Canine distemper virus persistence in demyelinating encephalitis by swift intracellular cell-to-cell spread in astrocytes is controlled by the viral attachment protein

The mechanism of viral persistence, the driving force behind the chronic progression of inflammatory demyelination in canine distemper virus (CDV) infection, is associated with non-cytolytic viral cell-to-cell spread. Here, we studied the molecular mechanisms of viral spread of a recombinant fluorescent protein-expressing virulent CDV in primary canine astrocyte cultures. Time-lapse video microscopy documented that CDV spread was very efficient using cell processes contacting remote target cells. Strikingly, CDV transmission to remote cells could occur in less than 6 h, suggesting that a complete viral cycle with production of extracellular free particles was not essential in enabling CDV to spread in glial cells. Titration experiments and electron microscopy confirmed a very low CDV particle production despite higher titers of membrane-associated viruses. Interestingly, confocal laser microscopy and lentivirus transduction indicated expression and functionality of the viral fusion machinery, consisting of the viral fusion (F) and attachment (H) glycoproteins, at the cell surface. Importantly, using a single-cycle infectious recombinant H-knockout, H-complemented virus, we demonstrated that H, and thus potentially the viral fusion complex, was necessary to enable CDV spread. Furthermore, since we could not detect CD150/SLAM expression in brain cells, the presence of a yet non-identified glial receptor for CDV was suggested. Altogether, our findings indicate that persistence in CDV infection results from intracellular cell-to-cell transmission requiring the CDV-H protein. Viral transfer, happening selectively at the tip of astrocytic processes, may help the virus to cover long distances in the astroglial network, “outrunning” the host’s immune response in demyelinating plaques, thus continuously eliciting new lesions

Spektroskopische Untersuchungen von Verunreinigungen am Belt-Pinch HECTOR

SIGLEDEGerman

Lipase inhibitor orlistat prevents hepatitis B virus infection by targeting an early step in the virus life cycle.

Hepatitis B Virus (HBV) is a strictly hepatotropic pathogen which is very efficiently targeted to the liver and into its host cell, the hepatocyte. The sodium taurocholate co-transporting polypeptide (NTCP) has been identified as a key virus entry receptor, but the early steps in the virus life cycle are still only barely understood. Here, we investigated the effect of lipase inhibition and lipoprotein uptake on HBV infection using differentiated HepaRG cells and primary human hepatocytes. We found that an excess of triglyceride rich lipoprotein particles in vitro diminished HBV infection and a reduced hepatic virus uptake in vivo if apolipoprotein E is lacking indicating virus transport along with lipoproteins to target hepatocytes. Moreover, we showed that HBV infection of hepatocytes was inhibited by the broadly active lipase inhibitor orlistat, approved as a therapeutic agent which blocks neutral lipid hydrolysis activity. Orlistat treatment targets HBV infection at a post-entry step and inhibited HBV infection during virus inoculation strongly in a dose-dependent manner. In contrast, orlistat had no effect on HBV gene expression or replication or when added after HBV infection. Taken together, our data indicate that HBV connects to the hepatotropic lipoprotein metabolism and that inhibition of cellular hepatic lipase(s) may allow to target early steps of HBV infection

N-(3-Cyanophenyl)-2-phenylacetamide, an effective inhibitor of morbillivirus-induced membrane fusion with low cytotoxicity

Based on the structural similarity of viral fusion proteins within the family Paramyxoviridae, we tested recently described and newly synthesized acetanilide derivatives for their capacity to inhibit measles virus (MV)-, canine distemper virus (CDV)- and Nipah virus (NiV)-induced membrane fusion. We found that N-(3-cyanophenyl)-2-phenylacetamide (compound 1) has a high capacity to inhibit MV- and CDV-induced (IC(50) muM), but not NiV-induced, membrane fusion. This compound is of outstanding interest because it can be easily synthesized and its cytotoxicity is low [50 % cytotoxic concentration (CC(50)) >/= 300 muM], leading to a CC(50)/IC(50) ratio of approximately 100. In addition, primary human peripheral blood lymphocytes and primary dog brain cell cultures (DBC) also tolerate high concentrations of compound 1. Infection of human PBMC with recombinant wild-type MV is inhibited by an IC(50) of approximately 20 muM. The cell-to-cell spread of recombinant wild-type CDV in persistently infected DBC can be nearly completely inhibited by compound 1 at 50 muM, indicating that the virus spread between brain cells is dependent on the activity of the viral fusion protein. Our findings demonstrate that this compound is a most applicable inhibitor of morbillivirus-induced membrane fusion in tissue culture experiments including highly sensitive primary cells

Clearance of Measles Virus from Persistently Infected Cells by Short Hairpin RNA▿

