Inhibition of Acid Sphingomyelinase Allows for Selective Targeting of CD4+ Conventional versus Foxp3+ Regulatory T Cells.

CD4+ Foxp3+ regulatory T cells (Tregs) depend on CD28 signaling for their survival and function, a receptor that has been previously shown to activate the acid sphingomyelinase (Asm)/ceramide system. In this article, we show that the basal and CD28-induced Asm activity is higher in Tregs than in conventional CD4+ T cells (Tconvs) of wild-type (wt) mice. In Asm-deficient (Smpd1-/-; Asm-/-) mice, as compared with wt mice, the frequency of Tregs among CD4+ T cells, turnover of the effector molecule CTLA-4, and their suppressive activity in vitro were increased. The biological significance of these findings was confirmed in our Treg-sensitive mouse model of measles virus (MV) CNS infection, in which we observed more infected neurons and less MV-specific CD8+ T cells in brains of Asm-/- mice compared with wt mice. In addition to genetic deficiency, treatment of wt mice with the Asm inhibitor amitriptyline recapitulated the phenotype of Asm-deficient mice because it also increased the frequency of Tregs among CD4+ T cells. Reduced absolute cell numbers of Tconvs after inhibitor treatment in vivo and extensive in vitro experiments revealed that Tregs are more resistant toward Asm inhibitor-induced cell death than Tconvs. Mechanistically, IL-2 was capable of providing crucial survival signals to the Tregs upon inhibitor treatment in vitro, shifting the Treg/Tconv ratio to the Treg side. Thus, our data indicate that Asm-inhibiting drugs should be further evaluated for the therapy of inflammatory and autoimmune disorders.

Measles Virus Defective Interfering RNAs Are Generated Frequently and Early in the Absence of C Protein and Can Be Destabilized by Adenosine Deaminase Acting on RNA-1-Like Hypermutations.

UNLABELLED: Defective interfering RNAs (DI-RNAs) of the viral genome can form during infections of negative-strand RNA viruses and outgrow full-length viral genomes, thereby modulating the severity and duration of infection. Here we document the frequent de novo generation of copy-back DI-RNAs from independent rescue events both for a vaccine measles virus (vac2) and for a wild-type measles virus (IC323) as early as passage 1 after virus rescue. Moreover, vaccine and wild-type C-protein-deficient (C-protein-knockout [CKO]) measles viruses generated about 10 times more DI-RNAs than parental virus, suggesting that C enhances the processivity of the viral polymerase. We obtained the nucleotide sequences of 65 individual DI-RNAs, identified breakpoints and reinitiation sites, and predicted their structural features. Several DI-RNAs possessed clusters of A-to-G or U-to-C transitions. Sequences flanking these mutation sites were characteristic of those favored by adenosine deaminase acting on RNA-1 (ADAR1), which catalyzes in double-stranded RNA the C-6 deamination of adenosine to produce inosine, which is recognized as guanosine, a process known as A-to-I RNA editing. In individual DI-RNAs the transitions were of the same type and occurred on both sides of the breakpoint. These patterns of mutations suggest that ADAR1 edits unencapsidated DI-RNAs that form double-strand RNA structures. Encapsidated DI-RNAs were incorporated into virus particles, which reduced the infectivity of virus stocks. The CKO phenotype was dominant: DI-RNAs derived from vac2 with a CKO suppressed the replication of vac2, as shown by coinfections of interferon-incompetent lymphatic cells with viruses expressing different fluorescent reporter proteins. In contrast, coinfection with a C-protein-expressing virus did not counteract the suppressive phenotype of DI-RNAs. IMPORTANCE: Recombinant measles viruses (MVs) are in clinical trials as cancer therapeutics and as vectored vaccines for HIV-AIDS and other infectious diseases. The efficacy of MV-based vectors depends on their replication proficiency and immune activation capacity. Here we document that copy-back defective interfering RNAs (DI-RNAs) are generated by recombinant vaccine and wild-type MVs immediately after rescue. The MV C protein interferes with DI-RNA generation and may enhance the processivity of the viral polymerase. We frequently detected clusters of A-to-G or U-to-C transitions and noted that sequences flanking individual mutations contain motifs favoring recognition by the adenosine deaminase acting on RNA-1 (ADAR1). The consistent type of transitions on the DI-RNAs indicates that these are direct substrates for editing by ADAR1. The ADAR1-mediated biased hypermutation events are consistent with the protein kinase R (PKR)-ADAR1 balancing model of innate immunity activation. We show by coinfection that the C-defective phenotype is dominant.

