Porcine reproductive and respiratory syndrome virus (PRRSV) infection spreads by cell-to-cell transfer in cultured MARC-145 cells, is dependent on an intact cytoskeleton, and is suppressed by drug-targeting of cell permissiveness to virus infection

Background

Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiologic agent of PRRS, causing widespread chronic infections which are largely uncontrolled by currently available vaccines or other antiviral measures. Cultured monkey kidney (MARC-145) cells provide an important tool for the study of PRRSV replication. For the present study, flow cytometric and fluorescence antibody (FA) analyses of PRRSV infection of cultured MARC-145 cells were carried out in experiments designed to clarify viral dynamics and the mechanism of viral spread. The roles of viral permissiveness and the cytoskeleton in PRRSV infection and transmission were examined in conjunction with antiviral and cytotoxic drugs.

Results

Flow cytometric and FA analyses of PRRSV antigen expression revealed distinct primary and secondary phases of MARC-145 cell infection. PRRSV antigen was randomly expressed in a few percent of cells during the primary phase of infection (up to about 20–22 h p.i.), but the logarithmic infection phase (days 2–3 p.i.), was characterized by secondary spread to clusters of infected cells. The formation of secondary clusters of PRRSV-infected cells preceded the development of CPE in MARC-145 cells, and both primary and secondary PRRSV infection were inhibited by colchicine and cytochalasin D, demonstrating a critical role of the cytoskeleton in viral permissiveness as well as cell-to-cell transmission from a subpopulation of cells permissive for free virus to secondary targets. Cellular expression of actin also appeared to correlate with PRRSV resistance, suggesting a second role of the actin cytoskeleton as a potential barrier to cell-to-cell transmission. PRRSV infection and cell-to-cell transmission were efficiently suppressed by interferon-γ (IFN-γ), as well as the more-potent experimental antiviral agent AK-2.

Conclusion

The results demonstrate two distinct mechanisms of PRRSV infection: primary infection of a relatively small subpopulation of innately PRRSV-permissive cells, and secondary cell-to-cell transmission to contiguous cells which appear non-permissive to free virus. The results also indicate that an intact cytoskeleton is critical for PRRSV infection, and that viral permissiveness is a highly efficient drug target to control PRRSV infection. The data from this experimental system have important implications for the mechanisms of PRRSV persistence and pathology, as well as for a better understanding of arterivirus regulation.     

Towards new antimalarial drugs: synthesis of non-hydrolyzable phosphate mimics as feed for a predictive QSAR study on 1-deoxy-D-xylulose-5-phosphate reductoisomerase inhibitors

The conversion of 1-deoxy-D-xylulose-5-phosphate (DOXP) to 2-C-methyl-D-erythritol-4-phosphate (MEP) is effectively blocked by 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr) inhibitors such as the natural antibiotic fosmidomycin. Prediction of binding affinities for closely related Dxr ligands as well as estimation of the affinities of structurally more distinct inhibitors within this class of non-hydrolyzable phosphate mimics relies on the synthesis of fosmidomycin derivatives with a broad range of target affinity. Maintaining the phosphonic acid moiety, linear modifications of the lead structure were carried out in an effort to expand the SAR of this physicochemically challenging class of compounds. Synthetic access to a set of phosphonic acids with inhibitory activity (IC(50)) in the range from 1 to >30 microM vs. E. coli Dxr and 0.4 to 20 microM against P. falciparum Dxr is reported.     

Vγ9/Vδ2 T cell activation induced by bacterial low molecular mass compounds depends on the 1-deoxy-d-xylulose 5-phosphate pathway of isoprenoid biosynthesis

Isopentenyl diphosphate (IPP), an important precursor of isoprenoid biosynthesis in prokaryotic and eukaryotic organisms, has been shown to activate Vgamma9/Vdelta2 T cells, the major subset of human gammadelta T cells. The biosynthesis of IPP has been first described as the acetate/mevalonate pathway. Recently, 1-deoxy-D-xylulose 5-phosphate (DOXP) and 2-C-methyl-D-erythritol 4-phosphate have been shown to be key metabolites in the DOXP pathway also leading to the formation of IPP in some eubacteria such as Escherichia coli. Here we report that the low molecular mass fraction of extracts from bacteria using the DOXP pathway induces Vgamma9/Vdelta2 T cell activation, while analogous preparations from bacteria using the classical mevalonate pathway fail to do so. Addition of 1-deoxy-D-xylulose potentiates the ability of E. coli extracts to activate Vgamma9/Vdelta2 T cells. As the amounts of IPP present in the bacterial preparations are not sufficient to induce significant Vgamma9/Vdelta2 T cell activation, our data suggest that compounds other than IPP associated with the DOXP pathway are responsible for Vgamma9/Vdelta2 T cell activation.     

