Rationally Designed Peptides as Efficient Inhibitors of Nucleic Acid Chaperone Activity of HIV-1 Nucleocapsid Protein.

Due to its essential roles in the viral replication cycle and to its highly conserved sequence, the nucleocapsid protein (NCp7) of the human immunodeficiency virus type 1 is a target of choice for inhibiting replication of the virus. Most NCp7 inhibitors identified so far are small molecules. A small number of short peptides also act as NCp7 inhibitors by competing with its nucleic acid (NA) binding and chaperone activities but exhibit antiviral activity only at relatively high concentrations. In this work, in order to obtain more potent NCp7 competitors, we designed a library of longer peptides (10-17 amino acids) whose sequences include most of the NCp7 structural determinants responsible for its specific NA binding and destabilizing activities. Using an in vitro assay, the most active peptide (pE) was found to inhibit the NCp7 destabilizing activity, with a 50% inhibitory concentration in the nanomolar range, by competing with NCp7 for binding to its NA substrates. Formulated with a cell-penetrating peptide (CPP), pE was found to accumulate into HeLa cells, with low cytotoxicity. However, either formulated with a CPP or overexpressed in cells, pE did not show any antiviral activity. In vitro competition experiments revealed that its poor antiviral activity may be partly due to its sequestration by cellular RNAs. The selected peptide pE therefore appears to be a useful tool for investigating NCp7 properties and functions in vitro, but further work will be needed to design pE-derived peptides with antiviral activity.

Iron Release from the Siderophore Pyoverdine in Pseudomonas aeruginosa Involves Three New Actors: FpvC, FpvG, and FpvH.

Siderophores are iron chelators produced by bacteria to access iron, an essential nutriment. Pyoverdine (PVDI), the major siderophore produced by Pseudomonas aeruginosa PAO1, consists of a fluorescent chromophore linked to an octapeptide. The ferric form of PVDI is transported from the extracellular environment into the periplasm by the outer membrane transporter, FpvA. Iron is then released from the siderophore in the periplasm by a mechanism that does not involve chemical modification of the chelator but an iron reduction step. Here, we followed the kinetics of iron release from PVDI, in vitro and in living cells, by monitoring its fluorescence (as apo PVDI is fluorescent, whereas PVDI-Fe(III) is not). Deletion of the inner membrane proteins fpvG (PA2403) and fpvH (PA2404) affected 55Fe uptake via PVDI and completely abolished PVDI-Fe dissociation, indicating that these two proteins are involved in iron acquisition via this siderophore. PVDI-Fe dissociation studies, using an in vitro assay, showed that iron release from this siderophore requires the presence of an iron reducer (DTT) and an iron chelator (ferrozine). In this assay, DTT could be replaced by the inner membrane protein, FpvG, and ferrozine by the periplasmic protein, FpvC, suggesting that FpvG acts as a reductase and FpvC as an Fe2+ chelator in the process of PVDI-Fe dissociation in the periplasm of P. aeruginosa cells. This mechanism of iron release from PVDI is atypical among Gram-negative bacteria but seems to be conserved among Pseudomonads.

A phenyl-thiadiazolylidene-amine derivative ejects zinc from retroviral nucleocapsid zinc fingers and inactivates HIV virions.

BACKGROUND: Sexual acquisition of the human immunodeficiency virus (HIV) through mucosal transmission may be prevented by using topically applied agents that block HIV transmission from one individual to another. Therefore, virucidal agents that inactivate HIV virions may be used as a component in topical microbicides. RESULTS: Here, we have identified 2-methyl-3-phenyl-2H-[1,2,4]thiadiazol-5-ylideneamine (WDO-217) as a low-molecular-weight molecule that inactivates HIV particles. Both HIV-1 and HIV-2 virions pretreated with this compound were unable to infect permissive cells. Moreover, WDO-217 was able to inhibit infections of a wide spectrum of wild-type and drug-resistant HIV-1, including clinical isolates, HIV-2 and SIV strains. Whereas the capture of virus by DC-SIGN was unaffected by the compound, it efficiently prevented the transmission of DC-SIGN-captured virus to CD4+ T-lymphocytes. Interestingly, exposure of virions to WDO-217 reduced the amount of virion-associated genomic RNA as measured by real-time RT-qPCR. Further mechanism-of-action studies demonstrated that WDO-217 efficiently ejects zinc from the zinc fingers of the retroviral nucleocapsid protein NCp7 and inhibits the cTAR destabilization properties of this protein. Importantly, WDO-217 was able to eject zinc from both zinc fingers, even when NCp7 was bound to oligonucleotides, while no covalent interaction between NCp7 and WDO-217 could be observed. CONCLUSION: This compound is a new lead structure that can be used for the development of a new series of NCp7 zinc ejectors as candidate topical microbicide agents.

Effects of tamoxifen on estrogen receptor-α level in immune cells and humoral specific response after immunization of C3H/He male mice with syngeneic testicular germ cells (TGC).

Estrogens and estrogen receptors (ERs) are potent regulators of the immune response. Disruption of ERα or modulation of its function by selective ligands during experimental autoimmune conditions changes the course of disease by influencing specific humoral and cellular responses. However, it is not known whether fluctuation in the ERα level and the variable accessibility to its ligands in immune cells influence the development of specific immune responses against auto-antigens. This study was designed to evaluate the expression level of ERα in splenic immune cells and the specific humoral immune response in male C3H/He/W mice immunized with syngeneic testicular germ cells (TGC) in the presence of tamoxifen. Levels of ERα protein in immune cell subpopulations of immunized mice (assessed by flow cytometry) increased in MHCII(+)CD86(+), MHCII(+)CD86(- ), F4/80(+)MHCII(+), immature macrophages (F4/80(+)/MHCII(- )), and CD3(+)CD4(+) T cells. Addition of tamoxifen decreased the level of ERα in MHCII(+)CD86(+), MHCII(+)CD86(- ), F4/80(+)MHCII(+), immature macrophages (F4/80(+)/MHCII(- )), and the CD19(+)CD3(- ) cell subpopulation of immunized mice. Therefore, immunization with syngeneic antigen and tamoxifen treatment evoked cell-type specific changes in the level of ERα. Irrespective of tamoxifen treatment the humoral response in immunized animals toward TGCs was similar, suggesting that modulation of the level of ERα in immune cells is not directly related to specific auto-antibody production.