Human plasmacytoid dendritic cells (PDC) are key sentinels alerting both innate and adaptive immune responses through production of huge amounts of alpha/beta interferon (IFN). IFN induction in PDC isSuccessful defense against invading pathogens involves rapid recognition of conserved danger signals through members of the Toll-like receptor (TLR) protein family (1) and induction of cytokines that activate both innate and adaptive immunity. A principal effector integrating early antiviral and immunostimulatory activities is the alpha/beta interferon (IFN) system, including the group of IFN-␣ isotypes and IFN-␤ (21). Although most types of cells can produce IFN through recognition of cytosolic double-stranded RNA (1a, 36, 44), or upon stimulation of TLR3 and TLR4 through double-stranded RNA or lipopolysaccharide, respectively (1), the vast amount of IFN upon entry of bacterial and viral pathogens is produced by a specialized cell population, plasmacytoid dendritic cells (PDC) (2, 6). Transcriptional induction of IFN genes is controlled by interferon regulatory factors (IRFs). IRF-3 mainly regulates IFN-␤ induction, whereas IRF-7 has the ability to activate IFN-␣ promoters (22,25,45). In contrast to other cell types, PDC constitutively express high levels of IRF-7 such that expression of IFN-␣ by PDC is independent of the IFN-␣ receptor-mediated positive feedback via IFN-␤ (3, 13, 16, 18), explaining in part the promptness of high-capacity IFN-␣ production.The TLR repertoire of human PDC is composed of TLR7 and TLR9, both located in the endosomal membrane. As shown recently, TLR7 and TLR8 recognize viral singlestranded RNA (8, 12) as well as imidazoquinolines such as imiquimod and resiquimod (R848) and guanosine analogs (reviewed in references 1 and 42). In contrast, TLR9 recognizes bacterial or viral DNA (1), including synthetic CpG oligodeoxynucleotides (ODN) (11). Indeed, recent work revealed IFN-␣ production in PDC after incubation with a variety of inactivated or live DNA and RNA viruses, including herpes simplex virus types 1 and 2 (16,19,23), murine cytomegalovirus (7), human immunodeficiency virus (46), influenza A virus (8,24), Sendai virus (14,16), and vesicular stomatitis virus (3,24). For herpes simplex virus (19,23), Influenza A virus (8,24), and vesicular stomatitis virus (24), the critical involvement of MyD88 adaptor-dependent TLR9 and TLR7 signaling has been demonstrated.In addition to perceiving external virus components through TLR7 and TLR9, human PDC have the means to sense cytosolic replicating RNA viruses. As we could show recently, respiratory syncytial virus (RSV) escapes from recognition by PDC TLRs (14). Nevertheless, infection with a particular laboratory strain of RSV (subtype A, strain Long), or cytosolic delivery of double-stranded RNA but not of poly(I:C) led to potent IFN-␣ induction in PDC in a TLR-and protein kinase R-independent manner (14).The considerable repertoire of tools for sensing pathogens combined with a tremendous capacity to produce IFN make human PDC the key sentinel...

Bovine respiratory syncytial virus (BRSV) is a major etiological agent of respiratory tract disease in calves and results in substantial economic loss (40,45). The immune response and pathology in calves mimic symptoms caused by human respiratory syncytial virus (HRSV), which remains the leading cause of serious bronchiolitis and pneumonia in infants and young children throughout the world (9). Molecular cloning has confirmed a very close relationship between BRSV and HRSV and has revealed substantial differences from other members of the Paramyxoviridae family, leading to the establishment of the Pneumovirus genus within the Paramyxoviridae family (36, 37).As with all members of the order Mononegavirales, the 15-kb genomic RNA of RSV is contained in a ribonucleoprotein (RNP) complex which serves as a template for sequential transcription of genes (25, 49). Eleven proteins are expressed from 10 transcription units, which are arranged in the order 3Ј-NS1-NS2-N-P-M-SH-G-F-M2-L-5Ј (5, 9, 30, 31). The proteins encoded include five RNP-associated proteins, namely, the nucleoprotein N, the phosphoprotein P, the large catalytic subunit L of the RNA polymerase, and a transcription elongation factor (M2-1) encoded by the first of two overlapping open reading frames of the M2 gene (8,17,27,38). The second open reading frame of the M2 transcription unit (M2-2) was reported to encode a nonessential protein (1) which is probably involved in the regulation of RNA synthesis (4, 28). Three viral proteins are associated with the viral envelope, namely, the fusion protein F, the putative attachment protein G, and a small hydrophobic protein SH.The presence of two nonstructural protein genes, NS1 and NS2, at the 3Ј-terminal position of the genome distinguishes pneumoviruses from all other members of the Mononegavirales. Due to the 3Ј-proximal location, the NS genes are abundantly transcribed. The encoded proteins have been demonstrated in infected cells (10,16). The BRSV NS1 and NS2 genes encode polypeptides of 136 and 124 amino acids, respectively. Comparison with NS proteins of HRSV subgroup A and B proteins revealed amino acid identities of 69 and 68% for NS1 proteins and 84 and 83%, for NS2 proteins, respectively (5, 34). The deduced sequences, however, did not provide obvious clues to the function of NS proteins in the virus life cycle. The HRSV NS1 protein was reported to be associated with the M protein, while the NS2 protein did not show any detectable association with RSV structural proteins, indicating distinct functions of NS1 and NS2 (16,47). An inhibitory function of NS1 in virus RNA transcription and RNP replication was recently suggested by experiments in which artificial HRSV minigenomes were grown in the absence or presence of NS1. In the same study, an inhibitory but far less pronounced effect was also observed for NS2 (3).Recently established protocols for recovery of infectious minus-strand RNA viruses from cDNA (11) have allowed the generation of recombinant HRSV (8, 29) and BRSV (5) and allowed researchers to...

