Fusion peptides comprise conserved hydrophobic domains absolutely required for the fusogenic activity of glycoproteins from divergent virus families. After 30 years of intensive research efforts, the structures and functions underlying their high degree of sequence conservation are not fully elucidated. The long-hydrophobic viral fusion peptide (VFP) sequences are structurally constrained to access three successive states after biogenesis. Firstly, the VFP sequence must fulfill the set of native interactions required for (meta) stable folding within the globular ectodomains of glycoprotein complexes. Secondly, at the onset of the fusion process, they get transferred into the target cell membrane and adopt specific conformations therein. According to commonly accepted mechanistic models, membrane-bound states of the VFP might promote the lipid bilayer remodeling required for virus-cell membrane merger. Finally, at least in some instances, several VFPs co-assemble with transmembrane anchors into membrane integral helical bundles, following a locking movement hypothetically coupled to fusion-pore expansion. Here we review different aspects of the three major states of the VFPs, including the functional assistance by other membrane-transferring glycoprotein regions, and discuss briefly their potential as targets for clinical intervention.

The nonstructural protein p7 of classical swine fever virus (CSFV) is a small hydrophobic polypeptide with an apparent molecular mass of 6 to 7 kDa. The protein contains two hydrophobic stretches of amino acids interrupted by a short charged segment that are predicted to form transmembrane helices and a cytosolic loop, respectively. Using reverse genetics, partial in-frame deletions of p7 were deleterious for virus growth, demonstrating that CSFV p7 function is critical for virus production in cell cultures. A panel of recombinant mutant CSFVs was created using alanine scanning mutagenesis of the p7 gene harboring sequential three- to six-amino-acid residue substitutions spanning the entire protein. These recombinant viruses allowed the identification of the regions within p7 that are critical for virus production in vitro. In vivo , some of these viruses were partially or completely attenuated in swine relative to the highly virulent parental CSFV Brescia strain, indicating a significant role of p7 in CSFV virulence. Structure-function analyses in model membranes emulating the endoplasmic reticulum lipid composition confirmed that CSFV p7 is a pore-forming protein, and that pore-forming activity resides in the C-terminal transmembrane helix. Therefore, p7 is a viroporin which is clearly involved in the process of CSFV virulence in swine.

Antibodies against the Membrane-Proximal External Region (MPER) of the Env gp41 subunit neutralize HIV-1 with exceptional breadth and potency. Due to the lack of knowledge on the MPER native structure and accessibility, different and exclusive models have been proposed for the molecular mechanism of MPER recognition by broadly neutralizing antibodies. Here, accessibility of antibodies to the native Env MPER on single virions has been addressed through STED microscopy. STED imaging of fluorescently labeled Fabs reveals a common pattern of native Env recognition for HIV-1 antibodies targeting MPER or the surface subunit gp120. In the case of anti-MPER antibodies, the process evolves with extra contribution of interactions with the viral lipid membrane to binding specificity. Our data provide biophysical insights into the recognition of the potent and broadly neutralizing MPER epitope on HIV virions, and as such is of importance for the design of therapeutic interventions.

RTX (Repeats in ToXin) pore-forming toxins constitute an expanding family of exoproteins secreted by many Gram-negative bacteria and involved in infectious diseases caused by said pathogens. Despite the relevance in the host/pathogen interactions, the structure and characteristics of the lesions formed by these toxins remain enigmatic. Here, we capture the first direct nanoscale pictures of lytic pores formed by an RTX toxin, the Adenylate cyclase (ACT), secreted by the whooping cough bacterium Bordetella pertussis. We reveal that ACT associates into growing-size oligomers of variable stoichiometry and heterogeneous architecture (lines, arcs, and rings) that pierce the membrane, and that, depending on the incubation time and the toxin concentration, evolve into large enough “holes” so as to allow the flux of large molecular mass solutes, while vesicle integrity is preserved. We also resolve ACT assemblies of similar variable stoichiometry in the cell membrane of permeabilized target macrophages, proving that our model system recapitulates the process of ACT permeabilization in natural membranes. Based on our data we propose a non-concerted monomer insertion and sequential mechanism of toroidal pore formation by ACT. A size-tunable pore adds a new regulatory element to ACT-mediated cytotoxicity, with different pore sizes being putatively involved in different physiological scenarios or cell types.

