For enveloped viruses, fusion of the viral envelope with a cellular membrane is critical for a productive infection to occur. This fusion process is mediated by at least three classes of fusion proteins (Class I, II, and III) based on the protein sequence and structure. For Rift Valley fever virus (RVFV), the glycoprotein Gc (Class II fusion protein) mediates this fusion event following entry into the endocytic pathway, allowing the viral genome access to the cell cytoplasm. Here, we show that peptides analogous to the RVFV Gc stem region inhibited RVFV infectivity in cell culture by inhibiting the fusion process. Further, we show that infectivity can be inhibited for diverse, unrelated RNA viruses that have Class I (Ebola virus), Class II (Andes virus), or Class III (vesicular stomatitis virus) fusion proteins using this single peptide. Our findings are consistent with an inhibition mechanism similar to that proposed for stem peptide fusion inhibitors of dengue virus in which the RVFV inhibitory peptide first binds to both the virion and cell membranes, allowing it to traffic with the virus into the endocytic pathway. Upon acidification and rearrangement of Gc, the peptide is then able to specifically bind to Gc and prevent fusion of the viral and endocytic membranes, thus inhibiting viral infection. These results could provide novel insights into conserved features among the three classes of viral fusion proteins and offer direction for the future development of broadly active fusion inhibitors.
Polysialic acid (PSA) is a major regulator of cell-cell interactions in the developing nervous system and in neural plasticity in the adult. As a polyanionic molecule with high water-binding capacity, PSA increases the intercellular space generating permissive conditions for cell motility. PSA enhances stem cell migration and axon path finding and promotes repair in the lesioned peripheral and central nervous systems, thus contributing to regeneration. As a next step in developing an improved PSA-based approach to treat nervous system injuries, we searched for small organic compounds that mimic PSA and identified as a PSA mimetic 5-nonyloxytryptamine oxalate, described as a selective 5-hydroxytryptamine receptor 1B (5-HT1B ) agonist. Similar to PSA, 5-nonyloxytryptamine binds to the PSA-specific monoclonal antibody 735, enhances neurite outgrowth of cultured primary neurons and process formation of Schwann cells, protects neurons from oxidative stress, reduces migration of astrocytes and enhances myelination in vitro. Furthermore, nonyloxytryptamine treatment enhances expression of the neural cell adhesion molecule (NCAM) and its polysialylated form PSA-NCAM and reduces expression of the microtubule-associated protein MAP2 in cultured neuroblastoma cells. These results demonstrate that 5-nonyloxytryptamine mimics PSA and triggers PSA-mediated functions, thus contributing to the repertoire of molecules with the potential to improve recovery in acute and chronic injuries of the mammalian peripheral and central nervous systems. Polysialic acid (PSA) plays important roles in nervous system development, as well as synaptic plasticity and regeneration in the adult. 5-Nonyloxytryptamine oxalate (5-NOT) mimics PSA and triggers PSA-mediated functions in neurons and glial cells. 5-NOT stimulates neuritogenesis, myelination and Schwann cell migration. This study sets the basis to develop a PSA-mediated therapy of acute and chronic nervous system diseases.
Type I interferons (IFNs) are potent mediators of the innate immune response to viral infection. IFNs released from infected cells bind to a receptor (IFNAR) on neighboring cells, triggering signaling cascades that limit further infection. Subtle variations in amino acids can alter IFNAR binding and signaling outcomes. We used a new gene crossbreeding method to generate hybrid, type I human IFNs with enhanced antiviral activity against four dissimilar, highly pathogenic viruses. Approximately 1400 novel IFN genes were expressed in plants, and the resultant IFN proteins were screened for antiviral activity. Comparing the gene sequences of a final set of 12 potent IFNs to those of parent genes revealed strong selection pressures at numerous amino acids. Using three-dimensional models based on a recently solved experimental structure of IFN bound to IFNAR, we show that many but not all of the amino acids that were highly selected for are predicted to improve receptor binding.