Sphingosine kinase 1 regulates measles virus replication.

Measles virus (MV) manipulates host factors to facilitate virus replication. Sphingosine kinase (SK) is an enzyme catalyzing the formation of sphingosine 1-phosphate and modulates multiple cellular processes including the host defense system. Here, we determined the role of SK1 in MV replication. Overexpression of SK1 enhanced MV replication. In contrast, inhibition of SK impaired viral protein expression and infectious virus production from cells expressing MV receptor, SLAM or Nectin-4. The inhibition of virus replication was observed when the cells were infected by vaccine strain or wild type MV or V/C gene-deficient MV. Importantly, SK inhibition suppressed MV-induced activation of NF-κB. The inhibitors specific to NF-κB signal pathway repressed the synthesis of MV proteins, revealing the importance of NF-κB activation for efficient MV replication. Therefore, SK inhibition restricts MV replication and modulates the NF-κB signal pathway, demonstrating that SK is a cellular factor critical for MV replication.

Adenosine deaminase acting on RNA 1 (ADAR1) suppresses the induction of interferon by measles virus.

ADAR1, the interferon (IFN)-inducible adenosine deaminase acting on RNA, catalyzes the C-6 deamination of adenosine (A) to produce inosine (I) in RNA substrates with a double-stranded character. Because double-stranded RNA is a known inducer of IFN, we tested the role of ADAR1 in IFN induction following virus infection. HeLa cells made stably deficient in ADAR1 (ADAR1(kd)) were compared to vector control (CON(kd)) and protein kinase PKR-deficient (PKR(kd)) cells for IFN-ß induction following infection with either parental (wild-type [WT]) recombinant Moraten vaccine strain measles virus (MV) or isogenic knockout mutants deficient for either V (V(ko)) or C (C(ko)) protein expression. We observed potent IFN-ß transcript induction in ADAR1(kd) cells by all three viruses; in contrast, in ADAR1-sufficient CON(kd) cells, only the C(ko) mutant virus was an effective inducer and the IFN-ß RNA induction was amplified by PKR. The enhanced IFN-ß transcript-inducing capacity of the WT and V(ko) viruses seen in ADAR1-deficient cells correlated with the enhanced activation of PKR, IFN regulatory factor IRF3, and activator of transcription ATF2, reaching levels similar to those seen in C(ko) virus-infected cells. However, the level of IFN-ß protein produced was not proportional to the level of IFN-ß RNA but rather correlated inversely with the level of activated PKR. These results suggest that ADAR1 functions as an important suppressor of MV-mediated responses, including the activation of PKR and IRF3 and the induction of IFN-ß RNA. Our findings further implicate a balanced interplay between PKR and ADAR1 in modulating IFN-ß protein production following virus infection.

Peroxiredoxin 1 is required for efficient transcription and replication of measles virus.

Measles is a highly contagious human disease caused by the measles virus (MeV). In this study, by proteomic analysis, we identified peroxiredoxin 1 (Prdx1) as a host factor that binds to the C-terminal region of the nucleoprotein (N; N(TAIL)) of MeV. Glutathione S-transferase (GST) pulldown experiments showed that the Prdx1-binding site overlapped with the MeV phosphoprotein (P)-binding site on N(TAIL) and that Prdx1 competed for the binding to N(TAIL) with the P protein, which is a component of RNA-dependent RNA polymerase (RdRp). Furthermore, RNA interference for Prdx1 resulted in a significant reduction in MeV growth in HEK293-SLAM cells. A minigenome assay indicated that Prdx1 suppression affected the viral RNA transcription and/or replication step. Relative quantification of viral RNA by real-time PCR (RT-PCR) showed that Prdx1 suppression not only reduced viral RNA transcription and replication but also enhanced polar attenuation in viral mRNA transcription. Surface plasmon resonance analysis showed that the binding affinity of Prdx1 to MeV-N was 40-fold lower than that of MeV-P to MeV-N, which suggested that Prdx1 might be involved in the early stage of MeV infection, when the expression level of Prdx1 was much higher than that of MeV-P. Since Prdx1 was expressed abundantly and constitutively in various cells, the results in this study indicate that Prdx1 is one of the inherent host factors implicated in MeV RNA synthesis.

Double-stranded RNA adenosine deaminase ADAR-1-induced hypermutated genomes among inactivated seasonal influenza and live attenuated measles virus vaccines.

