Contribution of endocytic motifs in the cytoplasmic tail of herpes simplex virus type 1 glycoprotein B to virus replication and cell-cell fusion

The use of endocytic pathways by viral glycoproteins is thought to play various functions during viral infection. We previously showed in transfection assays that herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is transported from the cell surface back to the trans-Golgi network (TGN) and that two motifs of gB cytoplasmic tail, YTQV and LL, function distinctly in this process. To investigate the role of each of these gB trafficking signals in HSV-1 infection, we constructed recombinant viruses in which each motif was rendered nonfunctional by alanine mutagenesis. In infected cells, wild-type gB was internalized from the cell surface and concentrated in the TGN. Disruption of YTQV abolished internalization of gB during infection, whereas disruption of LL induced accumulation of internalized gB in early recycling endosomes and impaired its return to the TGN. The growth of both recombinants was moderately diminished. Moreover, the fusion phenotype of cells infected with the gB recombinants differed from that of cells infected with the wild-type virus. Cells infected with the YTQV-mutated virus displayed reduced cell-cell fusion, whereas giant syncytia were observed in cells infected with the LL-mutated virus. Furthermore, blocking gB internalization or impairing gB recycling to the cell surface, using drugs or a transdominant negative form of Rab11, significantly reduced cell-cell fusion. These results favor a role for endocytosis in virus replication and suggest that gB intracellular trafficking is involved in the regulation of cell-cell fusion.     

Aminoglycoside binding to the HIV-1 RNA dimerization initiation site: thermodynamics and effect on the kissing-loop to duplex conversion

Owing to a striking, and most likely fortuitous, structural and sequence similarity with the bacterial 16 S ribosomal A site, the RNA kissing-loop complex formed by the HIV-1 genomic RNA dimerization initiation site (DIS) specifically binds 4,5-disubstituted 2-deoxystreptamine (2-DOS) aminoglycoside antibiotics. We used chemical probing, molecular modeling, isothermal titration calorimetry (ITC) and UV melting to investigate aminoglycoside binding to the DIS loop–loop complex. We showed that apramycin, an aminoglycoside containing a bicyclic moiety, also binds the DIS, but in a different way than 4,5-disubstituted 2-DOS aminoglycosides. The determination of thermodynamic parameters for various aminoglycosides revealed the role of the different rings in the drug–RNA interaction. Surprisingly, we found that the affinity of lividomycin and neomycin for the DIS (K_d ~ 30 nM) is significantly higher than that obtained in the same experimental conditions for their natural target, the bacterial A site (K_d ~ 1.6 µM). In good agreement with their respective affinity, aminoglycoside increase the melting temperature of the loop–loop interaction and also block the conversion from kissing-loop complex to extended duplex. Taken together, our data might be useful for selecting new molecules with improved specificity and affinity toward the HIV-1 DIS RNA.     

Native European eels as a potential biological control for invasive crayfish

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Molecular basis for mitochondrial localization of viral particles during beet necrotic yellow vein virus infection

During infection, Beet necrotic yellow vein virus (BNYVV) particles localize transiently to the cytosolic surfaces of mitochondria. To understand the molecular basis and significance of this localization, we analyzed the targeting and membrane insertion properties of the viral proteins. ORF1 of BNYVV RNA-2 encodes the 21-kDa major coat protein, while ORF2 codes for a 75-kDa minor coat protein (P75) by readthrough of the ORF1 stop codon. Bioinformatic analysis highlighted a putative mitochondrial targeting sequence (MTS) as well as a major (TM1) and two minor (TM3 and TM4) transmembrane regions in the N-terminal part of the P75 readthrough domain. Deletion and gain-of-function analyses based on the localization of green fluorescent protein (GFP) fusions showed that the MTS was able to direct a reporter protein to mitochondria but that the protein was not persistently anchored to the organelles. GFP fused either to MTS and TM1 or to MTS and TM3-TM4 efficiently and specifically associated with mitochondria in vivo. The actual role of the individual domains in the interaction with the mitochondria seemed to be determined by the folding of P75. Anchoring assays to the outer membranes of isolated mitochondria, together with in vivo data, suggest that the TM3-TM4 domain is the membrane anchor in the context of full-length P75. All of the domains involved in mitochondrial targeting and anchoring were also indispensable for encapsidation, suggesting that the assembly of BNYVV particles occurs on mitochondria. Further data show that virions are subsequently released from mitochondria and accumulate in the cytosol.     

