Kaposi's sarcoma associated herpesvirus tegument protein ORF75 is essential for viral lytic replication and plays a critical role in the antagonization of ND10-instituted intrinsic immunity.

Nuclear domain 10 (ND10) components are restriction factors that inhibit herpesviral replication. Effector proteins of different herpesviruses can antagonize this restriction by a variety of strategies, including degradation or relocalization of ND10 proteins. We investigated the interplay of Kaposi's Sarcoma-Associated Herpesvirus (KSHV) infection and cellular defense by nuclear domain 10 (ND10) components. Knock-down experiments in primary human cells show that KSHV-infection is restricted by the ND10 components PML and Sp100, but not by ATRX. After KSHV infection, ATRX is efficiently depleted and Daxx is dispersed from ND10, indicating that these two ND10 components can be antagonized by KSHV. We then identified the ORF75 tegument protein of KSHV as the viral factor that induces the disappearance of ATRX and relocalization of Daxx. ORF75 belongs to a viral protein family (viral FGARATs) that has homologous proteins in all gamma-herpesviruses. Isolated expression of ORF75 in primary cells induces a relocalization of PML and dispersal of Sp100, indicating that this viral effector protein is able to influence multiple ND10 components. Moreover, by constructing a KSHV mutant harboring a stop codon at the beginning of ORF75, we could demonstrate that ORF75 is absolutely essential for viral replication and the initiation of viral immediate-early gene expression. Using recombinant viruses either carrying Flag- or YFP-tagged variants of ORF75, we could further corroborate the role of ORF75 in the antagonization of ND10-mediated intrinsic immunity, and show that it is independent of the PML antagonist vIRF3. Members of the viral FGARAT family target different ND10 components, suggesting that the ND10 targets of viral FGARAT proteins have diversified during evolution. We assume that overcoming ND10 intrinsic defense constitutes a critical event in the replication of all herpesviruses; on the other hand, restriction of herpesviral replication by ND10 components may also promote latency as the default outcome of infection.

Comprehensive approach to study complement C4 in systemic lupus erythematosus: Gene polymorphisms, protein levels and functional activity.

Genetic variation of the genes encoding complement component C4 is strongly associated with systemic lupus erythematosus (SLE), a chronic multi-organ auto-immune disease. This study examined C4 and its isotypes on a genetic, protein, and functional level in 140 SLE patients and 104 healthy controls. Gene copy number (GCN) variation, silencing CT-insertion, and the retroviral HERV-K(C4) insertion) were analyzed with multiplex ligation-dependent probe amplification. Increased susceptibility to SLE was found for low GCN (≪2) of C4A. Serositis was the only clinical manifestation associated with low C4A GCN. One additional novel silencing mutation in the C4A gene was found by Sanger sequencing. This mutation causes a premature stop codon in exon 11. Protein concentrations of C4 isoforms C4A and C4B were determined with ELISA and were significantly lower in SLE patients compared to healthy controls. To study C4 isotypes on a functional level, a new C4 assay was developed, which distinguishes C4A from C4B by its binding capacity to amino or hydroxyl groups, respectively. This assay showed high correlation with ELISA and detected crossing over of Rodgers and Chido antigens in 3.2% (8/244) of individuals. The binding capacity of available C4 to its substrates was unaffected in SLE. Our study provides, for the first time, a complete overview of C4 in SLE from genetic variation to binding capacity using a novel test. As this test detects crossing over of Rodgers and Chido antigens, it will allow for more accurate measurement of C4 in future studies.

An outbreak of fulminant hepatitis B in immunocompromised hemodialysis patients.

