One of the characteristics of hepatitis C virus (HCV) is the high incidence of persistent infection. HCV core protein, in addition to forming the viral nucleocapsid, has multiple regulatory functions in host-cell transcription, apoptosis, cell transformation, and lipid metabolism and may play a role in suppressing host immune response. This protein is thought to be present in the bloodstream of the infected host as the nucleocapsid of infectious, enveloped virions. This study provides evidence that viral particles with the physicochemical, morphological, and antigenic properties of nonenveloped HCV nucleocapsids are present in the plasma of HCV-infected individuals. These particles have a buoyant density of 1.32 to 1.34 g/ml in CsCl, are heterogeneous in size (with predominance of particles 38 to 43 or 54 to 62 nm in diameter on electron microscopy), and express on their surface epitopes located in amino acids 24 to 68 of the core protein. Similar nucleocapsid-like particles are also produced in insect cells infected with recombinant baculovirus bearing cDNA for structural HCV proteins. HCV core particles isolated from plasma were used to generate anti-core monoclonal antibodies (MAbs). These MAbs stained HCV core in the cytoplasm of hepatocytes from experimentally infected chimpanzees in the acute phase of the infection. These chimpanzees had concomitantly HCV core antigen in serum. These findings suggest that overproduction of nonenveloped nucleocapsids and their release into the bloodstream are properties of HCV morphogenesis. The presence of circulating cores in serum and accumulation of the core protein in liver cells during the early phase of infection may contribute to the persistence of HCV and its many immunopathological effects in the infected host.

The possible role of candidate receptors in the cellular penetration of HCV from serum of infected patients remains unclear. SR-BI/Cla1 interacts with plasma HDL, native and modified LDL, and VLDL, and facilitates cellular cholesterol efflux to lipoprotein acceptors. SR-BI/Cla1 binds HCV E2 protein and interacts with HCV pseudotypes via the HVR1 of the E2 envelope glycoprotein. Our data reveal that functional SR-BI/Cla1 expressed on the surface of CHO cells mediates the binding and uptake of HCV from the sera of infected patients. Interaction between HCV and SR-BI/Cla1 is not sensitive to either anti-E2 or anti-HVR1 antibodies but is effectively inhibited by anti-betalipoprotein antibodies and competed out by apoB-containing lipoproteins and notably by VLDL. We interpret our data to indicate that VLDL associated with or incorporated into HCV plays a critical role in the primary interaction of HCV with SR-BI/Cla1, whereas the HCV E2 protein does not. In addition, our findings in hepatoma cell lines suggest that the interaction of HCV with human hepatocytes is equally mediated, at least in a part, by VLDL, and as such may represent an alternative pathway for infection. The association of HCV with ApoB-containing lipoproteins may promote cellular uptake of this virus in the presence of neutralizing antibodies.

Hepatitis C virus is a poor inducer of interferon (IFN), although its structured viral RNA can bind the RNA helicase RIG-I, and activate the IFN-induction pathway. Low IFN induction has been attributed to HCV NS3/4A protease-mediated cleavage of the mitochondria-adapter MAVS. Here, we have investigated the early events of IFN induction upon HCV infection, using the cell-cultured HCV JFH1 strain and the new HCV-permissive hepatoma-derived Huh7.25.CD81 cell subclone. These cells depend on ectopic expression of the RIG-I ubiquitinating enzyme TRIM25 to induce IFN through the RIG-I/MAVS pathway. We observed induction of IFN during the first 12 hrs of HCV infection, after which a decline occurred which was more abrupt at the protein than at the RNA level, revealing a novel HCV-mediated control of IFN induction at the level of translation. The cellular protein kinase PKR is an important regulator of translation, through the phosphorylation of its substrate the eIF2α initiation factor. A comparison of the expression of luciferase placed under the control of an eIF2α-dependent (IRESEMCV) or independent (IRESHCV) RNA showed a specific HCV-mediated inhibition of eIF2α-dependent translation. We demonstrated that HCV infection triggers the phosphorylation of both PKR and eIF2α at 12 and 15 hrs post-infection. PKR silencing, as well as treatment with PKR pharmacological inhibitors, restored IFN induction in JFH1-infected cells, at least until 18 hrs post-infection, at which time a decrease in IFN expression could be attributed to NS3/4A-mediated MAVS cleavage. Importantly, both PKR silencing and PKR inhibitors led to inhibition of HCV yields in cells that express functional RIG-I/MAVS. In conclusion, here we provide the first evidence that HCV uses PKR to restrain its ability to induce IFN through the RIG-I/MAVS pathway. This opens up new possibilities to assay PKR chemical inhibitors for their potential to boost innate immunity in HCV infection.