Phenol hydroxylase that catalyzes the conversion of phenol to catechol in Rhodococcus erythropolis UPV-1 was identified as a two-component flavin-dependent monooxygenase. The two proteins are encoded by the genes pheA1 and pheA2, located very closely in the genome. The sequenced pheA1 gene was composed of 1,629 bp encoding a protein of 542 amino acids, whereas the pheA2 gene consisted of 570 bp encoding a protein of 189 amino acids. The deduced amino acid sequences of both genes showed high homology with several two-component aromatic hydroxylases. The genes were cloned separately in cells of Escherichia coli M15 as hexahistidine-tagged proteins, and the recombinant proteins His(6)PheA1 and His(6)PheA2 were purified and its catalytic activity characterized. His(6)PheA1 exists as a homotetramer of four identical subunits of 62 kDa that has no phenol hydroxylase activity on its own. His(6)PheA2 is a homodimeric flavin reductase, consisting of two identical subunits of 22 kDa, that uses NAD(P)H in order to reduce flavin adenine dinucleotide (FAD), according to a random sequential kinetic mechanism. The reductase activity was strongly inhibited by thiol-blocking reagents. The hydroxylation of phenol in vitro requires the presence of both His(6)PheA1 and His(6)PheA2 components, in addition to NADH and FAD, but the physical interaction between the proteins is not necessary for the reaction.

Viroporins comprise a family of non-structural proteins that play significant and diverse roles during the replication cycle of many animal viruses. Consequently, they have become promising targets for inhibitory drug and vaccine development. Structure–function traits common to all members of the family are their small size (ca. 60–120 aa), high hydrophobicity, and the presence of helical domains that transverse the membrane and assemble into oligomeric-permeating structures therein. The possibility that viroporins show in particular conditions any kind of specificity in the transport of ions and small solutes remains a point of contention in the field. Here we have approached this issue using the Classical Swine Fever Virus (CSFV) protein p7 viroporin as a model. We have previously reported that CSFV-p7 induces release of ANTS (MW: 427.33) from lipid vesicles that emulate the Endoplasmic Reticulum (ER) membrane, and that this process is dependent on pH, modulated by the lipid composition, and recreated by a C-terminal transmembrane helix. Here we have assayed CSFV-p7 for its capacity to form ion-conducting channels in ER-like planar lipid membranes, and established whether this activity is subject to regulation by the same factors. The analysis of electrophysiological recordings in ER membrane surrogates suggests that CSFV-p7 forms pores wide enough to allow ANTS release. Moreover, we were able to discriminate between two pore structures with slightly different sizes and opposite ion selectivities. The fact that the relative abundances of each pore type depend crucially on membrane composition strengthens the view that the physicochemical properties of the lipid bilayers present in the cell endomembrane system modulate viroporin activity.

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We have previously shown that Classical Swine Fever Virus (CSFV) p7 is an essential nonstructural protein with a viroporin activity, a critical function in the progression of virus infection. We also identified p7 domains and amino acid residues critical for pore formation. Here, we describe how p7 specifically interacts with host protein CAMLG, an integral ER transmembrane protein involved in intracellular calcium release regulation and signal response generation. Detection of interaction as well as the identification of p7 areas mediating interaction with CAMLG was performed by yeast two-hybrid. p7-CAMLG interaction was further confirmed by confocal microscopy in eukaryotic cells, co-expressing both proteins. Mutant forms of p7 having substituted native residues identified as mediating interaction with CAMLG showed a decreased co-localization compared with the native forms of p7. Furthermore, it is shown that native p7, but not the mutated forms of p7 that fail to interact with CAMLG, efficiently mediates calcium permeability in the ER. Interestingly, viruses harboring some of those mutated forms of p7 have been previously shown to have a significantly decreased virulence in swine.