Plasmacytoid dendritic cells sense viral ssRNA or its degradation products via TLR7/8 and CpG motifs within viral DNA via TLR9. Although these two endosomal pathways operate independently of viral replication, little is known about the detection of actively replicating viruses in plasmacytoid dendritic cell (PDC). Replication and transcription of the viral genome of ssRNA viruses as well as many DNA viruses lead to the formation of cytosolic dsRNA absent in noninfected cells. In this study, we used human respiratory syncytial virus (HRSV) encoding a fusion (F) protein for direct cytosolic entry. Both HRSV infection and cytosolic delivery of a 65-nt dsRNA led to potent IFN-α induction in PDC, but not in myeloid dendritic cells. Inactivation of HRSV by UV irradiation abrogated IFN-α induction in PDC. The comparison of two respiratory syncytial virus (RSV) constructs carrying either the HRSV or the bovine RSV F protein revealed that F-mediated cytosolic entry of RSV was absolutely required for IFN-α induction in PDC. HRSV-induced IFN-α production was independent of endosomal acidification and of protein kinase R (PKR) kinase activity, as demonstrated with chloroquine and the PKR inhibitor 2-aminopurine, respectively. In contrast, the induction of IFN-α by the TLR7/8 ligand R848, by the TLR9 ligand CpG-A ODN 2216, and by inactivated influenza virus (TLR7/8 dependent) was completely blocked by 2-aminopurine. IFN-α induction by mouse pathogenic Sendai virus was not affected in PKR- and MyD88-deficient mice, confirming that a ssRNA virus, which is able to directly enter host cells via fusion at the plasma membrane, can be detected by PDC independently of PKR, TLR7/8, and TLR9.

A marked suppression of immune function has long been recognized as a major cause of the high morbidity and mortality rate associated with acute measles. As a hallmark of measles virus (MV)-induced immunosuppression, peripheral blood lymphocytes (PBLs) isolated from patients exhibit a significantly reduced capacity to proliferate in response to mitogens, allogens, or recall antigens. In an in vitro system we show that proliferation of naive PBLs [responder cells (RCs)] in response to a variety of stimuli was significantly impaired after cocultivation with MV-infected, UV-irradiated autologous PBLs [presenter cells (PCs)]. We further observed that a 50% reduction in proliferation of RCs could still be observed when the ratio of PC to RC was 1:100. The effect was completely abolished after physical separation of the two populations, which suggests that soluble factors were not involved. Proliferative inhibition of the RCs was observed after short cocultivation with MV-infected cells, which indicates that surface contact between one or more viral proteins and the RC population was required. We identified that the complex of both MV glycoproteins, F and H, is critically involved in triggering MV-induced suppression of mitogen-dependent proliferation, since the effect was not observed (i) using a recombinant MV in which F and H were replaced with vesicular stomatitis virus G or (ii) when either of these proteins was expressed alone. Coexpression of F and H, however, lead to a significant proliferative inhibition in the RC population. Our data indicate that a small number of MV-infected PBLs can induce a general nonresponsiveness in uninfected PBLs by surface contact, which may, in turn, account for the general suppression of immune responses observed in patients with acute measles.