Type I and type II interferons (IFNs) are cytokines that establish the cellular antiviral state through the induction of IFN-stimulated genes (ISGs). We sought to understand the basis of the antiviral activity induced by type I and II IFNs in relation to the functions of their ISGs. Based on gene expression studies, we systematically identified antiviral ISGs by performing blinded, functional screens on 288 type I and type II ISGs. We assessed and validated the antiviral activity of these ISGs against an RNA virus, vesicular stomatitis virus (VSV), and a DNA virus, murine gammaherpes virus (MHV-68). Overall, we identified 34 ISGs that elicited an antiviral effect on the replication of either one or both viruses. Fourteen ISGs have uncharacterized antiviral functions. We further defined ISGs that affect critical life-cycle processes in expression of VSV protein and MHV-68 immediate-early genes. Two previously undescribed antiviral ISGs, TAP1 and BMP2, were further validated. TAP1-deficient fibroblasts were more susceptible to VSV infection but less so to MHV-68 infection. On the other hand, exogenous BMP2 inhibits MHV-68 lytic growth but did not affect VSV growth. These results delineate common and distinct sets of type I and type II IFN-induced genes as well as identify unique ISGs that have either broad or specific antiviral effects on these viruses.

Kaposi's sarcoma-associated herpesvirus encodes two transmembrane proteins (modulator of immune recognition [MIR]1 and MIR2) that downregulate cell surface molecules (MHC-I, B7.2, and ICAM-1) involved in the immune recognition of infected cells. This downregulation results from enhanced endocytosis and subsequent endolysosomal degradation of the target proteins. Here, we show that expression of MIR1 and MIR2 leads to ubiquitination of the cytosolic tail of their target proteins and that ubiquitination is essential for their removal from the cell surface. MIR1 and MIR2 both contain cytosolic zinc fingers of the PHD subfamily, and these structures are required for this activity. In vitro, addition of a MIR2–glutathione S-transferase (GST) fusion protein to purified E1 and E2 enzymes leads to transfer of ubiquitin (Ub) to GST-containing targets in an ATP- and E2-dependent fashion; this reaction is abolished by mutation of the Zn-coordinating residues of the PHD domain. Thus, MIR2 defines a novel class of membrane-bound E3 Ub ligases that modulates the trafficking of host cell membrane proteins.

Summary A conserved herpesviral kinase has been shown to play multiple vital roles in the life cycle of herpesviruses. ORF36, the kinase of murine gamma-herpesvirus 68 (MHV-68), was identified to counteract antiviral type I interferon (IFN) response through the screening of mutant viruses. ORF36 binds to activated interferon regulatory factor 3 (IRF-3) in the nucleus and inhibits the interaction between the IRF-3 and the co-transcriptional activator CBP, thereby suppressing the recruitment of RNA polymerase II to interferon beta promoter. Although the conserved kinase activity is not absolutely required for this interaction, the anti-IFN function of ORF36 is conserved among all herpesvirus subfamilies. Mutant viruses without ORF36 induce more interferon response and are attenuated both in vitro and in vivo. Our data suggest that herpesviruses have evolved an inhibitor of antiviral IFN defense within their conserved kinase, which is critical for herpesvirus to evade host immune control and persist in a host.

Type I Interferons are cytokines of the innate immune system that induce antiviral protein expression in response to viral infection. Various proteins and pathways have been shown to recognize nucleic acids ligands especially from RNA viruses. Here, we will review recent developments including transcription of DNA virus genomes into RNA ligands, and the recognition of viruses by TLR2 for interferon induction. The induced IFNs activate many interferon stimulated genes (ISGs) that have direct anti-viral effects. Recent studies have identified IFITM proteins as the first ISG to inhibit viral entry processes and revealed mechanistic understanding of known anti-viral ISGs such as ISG15 and Viperin.

Langerhans cells (LC) are a unique subset of dendritic cells (DC), present in the epidermis and serving as the first line of defense against pathogens invading the skin. To investigate the role of human LCs in innate immune responses, we examined TLR expression and function of LC-like DCs derived from CD34+ progenitor cells and compared them to DCs derived from peripheral blood monocytes (monocyte-derived DC; Mo-DC). LC-like DCs and Mo-DCs expressed TLR1–10 mRNAs at comparable levels. Although many of the TLR-induced cytokine patterns were similar between the two cell types, stimulation with the TLR3 agonist poly(I:C) triggered significantly higher amounts of the IFN-inducible chemokines CXCL9 (monokine induced by IFN-γ) and CXCL11 (IFN-γ-inducible T cell α chemoattractant) in LC-like DCs as compared with Mo-DCs. Supernatants from TLR3-activated LC-like DCs reduced intracellular replication of vesicular stomatitis virus in a type I IFN-dependent manner. Finally, CXCL9 colocalized with LCs in skin biopsy specimens from viral infections. Together, our data suggest that LCs exhibit a direct antiviral activity that is dependent on type I IFN as part of the innate immune system.

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Kaposi's sarcoma-associated herpesvirus encodes two related proteins, MIR1 and MIR2, that lead to reduction of the cell surface levels of major histocompatibility complex class I and other polypeptides involved in immune recognition. MIR1 and MIR2 do not affect the assembly or transport of their target proteins through the secretory pathway; rather, they act to enhance the selective endocytosis of target chains from the cell surface. Sequence inspection reveals that the modulator of immune recognition (MIR) proteins contain an NH 2 -terminal zinc finger of the plant homeodomain (PHD) subfamily, two transmembrane (TM) domains, and a C-terminal conserved region (CR). Here we examine the transmembrane topology and functional organization of MIR2. Both the PHD domain and the CR are disposed cytosolically and are essential for MIR-mediated endocytosis. MIR proteins form homo-oligomers; this activity is independent of the PHD and CR elements and maps instead to the TM regions. Analysis of chimeras between MIR1 and MIR2 reveals that the TM regions also mediate target selectivity. Mutations that ablate the PHD or CR regions generate dominant negative phenotypes for major histocompatibility complex class I endocytosis. These findings suggest a domain organization for the MIR proteins, with the TM regions involved in target selection and the cytosolic PHD and CR domains involved in the possible recruitment of cellular machinery that directly or indirectly regulates internalization of target molecules.Herpesviruses are a family of large DNA viruses that are able to induce a persistent (usually lifelong) infection. To facilitate the production of such long term infections, herpesviruses have evolved multiple strategies to evade immune detection. Most commonly, this is achieved by interrupting the synthesis, assembly, or function of major histocompatibility complex class I (MHC-I) 1 molecules, key proteins involved in the recognition of infected cells by cytotoxic T lymphocytes (1, 2). Kaposi's sarcoma-associated herpesvirus (also known as human herpesvirus 8) is the etiologic agent of Kaposi's sarcoma and several other AIDS-related proliferative disorders (1, 2). We (3) and others (4 -6) have shown that Kaposi's sarcoma-associated herpesvirus possesses two genes, K3 and K5, that encode protein products termed MIR1 and MIR2, respectively (for modulator of immune recognition). MIR1 and MIR2 are homologous proteins that are localized predominantly in the endoplasmic reticulum (ER) and lead to reduction of the levels of MHC-I chains present at the cell surface (3). Despite their predominantly ER localization, MIR1 and MIR2 do not affect the assembly, glycosylation, or transport of MHC-I chains in that organelle. Rather, they act to enhance the endocytosis of MHC-I chains from the cell surface, with the endocytosed chains subsequently targeted to the lysosome for proteolytic destruction (3).This enhanced endocytosis of MHC-I does not reflect a generalized induction of endocytosis, since many other surface proteins known to under...