Conformational and inframolecular studies of the protonation of adenophostin analogues lacking the adenine moiety

Four adenophostin analogues lacking the adenine moiety were subjected to 31P- and 1H-NMR titrations in order to determine the acid-base behaviour of the individual ionisable groups of the molecules and the complex interplay of intramolecular interactions resulting from the protonation process. For the two trisphosphorylated compounds, the curve pattern of the phosphorus nuclei corresponds to the superimposition of the titration curves of a monophosphorylated polyol and a polyol carrying two vicinal phosphates, suggesting that the two phosphate moieties behave independently. Also, the general shape of 1H-NMR titration curves of the studied compounds is very close to that of adenophostin A, indicating that the adenine moiety does not specifically interact with the phosphorylated sugar moieties. The curves show, however, that both trisphosphorylated compounds adopt slightly different preferential conformations which could contribute to explain the difference in their affinity for Ins(1,4,5)P3 receptor. Their macroscopic as well as the microscopic protonation constants are higher than those of adenophostin A, indicating that the adenine moiety plays a base-weakening effect on the phosphate groups. Further analysis of the microscopic protonation constants confirms that the compound whose conformation is the closest to that of adenophostin A also shows the highest biological activity. The two bisphosphorylated analogues studied behave very similarly, suggesting that the deletion of the hydroxymethyl group on the pentafuranosyl ring only weakly influences the protonation process of the phosphate groups that bear the glucopyranose moiety.     

An interaction between U2AF 65 and CF I(m) links the splicing and 3' end processing machineries

The protein factor U2 snRNP Auxiliary Factor (U2AF) 65 is an essential component required for splicing and involved in the coupling of splicing and 3' end processing of vertebrate pre-mRNAs. Here we have addressed the mechanisms by which U2AF 65 stimulates pre-mRNA 3' end processing. We identify an arginine/serine-rich region of U2AF 65 that mediates an interaction with an RS-like alternating charge domain of the 59 kDa subunit of the human cleavage factor I (CF I(m)), an essential 3' processing factor that functions at an early step in the recognition of the 3' end processing signal. Tethered functional analysis shows that the U2AF 65/CF I(m) 59 interaction stimulates in vitro 3' end cleavage and polyadenylation. These results therefore uncover a direct role of the U2AF 65/CF I(m) 59 interaction in the functional coordination of splicing and 3' end processing.     

A simple theoretical model for fluorescence polarization binding assay development

Fluorescence polarization is a screening technology that is radioactivity free, homogeneous, and ratiometric. The signal measured with this technology is a weighted value of free and bound ligand. As a consequence, saturation curves are accessible only after calculation of the corresponding concentrations of free and bound ligand. To make this technology more accessible to assay development, the authors propose a simple mathematical model that predicts fluorescence polarization values from ligand and receptor total concentrations, depending on the corresponding dissociation constant. This model was validated using data of Bodipy-NDP-alphaMSH binding to MC(5), obtained after either ligand saturation of a receptor preparation or, conversely, receptor saturation of a ligand solution. These experimental data were also used to calculate the actual concentration of free and bound ligand and receptor and to obtain pharmacological constants by Scatchard analysis. A general method is proposed, which facilitates the design of fluorescence polarization binding assays by relying on the representation of theoretical polarization values. This approach is illustrated by the application to 2 systems of very different affinities.