Fungal mating types in self-incompatible Pezizomycotina are specified by one of two alternate sequences occupying the same locus on corresponding chromosomes. One sequence is characterized by a gene encoding an HMG protein, while the hallmark of the other is a gene encoding a protein with an ?1 domain showing similarity to the Mat?1p protein of Saccharomyces cerevisiae. DNA-binding HMG proteins are ubiquitous and well characterized. In contrast, ?1 domain proteins have limited distribution and their evolutionary origin is obscure, precluding a complete understanding of mating-type evolution in Ascomycota. Although much work has focused on the role of the S. cerevisiae Mat?1p protein as a transcription factor, it has not yet been placed in any of the large families of sequence-specific DNA-binding proteins.
Potyvirus resistance in Capsicum spp. has been attributed to amino acid substitutions at the pvr1 locus that cause conformational shifts in eukaryotic translation initiation factor eIF4E. The viral genome-linked protein (VPg) sequence was isolated and compared from three Tobacco etch virus (TEV) strains, highly aphid-transmissible (HAT), Mex21, and N, which differentially infect Capsicum genotypes encoding Pvr1(+), pvr1, and pvr1(2). Viral chimeras were synthesized using the TEV-HAT genome, replacing HAT VPg with Mex21 or N VPg. TEV HAT did not infect pepper plants homozygous for either the pvr1 or pvr1(2) allele. However, the novel chimeric TEV strains, TEVHAT(Mex21-VPg) and TEV-HAT(N-VPg), infected pvr1 and pvr1(2) pepper plants, respectively, demonstrating that VPg is the virulence determinant in this pathosystem. Three dimensional structural models predicted interaction between VPg and the susceptible eIF4E genotype in every case, while resistant genotypes were never predicted to interact. To determine whether there is a correlation between physical interaction of VPg with eIF4E and infectivity, the effects of amino acid variation within VPg were assessed. Interaction between pvr1(2) eIF4E and N VPg was detected in planta, implying that the six amino acid differences in N VPg relative to HAT VPg are responsible for restoring the physical interaction and infectivity.
Quantitatively predicting changes in drug sensitivity associated with residue mutations is a major challenge in structural biology. By expanding the limits of free energy calculations, we successfully identified mutations in influenza neuraminidase (NA) that confer drug resistance to two antiviral drugs, zanamivir and oseltamivir. We augmented molecular dynamics (MD) with Hamiltonian Replica Exchange and calculated binding free energy changes for H274Y, N294S, and Y252H mutants. Based on experimental data, our calculations achieved high accuracy and precision compared with results from established computational methods. Analysis of 15 micros of aggregated MD trajectories provided insights into the molecular mechanisms underlying drug resistance that are at odds with current interpretations of the crystallographic data. Contrary to the notion that resistance is caused by mutant-induced changes in hydrophobicity of the binding pocket, our simulations showed that drug resistance mutations in NA led to subtle rearrangements in the protein structure and its dynamics that together alter the active-site electrostatic environment and modulate inhibitor binding. Importantly, different mutations confer resistance through different conformational changes, suggesting that a generalized mechanism for NA drug resistance is unlikely.
We analyzed the thermodynamic and structural determinants of indolicidin interactions with eukaryotic and prokaryotic cell membranes using a series of atomistically detailed molecular dynamics simulations. We used quartz-supported bilayers with two different compositions of zwitterionic and anionic phospholipids as model eukaryotic and prokaryotic cell membranes. Indolicidin was preferentially attracted to the model prokaryotic cell membrane in contrast to the weak adsorption on the eukaryotic membrane. The nature of the indolicidin surface adsorption depended on an electrostatic guiding component, an attractive enthalpic component derived from van der Waals interactions, and a balance between entropic factors related to peptide confinement at the interface and counterion release from the bilayer surface. Thus, whereas we attributed the specificity of the indolicidin/membrane interaction to electrostatics, these interactions were not the sole contributors to the free energy of adsorption. Instead, a balance between an attractive van der Waals enthalpic component and a repulsive entropic component determined the overall strength of indolicidin adsorption.
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