Skin and chronic wound infections caused by highly antibiotic resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) are an increasing and urgent health problem worldwide, particularly with sharp increases in obesity and diabetes. New Zealand manuka honey has potent broad-spectrum antimicrobial activity, has been shown to inhibit the growth of MRSA strains, and bacteria resistant to this honey have not been obtainable in the laboratory. Combinational treatment of chronic wounds with manuka honey and common antibiotics may offer a wide range of advantages including synergistic enhancement of the antibacterial activity, reduction of the effective dose of the antibiotic, and reduction of the risk of antibiotic resistance. The aim of this study was to investigate the effect of Medihoney in combination with the widely used antibiotic rifampicin on S. aureus. Using checkerboard microdilution assays, time-kill curve experiments and agar diffusion assays, we show a synergism between Medihoney and rifampicin against MRSA and clinical isolates of S. aureus. Furthermore, the Medihoney/rifampicin combination stopped the appearance of rifampicin-resistant S. aureus in vitro. Methylglyoxal (MGO), believed to be the major antibacterial compound in manuka honey, did not act synergistically with rifampicin and is therefore not the sole factor responsible for the synergistic effect of manuka honey with rifampicin. Our findings support the idea that a combination of honey and antibiotics may be an effective new antimicrobial therapy for chronic wound infections.

Cardiolipin (CL) is the signature phospholipid of the mitochondrial inner membrane. In animals and yeast (Saccharomyces cerevisiae), CL depletion affects the stability of respiratory supercomplexes and is thus crucial to the energy metabolism of obligate aerobes. In eukaryotes, the last step of CL synthesis is catalyzed by CARDIOLIPIN SYNTHASE (CLS), encoded by a single-copy gene. Here, we characterize a cls mutant in Arabidopsis thaliana, which is devoid of CL. In contrast to yeast cls, where development is little affected, Arabidopsis cls seedlings are slow developing under short-day conditions in vitro and die if they are transferred to long-day (LD) conditions. However, when transferred to soil under LD conditions under low light, cls plants can reach the flowering stage, but they are not fertile. The cls mitochondria display abnormal ultrastructure and reduced content of respiratory complex I/complex III supercomplexes. The marked accumulation of tricarboxylic acid cycle derivatives and amino acids demonstrates mitochondrial dysfunction. Mitochondrial and chloroplastic antioxidant transcripts are overexpressed in cls leaves, and cls protoplasts are more sensitive to programmed cell death effectors, UV light, and heat shock. Our results show that CLS is crucial for correct mitochondrial function and development in Arabidopsis under both optimal and stress conditions.

