Albrecht von Brunn

Evolutionarily conserved herpesviral protein interaction networks.

Herpesviruses constitute a family of large DNA viruses widely spread in vertebrates and causing a variety of different diseases. They possess dsDNA genomes ranging from 120 to 240 kbp encoding between 70 to 170 open reading frames. We previously reported the protein interaction networks of two herpesviruses, varicella-zoster virus (VZV) and Kaposis sarcoma-associated herpesvirus (KSHV). In this study, we systematically tested three additional herpesvirus species, herpes simplex virus 1 (HSV-1), murine cytomegalovirus and Epstein-Barr virus, for protein interactions in order to be able to perform a comparative analysis of all three herpesvirus subfamilies. We identified 735 interactions by genome-wide yeast-two-hybrid screens (Y2H), and, together with the interactomes of VZV and KSHV, included a total of 1,007 intraviral protein interactions in the analysis. Whereas a large number of interactions have not been reported previously, we were able to identify a core set of highly conserved protein interactions, like the interaction between HSV-1 UL33 with the nuclear egress proteins UL31/UL34. Interactions were conserved between orthologous proteins despite generally low sequence similarity, suggesting that function may be more conserved than sequence. By combining interactomes of different species we were able to systematically address the low coverage of the Y2H system and to extract biologically relevant interactions which were not evident from single species.

Analysis of Intraviral Protein-Protein Interactions of the SARS Coronavirus ORFeome

The severe acute respiratory syndrome coronavirus (SARS-CoV) genome is predicted to encode 14 functional open reading frames, leading to the expression of up to 30 structural and non-structural protein products. The functions of a large number of viral ORFs are poorly understood or unknown. In order to gain more insight into functions and modes of action and interaction of the different proteins, we cloned the viral ORFeome and performed a genome-wide analysis for intraviral protein interactions and for intracellular localization. 900 pairwise interactions were tested by yeast-two-hybrid matrix analysis, and more than 65 positive non-redundant interactions, including six self interactions, were identified. About 38% of interactions were subsequently confirmed by CoIP in mammalian cells. Nsp2, nsp8 and ORF9b showed a wide range of interactions with other viral proteins. Nsp8 interacts with replicase proteins nsp2, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp13 and nsp14, indicating a crucial role as a major player within the replication complex machinery. It was shown by others that nsp8 is essential for viral replication in vitro, whereas nsp2 is not. We show that also accessory protein ORF9b does not play a pivotal role for viral replication, as it can be deleted from the virus displaying normal plaque sizes and growth characteristics in Vero cells. However, it can be expected to be important for the virus-host interplay and for pathogenicity, due to its large number of interactions, by enhancing the global stability of the SARS proteome network, or play some unrealized role in regulating protein-protein interactions. The interactions identified provide valuable material for future studies.

The SARS-Coronavirus-Host Interactome: Identification of Cyclophilins as Target for Pan-Coronavirus Inhibitors

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.

Principal neutralizing domain of HIV-1 is highly immunogenic when expressed on the surface of hepatitis B core particles

The development of subunit vaccines against HIV requires the identification of immunologically relevant antigens and a suitable method of antigen delivery. Ideally, defined epitopes with neutralizing activity should be included in a vaccine preparation. The carrier for such peptide sequences should enhance the immunogenicity of the selected epitopes. In this study hepatitis B virus core antigen (HBcAg) was used as a carrier moiety for the principal neutralizing domain (PND, V3-loop) of HIV-1. A 25 amino acid V3-loop sequence was fused to HBcAg at various positions by genetic engineering. The resulting hybrid HBcAg/HIV polypeptides were analysed for particle formation and immunogenicity. Fusion of the PND to an internal position replacing an immunodominant antibody-binding region of HBcAg or to a C-terminally truncated HBcAg resulted in the formation of hybrid particles with biochemical and biophysical properties similar to those of wild-type HBcAg particles. Both types of hybrids are recognized by monoclonal and polyclonal antisera raised against PND peptides of various HIV-1 isolates. Hybrid particles with a C-terminal fusion but not an internal fusion are also recognized by a polyvalent anti-HBcAg serum. In both cases the V3 domain is surface accessible. Immunization of mice with hybrid particles induces an enhanced antibody response against the V3 sequence. The internal fusion is more immunogenic than the C-terminal fusion.