BACKGROUND: We aimed to clarify the pathogenesis of an outbreak of fulminant hepatitis B in hemodialysis (HD) patients whose compromised cell-mediated immunity in turn contributed to chronic hepatitis B virus (HBV) carriage. METHODS: Five consecutive adult HD patients with acute hepatitis B were evaluated. Viral genotype, mutations, and HBV-DNA levels were studied in relation to viral clearance, liver disease severity, and liver histology by immunostaining. RESULTS: All five patients had hepatitis B surface antigen (HBsAg) genotype C, a G-to-A stop codon mutation at nucleotide (nt) 1896 in the precore region, an A-to-T mutation at nt 1762 and an G-to-A mutation at nt 1764 in the basal core promoter. The possible index patient, who suffered from liver cirrhosis, had HBsAg genotype C, anti-hepatitis B envelope (HBe), and these mutations. The level of HBV-DNA declined by about 10 percent per week and no difference in viral kinetics between the patients who died and the survivor was found, irrespective of therapies. The amount of liver cell apoptosis, as assessed by single-stranded DNA, was scarce. The risk of fulminant hepatic failure did not correlate with the preexistent liver histopathological changes. Acute HBV superinfection was associated with hepatitis C virus (HCV) elimination and increased mortality. CONCLUSIONS: This outbreak of fulminant hepatitis B suggests that HD patients can foster highly virulent HBV strains (possibly owing to their compromised immune responses), which may place others at risk of severe, life-threatening acute liver damage and at increased risk of mortality if chronic carriers of HCV should be infected. We aimed to clarify the pathogenesis of an outbreak of fulminant hepatitis B in hemodialysis (HD) patients whose compromised cell-mediated immunity in turn contributed to chronic hepatitis B virus (HBV) carriage.

The antiviral factor APOBEC3G improves CTL recognition of cultured HIV-infected T cells.

The cytidine deaminase APOBEC3G (A3G) enzyme exerts an intrinsic anti-human immunodeficiency virus (HIV) defense by introducing lethal G-to-A hypermutations in the viral genome. The HIV-1 viral infectivity factor (Vif) protein triggers degradation of A3G and counteracts this antiviral effect. The impact of A3G on the adaptive cellular immune response has not been characterized. We examined whether A3G-edited defective viruses, which are known to express truncated or misfolded viral proteins, activate HIV-1-specific (HS) CD8+ cytotoxic T lymphocytes (CTLs). To this end, we compared the immunogenicity of cells infected with wild-type or Vif-deleted viruses in the presence or absence of the cytidine deaminase. The inhibitory effect of A3G on HIV replication was associated with a strong activation of cocultivated HS-CTLs. CTL activation was particularly marked with Vif-deleted HIV and with viruses harboring A3G. Enzymatically inactive A3G mutants failed to enhance CTL activation. We also engineered proviruses bearing premature stop codons in their genome as scars of A3G editing. These viruses were not infectious but potently activated HS-CTLs. Therefore, the pool of defective viruses generated by A3G represents an underestimated source of viral antigens. Our results reveal a novel function for A3G, acting not only as an intrinsic antiviral factor but also as an inducer of the adaptive immune system.

Homozygosity for premature stop codon of the MHC class I chain-related gene A (MIC-A) is associated with early activation of islet autoimmunity of DR3/4-DQ2/8 high risk DAISY relatives.

We hypothesized that homozygosity for the major histocompatibility complex (MHC) class I chain-related gene A (MIC-A)5.1 allele with premature stop codon would increase diabetes risk of individuals followed from infancy in the DAISY study (Diabetes Autoimmunity Study in the young). Forty five percent (10/22) of relatives (siblings and offspring cohort, SOC) who developed anti-islet autoantibodies were MIC-A5.1/5.1 homozygous. Of SOC individuals without autoantibodies, 12/58 (19%, p = 0.02) were MIC-A5.1 homozygous. By life table analysis of expression of autoantibodies, DR3-DQ2/ DR4-DQ8 more than 50% of MIC-A5.1 homozygous children became autoantibody positive by 7 years of age, compared to delayed development of autoantibodies for non-MIC-A5.1/5.1 DR3-DQ2/ DR4-DQ8 children (p = 0.005). For DR3-DQ2/DR4-DQ8 nonrelatives, the risk of activating anti-islet autoimmunity remained low even with MIC-A5.1 homozygosity suggesting that there are additional factors contributing to the marked risk of relatives compared to the general population with the DR3-DQ2/DR4-DQ8 genotype.