Synthesis of beta- and gamma-oxa isosteres of fosmidomycin and FR900098 as antimalarial candidates

To expand the structure-activity relationships of fosmidomycin and FR900098, two potent antimalarials interfering with the MEP-pathway, we decided to replace a methylene group in beta-position of the phosphonate moiety of these leads by an oxygen atom. beta-oxa-FR900098 (11) proved equally active as the parent compound. When applied to 4-[hydroxyl(methyl)amino]-4-oxobutyl phosphonic acid, featuring a hydroxamate instead of the retrohydroxamate moiety, a beta-oxa modification yielded a derivative (13) with superior activity against the Plasmodium falciparum 3D7 strain than fosmidomycin, while a gamma-oxa modification resulted in less active derivatives. A bis(pivaloyloxymethyl)ester of phosphonate 13 proved twice as active in inhibiting cultured parasites as a similar prodrug of FR900098.     

Hypoxia. Hypoxia in the pathogenesis of systemic sclerosis

Autoimmunity, microangiopathy and tissue fibrosis are hallmarks of systemic sclerosis (SSc). Vascular alterations and reduced capillary density decrease blood flow and impair tissue oxygenation in SSc. Oxygen supply is further reduced by accumulation of extracellular matrix (ECM), which increases diffusion distances from blood vessels to cells. Therefore, severe hypoxia is a characteristic feature of SSc and might contribute directly to the progression of the disease. Hypoxia stimulates the production of ECM proteins by SSc fibroblasts in a transforming growth factor-β-dependent manner. The induction of ECM proteins by hypoxia is mediated via hypoxia-inducible factor-1α-dependent and -independent pathways. Hypoxia may also aggravate vascular disease in SSc by perturbing vascular endothelial growth factor (VEGF) receptor signalling. Hypoxia is a potent inducer of VEGF and may cause chronic VEGF over-expression in SSc. Uncontrolled over-expression of VEGF has been shown to have deleterious effects on angiogenesis because it leads to the formation of chaotic vessels with decreased blood flow. Altogether, hypoxia might play a central role in pathogenesis of SSc by augmenting vascular disease and tissue fibrosis.     

Synthesis of alpha-substituted fosmidomycin analogues as highly potent Plasmodium falciparum growth inhibitors

In view of the promising antimalarial activity of fosmidomycin or its N-acetyl homologue FR900098, the objective of this work was to investigate the influence of aromatic substituents in the alpha-position of the phosphonate moiety. The envisaged analogues were prepared using a linear route involving a 3-aryl-3-phosphoryl propanal intermediate. The activities of all compounds were evaluated on Eschericia coli 1-deoxy-d-xylulose 5-phosphate reductoisomerase and against two Plasmodium falciparum strains. Compared with fosmidomycin, several analogues displayed enhanced activity towards the P. falciparum strains. Compound 1e with a 3,4-dichlorophenyl substitution in the alpha-position of fosmidomycin emerged as the most potent analogue of this series. It is approximately three times more potent in inhibiting the growth of P. falciparum than FR900098, the most potent representative of this class reported so far.     

Structure of the E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate synthase (GcpE) from Thermus thermophilus

Isoprenoids are biosynthesized via the mevalonate or the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways the latter being used by most pathogenic bacteria, some parasitic protozoa, plant plastids, but not by animals. We determined the X-ray structure of the homodimeric [4Fe–4S] cluster carrying E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate synthase (GcpE) of Thermus thermophilus which catalyzes the penultimate reaction of the MEP pathway and is therefore an attractive target for drug development. The [4Fe–4S] cluster ligated to three cysteines and one glutamate is encapsulated at the intersubunit interface. The substrate binding site lies in front of an (αβ)₈ barrel. The great [4Fe–4S] cluster-substrate distance implicates large-scale domain rearrangements during the reaction cycle.

Structured summary

gcpEbinds to gcpE by x-ray crystallography (View interaction)     

Acyloxyalkyl ester prodrugs of FR900098 with improved in vivo anti-malarial activity

FR900098 represents an improved derivative of the new antimalarial drug fosmidomycin and acts through inhibition of the 1-deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase, an essential enzyme of the mevalonate independent pathway of isoprenoid biosynthesis. Prodrugs with increased activity after oral administration were obtained by chemical modification of the phosphonate moiety to yield acyloxyalkyl esters. The most successful compound demonstrated 2-fold increased activity in mice infected with the rodent malaria parasite Plasmodium vinckei.     

Discovery of a novel lead structure for anti-malarials

From a library of 61 compounds available from former studies 2,5-bis-acylaminobenzophenone 7p was identified as a lead structure for a novel class of anti-malaria agents active against multi-resistant Plasmodium falciparum strain Dd2. Some structural modifications of this initial lead demonstrated the potential for further improvement of the anti-plasmodial activity of this novel class of anti-malarials.     

Vascular alterations upon activation of TGFβ signaling in fibroblasts - implications for systemic sclerosis

Tissue fibrosis and vascular disease are hallmarks of systemic sclerosis (SSc). Transforming growth factor β (TGFβ) is a key-player in fibroblast activation and tissue fibrosis in SSc. In contrast to fibrosis, evidence for a role of TGFβ in vascular disease of SSc is scarce. Using a transgenic mouse model with fibroblast-specific expression of a kinase-deficient TGFβ receptor type II, Derrett-Smith and colleagues demonstrate that aberrant TGFβ signaling in fibroblasts might result in activation of vascular smooth muscle cells and architectural changes of the vessel wall of the aorta.