Is codon 129 of prion protein polymorphic in human beings but not in animals

2, not significant), respectively. sACE (U/L) tracked, codominantly, with the D allele in both groups (sarcoidosis group sACE against genotype p=0·01, control group p<0·0001, Mantel-Haenszel 2 test for linear association). Patients had greater sACE than controls when assessed according to genotype (figure). There was no significant effect of age or sex on sACE in either group. A new GBNR was defined on the basis of sACE values, two SDs from the mean in our control population. The mean (range) for the three genotypes (GBNR) were: II 17·6 (4·6‐30·6); ID 28·8 (10·0‐47·6); DD 41·1 (17·9‐64·3). With this new GBNR, 69% of sACE values were categorised as abnormally high, compared with 51·7% with our previous non-GBNR (15‐70 U/L). The overall type-1 error (false positive) of our new GBNR was 2·7% compared with 0% with our previous non-GBNR of 15‐70 U/L, whereas the overall type 2 error (false negative) was 32·3% compared with 47·4% with our previous normal range. Finally, we compared the logistic regression of disease presence versus (i) sACE levels alone (log10 transformed) and (ii) sACE plus ACE genotype. Addition of ACE genotype resulted in a highly significant improvement in prediction of disease (p<0·0001, likelihood 2 test) with our new GBNR. The new GBNR detects 69·0% of abnormally high sACE values and is an improvement of 33·5% on the previous normal range which would have identified 51·7% of our sarcoidosis patients. This improvement in sensitivity is most pronounced in patients who have at least one copy of the I allele (around 75% of sarcoidosis patients). A small increase in the type 1 error with the new GBNR is compensated for by the loss in type 2 error and the increase in sensitivity. Notably, the influence of the DD genotype on sACE in healthy controls was amplified in the sarcoidosis group, which suggests that the elusive functional variant in the ACE gene, presumed to exist in linkage disequilibrium with ACE-I/D, has a regulatory region that contains a response element to either the causal agent of sarcoidosis itself, or to a mediator that this agent releases. If our results are confirmed, GBNR for sACE should be determined locally (including for different racial populations) 1

Productive infection of in vitro generated haemopoietic progenitor cells from normal human adult peripheral blood with parvovirus B19: studies by morphology, immunocytochemistry, flow-cytometry and DNA-hybridization.

Parvovirus B19 exerts a highly selective cytopathic effect on erythroid progenitor cells. Studies so far on the pathogenesis of B19‐infection have been performed using bone marrow samples providing large amounts of erythroid progenitor cells. Extensive study, however, has been hampered by the limited access to bone marrow samples. We have designed a liquid culture method allowing the generation of large numbers of erythroid progenitor cells, initiating cultures with CD3‐ and CD14‐poor peripheral blood mononuclear cells. Following a 12 d preincubation in liquid cultures containing recombinant human interleukin 3 (rhll‐3) and recombinant human erythropoietin (rhEpo), cells harvested from the liquid cultures were exposed to B19‐containing plasma, followed by a further cultivation in liquid culture for up to 96 h. Cells expressing the CD13 and the glycophorin A (GlyA) antigens, respectively, were monitored sequentially by flow‐cytometry, demonstrating a selective inhibition of GlyA‐positive cells following B19‐inoculation. Typical morphological changes were observed on cytocentrifuge‐spots, and typical giant‐cells were identified as staining for GlyA. Productive infection by B19 was demonstrable, as B19‐DNA increased by about x 100 after 72 h of culture.