We sought to examine ADAR-1 editing of measles and influenza virus genomes derived from inactivated seasonal influenza and live attenuated measles virus vaccines grown on chicken cells as the culture substrate. Using highly sensitive 3DI-PCR (R. Suspène et al., Nucleic Acids Res. 36:e72, 2008), it was possible to show that ADAR-1 could hyperdeaminate adenosine residues in both measles virus and influenza virus A genomes. Detailed analysis of the dinucleotide editing context showed preferences for 5'ArA and 5'UrA, which is typical of editing in mammalian cells. The hyperedited mutant frequency, including genomes and antigenomes, was a log greater for influenza virus compared to measles virus, suggesting a greater sensitivity to restriction by ADAR-1.

Apoptosis of human peripheral blood mononuclear cells by wild-type measles virus infection is induced by interaction of hemagglutinin protein and cellular receptor, SLAM via caspase-dependent pathway.

Although MV infection causes lymphopenia and degradation of cell-mediated immunity, the mechanisms are poorly known. MV interacts with cellular receptors which mediate virus binding and uptake and are on the surface of PBMC. In this study, apoptosis of MV-infected PBMC in vitro was analyzed. Both PBMC treated with UV-inactivated viruses and those infected with live MV underwent apoptosis. Apoptosis of wild-type MV-infected PBMC was blocked by anti-SLAM and anti-MV hemagglutinin antibodies, respectively. Furthermore, addition of soluble MV hemagglutinin recombinant protein induced apoptosis in PBMC. These data suggest that induction of apoptosis in MV-infected PBMC is triggered by interaction between hemagglutinin protein of MV and receptor, without other viral components. To further determine the mechanisms of apoptosis, caspase activity was analyzed by Western blotting. Wild-type virus Yonekawa strain-induced apoptosis was blocked by pretreatment with pan-caspase inhibitor (Z-VAD-fmk). Intriguingly, the laboratory-adapted Nagahata strain-induced apoptosis was not blocked by Z-VAD-fmk, indicating that there may be different apoptosis pathways which depend on the viral receptors, SLAM and CD46. Both extrinsic and intrinsic apoptotic pathways, including activation of caspase-3, -8 and -9, are involved in Yonekawa strain-induced apoptosis. Taken together, the findings of this study could open up a new avenue for understanding the molecular mechanisms of MV-induced PBMC apoptosis and immunosuppression.

RNA-specific adenosine deaminase ADAR1 suppresses measles virus-induced apoptosis and activation of protein kinase PKR.

ADAR1 (adenosine deaminase acting on RNA) catalyzes the conversion of adenosine to inosine, a process known as A-to-I editing. Extensive A-to-I editing has been described in viral RNAs isolated from the brains of patients persistently infected with measles virus, although the precise role of ADAR during measles virus infection remains unknown. We generated human HeLa cells stably deficient in ADAR1 ("ADAR1(kd) cells") through short hairpin RNA-mediated knockdown, and using these cells, we tested the effect of ADAR1 deficiency on measles virus (MVvac strain) growth and virus-induced cell death. We found that the growth of mutant viruses lacking expression of the viral accessory proteins V and C (V(ko) and C(ko), respectively) was decreased in ADAR1-deficient cells compared with ADAR1-sufficient cells. In addition, apoptosis was enhanced in ADAR1-deficient cells following infection with wild type and V(ko) virus but not following infection with C(ko) virus or treatment with tumor necrosis factor-alpha or staurosporine. Furthermore, in C(ko)-infected ADAR1-sufficient cells when ADAR1 did not protect against apoptosis, caspase cleavage of the ADAR1 p150 protein was detected. Finally, enhanced apoptosis in ADAR1(kd) cells following infection with wild type and V(ko) virus correlated with enhanced activation of PKR kinase and interferon regulatory factor IRF-3. Taken together, these results demonstrate that ADAR1 is a proviral, antiapoptotic host factor in the context of measles virus infection and suggest that the antiapoptotic activity of ADAR1 is achieved through suppression of activation of proapoptotic and double-stranded RNA-dependent activities, as exemplified by PKR and IRF-3.

Clinicopathological study on liver dysfunction in measles.

We analyzed the clinical course of eight patients with liver dysfunction in measles. All of the patients showed an elevation of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH), but no jaundice. These levels returned to normal about 3 weeks after the onset of the rash. A percutaneous liver biopsy was done in two cases. Histological examination showed slight necrosis of liver cells but no significant changes in portal area. On electron microscopy, virus particles were not detected. We detected measles virus RNA in the liver specimen by RT-PCR, which suggests that the measles virus affects liver cells directly in measles.