Subacute sclerosing panencephalitis (SSPE) is a demyelinating central nervous system disease caused by a persistent measles virus (MV) infection of neurons and glial cells. There is still no specific therapy available, and in spite of an intact innate and adaptive immune response, SSPE leads inevitably to death. In order to select effective antiviral short interfering RNAs (siRNAs), we established a plasmid-based test system expressing the mRNA of DsRed2 fused with mRNA sequences of single viral genes, to which certain siRNAs were directed. siRNA sequences were expressed as short hairpin RNA (shRNA) from a lentiviral vector additionally expressing enhanced green fluorescent protein (EGFP) as an indicator. Evaluation by flow cytometry of the dual-color system (DsRed and EGFP) allowed us to find optimal shRNA sequences. Using the most active shRNA constructs, we transduced persistently infected human NT2 cells expressing virus-encoded HcRed (piNT2-HcRed) as an indicator of infection. shRNA against N, P, and L mRNAs of MV led to a reduction of the infection below detectable levels in a high percentage of transduced piNT2-HcRed cells within 1 week. The fraction of virus-negative cells in these cultures was constant over at least 3 weeks posttransduction in the presence of a fusion-inhibiting peptide (Z-Phe-Phe-Gly), preventing the cell fusion of potentially cured cells with persistently infected cells. Transduced piNT2 cells that lost HcRed did not fuse with underlying Vero/hSLAM cells, indicating that these cells do not express viral proteins any more and are “cured.” This demonstrates in tissue culture that NT2 cells persistently infected with MV can be cured by the transduction of lentiviral vectors mediating the long-lasting expression of anti-MV shRNA

Matrix conditions and KLF2-dependent induction of heme oxygenase-1 modulate inhibition of HCV replication by fluvastatin.

BACKGROUND & AIMS: HMG-CoA-reductase-inhibitors (statins) have been shown to interfere with HCV replication in vitro. We investigated the mechanism, requirements and contribution of heme oxygenase-1(HO-1)-induction by statins to interference with HCV replication. METHODS: HO-1-induction by fluva-, simva-, rosuva-, atorva- or pravastatin was correlated to HCV replication, using non-infectious replicon systems as well as the infectious cell culture system. The mechanism of HO-1-induction by statins as well as its relevance for interference with HCV replication was investigated using transient or permanent knockdown cell lines. Polyacrylamide(PAA) gels of different density degrees or the Rho-kinase-inhibitor Hydroxyfasudil were used in order to mimic matrix conditions corresponding to normal versus fibrotic liver tissue. RESULTS: All statins used, except pravastatin, decreased HCV replication and induced HO-1 expression, as well as interferon response in vitro. HO-1-induction was mediated by reduction of Bach1 expression and induction of the Nuclear factor (erythroid-derived 2)-like 2 (NRF2) cofactor Krueppel-like factor 2 (KLF2). Knockdown of KLF2 or HO-1 abrogated effects of statins on HCV replication. HO-1-induction and anti-viral effects of statins were more pronounced under cell culture conditions mimicking advanced stages of liver disease. CONCLUSIONS: Statin-mediated effects on HCV replication seem to require HO-1-induction, which is more pronounced in a microenvironment resembling fibrotic liver tissue. This implicates that certain statins might be especially useful to support HCV therapy of patients at advanced stages of liver disease

A Broad-Spectrum Antiviral Peptide Blocks Infection of Viruses by Binding to Phosphatidylserine in the Viral Envelope

The ongoing threat of viral infections and the emergence of antiviral drug resistance warrants a ceaseless search for new antiviral compounds. Broadly-inhibiting compounds that act on elements shared by many viruses are promising antiviral candidates. Here, we identify a peptide derived from the cowpox virus protein CPXV012 as a broad-spectrum antiviral peptide. We found that CPXV012 peptide hampers infection by a multitude of clinically and economically important enveloped viruses, including poxviruses, herpes simplex virus-1, hepatitis B virus, HIV-1, and Rift Valley fever virus. Infections with non-enveloped viruses such as Coxsackie B3 virus and adenovirus are not affected. The results furthermore suggest that viral particles are neutralized by direct interactions with CPXV012 peptide and that this cationic peptide may specifically bind to and disrupt membranes composed of the anionic phospholipid phosphatidylserine, an important component of many viral membranes. The combined results strongly suggest that CPXV012 peptide inhibits virus infections by direct interactions with phosphatidylserine in the viral envelope. These results reiterate the potential of cationic peptides as broadly-acting virus inhibitors

A Broad-Spectrum Antiviral Peptide Blocks Infection of Viruses by Binding to Phosphatidylserine in the Viral Envelope

The ongoing threat of viral infections and the emergence of antiviral drug resistance warrants a ceaseless search for new antiviral compounds. Broadly-inhibiting compounds that act on elements shared by many viruses are promising antiviral candidates. Here, we identify a peptide derived from the cowpox virus protein CPXV012 as a broad-spectrum antiviral peptide. We found that CPXV012 peptide hampers infection by a multitude of clinically and economically important enveloped viruses, including poxviruses, herpes simplex virus-1, hepatitis B virus, HIV-1, and Rift Valley fever virus. Infections with non-enveloped viruses such as Coxsackie B3 virus and adenovirus are not affected. The results furthermore suggest that viral particles are neutralized by direct interactions with CPXV012 peptide and that this cationic peptide may specifically bind to and disrupt membranes composed of the anionic phospholipid phosphatidylserine, an important component of many viral membranes. The combined results strongly suggest that CPXV012 peptide inhibits virus infections by direct interactions with phosphatidylserine in the viral envelope. These results reiterate the potential of cationic peptides as broadly-acting virus inhibitors