Bacteriophage Protein–Protein Interactions

Bacteriophages T7, λ, P22, and P2/P4 (from Escherichia coli), as well as ϕ29 (from Bacillus subtilis), are among the best-studied bacterial viruses. This chapter summarizes published protein interaction data of intraviral protein interactions, as well as known phage-host protein interactions of these phages retrieved from the literature. We also review the published results of comprehensive protein interaction analyses of Pneumococcus phages Dp-1 and Cp-1, as well as coliphages λ and T7. For example, the ≈55 proteins encoded by the T7 genome are connected by ≈43 interactions with another ≈15 between the phage and its host. The chapter compiles published interactions for the well-studied phages λ (33 intra-phage/22 phage-host), P22 (38/9), P2/P4 (14/3), and ϕ29 (20/2). We discuss whether different interaction patterns reflect different phage lifestyles or whether they may be artifacts of sampling. Phages that infect the same host can interact with different host target proteins, as exemplified by E. coli phage λ and T7. Despite decades of intensive investigation, only a fraction of these phage interactomes are known. Technical limitations and a lack of depth in many studies explain the gaps in our knowledge. Strategies to complete current interactome maps are described. Although limited space precludes detailed overviews of phage molecular biology, this compilation will allow future studies to put interaction data into the context of phage biology.

Epitopes of the human malaria parasite P. falciparum carried on the surface of HBsAg particles elicit an immune response against the parasite

The development of recombinant subunit vaccines against pathogenic organisms requires not only the identification of epitopes eliciting a protective immune response but also suitable carriers with adjuvant function. B- and T-cell epitopes of the malaria vaccine candidate gp190 were selected on the basis of a systematic search along the gp190 molecule and by computer prediction based on the amino acid sequence. Using some of the epitopes identified, we have redesigned the surface of the hepatitis B surface antigen lipoprotein particles by replacing the major antigenic determinants with malaria-specific sequences of up to 61 amino acids in length. Upon expression via vaccinia virus the hybrid particles elicit an anti-gp190 immune response in animals. Monoclonal antibodies derived from such infections recognize the native parasite.

Virus–host interactomes — antiviral drug discovery

One of the key questions in virology is how viruses, encoding relatively few genes, gain temporary or constant control over their hosts. To understand pathogenicity of a virus it is important to gain knowledge on the function of the individual viral proteins in the host cell, on their interactions with viral and cellular proteins and on the consequences of these interactions on cellular signaling pathways. A combination of transcriptomics, proteomics, high-throughput technologies and the bioinformatical analysis of the respective data help to elucidate specific cellular antiviral drug target candidates. In addition, viral and human interactome analyses indicate that different viruses target common, central human proteins for entering cellular signaling pathways and machineries which might constitute powerful broad-spectrum antiviral targets.

Comprehensive Analysis of Varicella-Zoster Virus Proteins Using a New Monoclonal Antibody Collection

ABSTRACT Varicella-zoster virus (VZV) is the etiological agent of chickenpox and shingles. Due to the viruss restricted host and cell type tropism and the lack of tools for VZV proteomics, it is one of the least-characterized human herpesviruses. We generated 251 monoclonal antibodies (MAbs) against 59 of the 71 (83%) currently known unique VZV proteins to characterize VZV protein expression in vitro and in situ. Using this new set of MAbs, 44 viral proteins were detected by Western blotting (WB) and indirect immunofluorescence (IF); 13 were detected by WB only, and 2 were detected by IF only. A large proportion of viral proteins was analyzed for the first time in the context of virus infection. Our study revealed the subcellular localization of 46 proteins, 14 of which were analyzed in detail by confocal microscopy. Seven viral proteins were analyzed in time course experiments and showed a cascade-like temporal gene expression pattern similar to those of other herpesviruses. Furthermore, selected MAbs tested positive on human skin lesions by using immunohistochemistry, demonstrating the wide applicability of the MAb collection. Finally, a significant portion of the VZV-specific antibodies reacted with orthologs of simian varicella virus (SVV), thus enabling the systematic analysis of varicella in a nonhuman primate model system. In summary, this study provides insight into the potential function of numerous VZV proteins and novel tools to systematically study VZV and SVV pathogenesis.