Non-expression of HLA-B*5111N is caused by an insertion into the cytosine island at exon 4 creating a frameshift stop codon.

The identification of the "blank" allele HLA-B*5111N, which was detected in German and Czech individuals, is described. In the pedigree analysis this new allele segregates with the serological haplotype HLA-A2; B-; DR4 which is frequent in Czech population. The non-expression of B*5111N is caused by the insertion of an additional cytosine molecule at the cytosine island between the nucleotides 621-626 (codons 183-185, first three codons of exon 4) leading to a frame shift that creates a stop codon at codon 196. This insertion may be explained either by conversion with the pseudogene HLA-J or by slipped-strand mispairing. In order not to overlook the presence of alleles with altered expression in case of hematopoietic stem cell transplantation, both serological and DNA-based typing should be performed (Note).

Increased transcription levels induce higher mutation rates in a hypermutating cell line.

Somatic hypermutation, in addition to V(D)J recombination, is the other major mechanism that generates the vast diversity of the Ab repertoire. Point mutations are introduced in the variable region of the Ig genes at a million-fold higher rate than in the rest of the genome. We have used a green fluorescent protein (GFP)-based reversion assay to determine the role of transcription in the mutation mechanism of the hypermutating cell line 18-81. A GFP transgene containing a premature stop codon is transcribed from the inducible tet-on operon. Using the inducible promoter enables us to study the mutability of the GFP transgene at different transcription levels. By analyzing stable transfectants of a hypermutating cell line with flow cytometry, the mutation rate at the premature stop codon can be measured by the appearance of GFP-positive revertant cells. Here we show that the mutation rate of the GFP transgene correlates with its transcription level. Increased transcription levels of the GFP transgene caused an increased point mutation rate at the premature stop codon. Treating a hypermutating transfection clone with trichostatin A, a specific inhibitor of histone deacetylase, caused an additional 2-fold increase in the mutation rate. Finally, using Northern blot analysis we show that the activation-induced cytidine deaminase, an essential trans-factor for the in vivo hypermutation mechanism, is transcribed in the hypermutating cell line 18-81.

The destabilization of IL-2 mRNA by a premature stop codon and its differential stabilization by trans-acting inhibitors of protein synthesis do not support a role for active translation in mRNA stability.

To investigate the role that translation plays in the stabilization of the IL-2 mRNA, we inhibited protein synthesis in both cis and trans. To block translation in trans, we utilized the inhibitors puromycin (PUR) and cycloheximide (CHX), which differentially effect polysome structure. We found that CHX enhances the stability of IL-2 mRNA in cells stimulated with anti-TCR Ab alone, but it inhibits CD28-induced message stabilization in costimulated cells. In contrast, PUR had a minimal effect on IL-2 mRNA stability in either the presence or absence of costimulation. The differential effects of these two inhibitors suggest that: 1) CHX is unlikely to stabilize the IL-2 mRNA by inhibiting the expression of a labile RNase; 2) CD28-mediated IL-2 mRNA stabilization does not require translation; and 3) IL-2 mRNA decay is not coupled to translation. To block translation in cis, we generated sequence-tagged IL-2 genomic reporters that contain a premature termination codon (PTC). In both the presence and absence of costimulation, these PTC-containing mRNAs exhibit drastically diminished stability. Interestingly, the addition of CHX but not PUR completely restored CD28-mediated stabilization, suggesting that CHX can block the enhanced decay induced by a PTC. Finally, CHX was able to superinduce IL-2 mRNA levels in anti-TCR Ab-stimulated cells but not in CD28-costimulated cells, suggesting that CHX may also act by other mechanisms.