the interactions in question. Among other procedures to measure and monitor protein-protein interactions, the PCA, evolved from the classical yeast two-hybrid approach (8), detects protein-protein interactions by a direct readou...

Background: The nuclear lamina is a protein meshwork lining the inner nuclear membrane, which contains a polymer of nuclear lamins associated with transmembrane proteins of the inner nuclear membrane. The lamina is involved in nuclear structure, gene expression, and association of the cytoplasmic cytoskeleton with the nucleus. We previously identified a group of 67 novel putative nuclear envelope transmembrane proteins (NETs) in a large-scale proteomics analysis. Because mutations in lamina proteins have been linked to several human diseases affecting skeletal muscle, we examined NET expression during differentiation of C2C12 myoblasts. Our goal was to identify new nuclear envelope and lamina components whose expression is coordinated with muscle differentiation.

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