SummaryThe host-virus interactions leading to cell infection with hepatitis C virus (HCV) are not fully understood. The tetraspanin CD-81 and human scavenger receptor SR-BI/Cla1 are major receptors mediating virus cell entry. However, HCV in patients' sera is associated with lipoproteins and infectious potential of the virus depends on lipoproteins associated to virus particles. We show here that lipoprotein lipase (LPL), targeting triglyceride-rich lipoproteins (TRL) to the liver, mediates binding and internalization of HCV to different types of cells, acting as a bridge between virusassociated lipoproteins and cell surface heparan sulfate proteoglycans (HSPG). The dimeric structure and catalytic activity of LPL are required for LPLmediated HCV uptake to cells. Unexpectedly, exogenous LPL significantly inhibits HCVcc infection in vitro. This effect is prevented by anti-LPL antibodies and by tetrahydrolipstatin (THL) a specific inhibitor of LPL enzymatic activity. In addition, we show that antibodies directed to apolipoprotein B (ApoB)-containing lipoproteins efficiently inhibits HCVcc infection. Our findings suggest that LPL mediates HCV cell entry by a mechanism similar to hepatic clearance of TRL from the circulation, promoting a non-productive virus uptake. These data provide new insight into mechanisms of HCV cell entry and suggest that LPL could modulate HCV infectivity in vivo.

Early events leading to the establishment of hepatitis C virus (HCV) infection are not completely understood. We show that intact and dynamic microtubules play a key role in the initiation of productive HCV infection. Microtubules were required for virus entry into cells, as evidenced using virus pseudotypes presenting HCV envelope proteins on their surface. Studies carried out using the recent infectious HCV model revealed that microtubules also play an essential role in early, postfusion steps of the virus cycle. Moreover, low concentrations of vinblastin and nocodazol, microtubule-affecting drugs, and paclitaxel, which stabilizes microtubules, inhibited infection, suggesting that microtubule dynamic instability and/or treadmilling mechanisms are involved in HCV internalization and early transport. By protein chip and direct core-dependent pull-down assays, followed by mass spectrometry, we identified ␤-and ␣-tubulin as cellular partners of the HCV core protein. Surface plasmon resonance analyses confirmed that core directly binds to tubulin with high affinity via amino acids 2-117. The interaction of core with tubulin in vitro promoted its polymerization and enhanced the formation of microtubules. Immune electron microscopy showed that HCV core associates, at least temporarily, with microtubules polymerized in its presence. Studies by confocal microscopy showed a juxtaposition of core with microtubules in HCV-infected cells. In summary, we report that intact and dynamic microtubules are required for virus entry into cells and for early postfusion steps of infection. HCV may exploit a direct interaction of core with tubulin, enhancing microtubule polymerization, to establish efficient infection and promote virus transport and/or assembly in infected cells.

Skin infections caused by antibiotic resistant Staphylococcus aureus are a significant health problem worldwide; often associated with high treatment cost and mortality rate. Complex natural products like New Zealand (NZ) manuka honey have been revisited and studied extensively as an alternative to antibiotics due to their potent broad-spectrum antimicrobial activity, and the inability to isolate honey-resistant S. aureus. Previous studies showing synergistic effects between manuka-type honeys and antibiotics have been demonstrated against the growth of one methicillin-resistant S. aureus (MRSA) strain. We have previously demonstrated strong synergistic activity between NZ manuka-type honey and rifampicin against growth and biofilm formation of multiple S. arueus strains. Here, we have expanded our investigation using multiple S. aureus strains and four different antibiotics commonly used to treat S. aureus-related skin infections: rifampicin, oxacillin, gentamicin, and clindamycin. Using checkerboard microdilution and agar diffusion assays with S. aureus strains including clinical isolates and MRSA we demonstrate that manuka-type honey combined with these four antibiotics frequently produces a synergistic effect. In some cases when synergism was not observed, there was a significant enhancement in antibiotic susceptibility. Some strains that were highly resistant to an antibiotic when present alone become sensitive to clinically achievable concentrations when combined with honey. However, not all of the S. aureus strains tested responded in the same way to these combinational treatments. Our findings support the use of NZ manuka-type honeys in clinical treatment against S. aureus-related infections and extend their potential use as an antibiotic adjuvant in combinational therapy. Our data also suggest that manuka-type honeys may not work as antibiotic adjuvants for all strains of S. aureus, and this may help determine the mechanistic processes behind honey synergy.