Pancreatic enzyme elevation in measles.

During an outbreak of measles in the period from May 1993 through February 1994, a 23-year-old woman with measles was admitted because of abdominal pain and vomiting. Moderately elevated levels of serum and urinary amylase were found. We investigated prospectively the next nine consecutive young adults hospitalized with severe measles. Pancreatic and other organ involvement was determined by serum and urinary amylase, serum lipase, and additional appropriate biochemical and hematological data. Four patients had elevated amylase levels in both serum and urine, whereas in one, serum amylase alone was increased. Serum lipase determined in eight patients was elevated in seven. In all patients elevated serum levels of aspartate aminotransferase and alanine aminotransferase or lactate dehydrogenase were found. In seven patients serum calcium concentrations were below the lower limit of normal. Four patients had mild to moderate thrombocytopenia. This is the first detailed report of pancreatic involvement in young adults with measles. This abnormal finding, its possible underlying mechanisms, and the clinical significance are discussed.

[Measles in the young adult. Clinical features of 201 cases]. / Sarampión en el adulto joven. Características clínicas en 201 casos.

The clinical and laboratory characteristics of 201 cases of measles and its associations with complications were analyzed during an outbreak of this disease in Mexico City. The complications were hepatitis (45%), bacterial pneumonia (17%), oral candidiasis (13%), upper gastrointestinal tract hemorrhage (13%), epistaxis (8%), encephalitis (4%), subcutaneous emphysema (2%), and hypocalcemic tetany (1%). In a subgroup of 20 consecutive patients hypocalcemia was found in 14 cases (70%), associated with high levels of calcitonin in three cases. An increased lactate dehydrogenase (LDH) was observed in 83% of the patients, showing a significant association with the occurrence of complications (p = 0.04) especially in the patients with values of LDH above 750 IU/mL (odds ratio of 6.4). Two patients died (1% mortality). The young patients with measles can develop serious complications, and an increased level of LDH may be a prognostic indicative of these complications.

Increased alpha-hydroxybutyrate dehydrogenase in serum from children with measles.

For treatment and prognostic evaluation of patients with measles, it is important to determine the source of the increase in serum alpha-hydroxybutyrate dehydrogenase (HBD; EC 1.1.99.6). Here we measured HBD and L-lactate dehydrogenase (LD; 1.1.1.27) activities in the serum of 18 children with measles and in 18 age-matched control patients with bronchopneumonia. HBD and LD activities in the measles patients were significantly higher than those in control subjects (P less than 0.001). The HBD/LD ratio in the measles patients two to three days after the onset of rash was significantly lower than that in control subjects (P less than 0.001). The low HBD/LD ratio was similar to the ratio found in lymphocytes. The number of peripheral lymphocytes decreased markedly after the onset of rash. Our results suggest that increased serum HBD and LD activity is common in measles infection and that the increase originates from the destruction of infected lymphocytes rather than from myocardiac injury.

Increased cytosol aminopeptidase and lactate dehydrogenase in serum originating from lymphocytes in measles and rubella infection.

We determined cytosol aminopeptidase (c-AP; EC 3.4.11.1) and lactate dehydrogenase (LDH) levels in serum; these enzymes are known to originate from lymphocytes in patients with measles and rubella. In patients with measles (n = 19), both enzyme levels increased markedly with the onset of rash: mean (+/- SD) c-AP was 269.7 +/- 103.5 U/L and LDH was 1149.5 +/- 255.2 U/L. In patients with rubella, activities of both enzymes increased mildly: c-AP (n = 18) was 81.6 +/- 24.4 U/L and LDH (n = 13) was 674.0 +/- 168.8 U/L. Increased c-AP and LDH levels in patients with measles and rubella presumably originate from the destruction of infected, activated lymphocytes, especially T lymphocytes.

Increased lactate dehydrogenase in serum in measles infection.

We determined lactate dehydrogenase (LD; EC 1.1.1.27) in serum from 75 sequential measles patients and 105 control patients. In the measles patients, LD was increased to 1147.9 +/- 43.8 (mean +/- SE) U/L, markedly more than in the control group (517.2 +/- 15.0 U/L, p less than 0.001). Both LD-3 and LD-4 isoenzymes were increased, but the concentration of LD-5 was normal. Patients with high LD values (greater than 1500 U/L) had longer hospital stays than did those with lower values (p less than 0.01). Our results suggest that increased LD in serum is a common finding in measles infection and presumably originates from lymphocytes.