Molecular Characterization of the Viroporin Function of Foot-and-Mouth Disease Virus Nonstructural Protein 2B.

Nonstructural protein 2B of foot-and-mouth disease (FMD) virus (FMDV) is comprised of a small, hydrophobic, 154-amino-acid protein. Structure-function analyses demonstrated that FMDV 2B is an ion channel-forming protein. Infrared spectroscopy measurements using partially overlapping peptides that spanned regions between amino acids 28 and 147 demonstrated the adoption of helical conformations in two putative transmembrane regions between residues 60 and 78 and between residues 119 and 147 and a third transmembrane region between residues 79 and 106, adopting a mainly extended structure. Using synthetic peptides, ion channel activity measurements in planar lipid bilayers and imaging of single giant unilamellar vesicles (GUVs) revealed the existence of two sequences endowed with membrane-porating activity: one spanning FMDV 2B residues 55 to 82 and the other spanning the C-terminal region of 2B from residues 99 to 147. Mapping the latter sequence identified residues 119 to 147 as being responsible for the activity. Experiments to assess the degree of insertion of the synthetic peptides in bilayers and the inclination angle adopted by each peptide regarding the membrane plane normal confirm that residues 55 to 82 and 119 to 147 of 2B actively insert as transmembrane helices. Using reverse genetics, a panel of 13 FMD recombinant mutant viruses was designed, which harbored nonconservative as well as alanine substitutions in critical amino acid residues in the area between amino acid residues 28 and 147. Alterations to any of these structures interfered with pore channel activity and the capacity of the protein to permeabilize the endoplasmic reticulum (ER) to calcium and were lethal for virus replication. Thus, FMDV 2B emerges as the first member of the viroporin family containing two distinct pore domains.IMPORTANCE FMDV nonstructural protein 2B is able to insert itself into cellular membranes to form a pore. This pore allows the passage of ions and small molecules through the membrane. In this study, we were able to show that both current and small molecules are able to pass though the pore made by 2B. We also discovered for the first time a virus with a pore-forming protein that contains two independent functional pores. By making mutations in our infectious clone of FMDV, we determined that mutations in either pore resulted in nonviable virus. This suggests that both pore-forming functions are independently required during FMDV infection.

Alteration of a Second Putative Fusion Peptide of Structural Glycoprotein E2 of Classical Swine Fever Virus Alters Virus Replication and Virulence in Swine.

E2, the major envelope glycoprotein of classical swine fever virus (CSFV), is involved in several critical virus functions, including cell attachment, host range susceptibility, and virulence in natural hosts. Functional structural analysis of E2 based on a Wimley-White interfacial hydrophobicity distribution predicted the involvement of a loop (residues 864 to 881) stabilized by a disulfide bond (869CKWGGNWTCV878, named FPII) in establishing interactions with the host cell membrane. This loop further contains an 872GG873 dipeptide, as well as two aromatic residues (871W and 875W) accessible to solvent. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how amino acid substitutions within FPII may affect replication of BICv in vitro and virus virulence in swine. Recombinant CSFVs containing mutations in different residues of FPII were constructed. A particular construct, harboring amino acid substitutions W871T, W875D, and V878T (FPII.2), demonstrated a significantly decreased ability to replicate in a swine cell line (SK6) and swine macrophage primary cell cultures. Interestingly, mutated virus FPII.2 was completely attenuated in pigs. Also, animals infected with FPII.2 virus were protected against virulent challenge with Brescia virus at 21 days postvaccination. Supporting a role for the E2 the loop from residues 864 to 881 in membrane fusion, only synthetic peptides that were based on the native E2 functional sequence were competent for insertion into model membranes and perturbation of their integrity, and this functionality was lost in synthetic peptides harboring amino acid substitutions W871T, W875D, and V878T in FPII.2. IMPORTANCE: This report describes the identification and characterization of a putative fusion peptide (FP) in the major structural protein E2 of classical swine fever virus (CSFV). The FP identification was performed by functional structural analysis of E2. We characterized the functional significance of this FP by using artificial membranes. Replacement of critical amino acid residues within the FP radically alters how it interacts with the artificial membranes. When we introduced the same mutations into the viral sequence, there was a reduction in replication in cell cultures, and when we infected domestic swine, the natural host of CSFV host, we observed that the virus was now completely attenuated in swine. In addition, the virus mutant that was attenuated in vivo efficiently protected pigs against wild-type virus. These results provide the proof of principle to support as a strategy for vaccine development the discovery and manipulation of FPs.

Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication.

E2, along with E(rns) and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions: cell attachment, host range susceptibility and virulence in natural hosts. Here we evaluate the role of a specific E2 region, (818)CPIGWTGVIEC(828), containing a putative fusion peptide (FP) sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how individual amino acid substitutions within this region of E2 may affect replication of BICv. A synthetic peptide representing the complete E2 FP amino acid sequence adopted a ß-type extended conformation in membrane mimetics, penetrated into model membranes, and perturbed lipid bilayer integrity in vitro. Similar peptides harboring amino acid substitutions adopted comparable conformations but exhibited different membrane activities. Therefore, a preliminary characterization of the putative FP (818)CPIGWTGVIEC(828) indicates a membrane fusion activity and a critical role in virus replication.

[Experimental renal insufficiency in rabbits produced by the intrarenal injection of cyanoacrylic ester]. / Insuficiencia renal experimental en conejos por inyección intrarrenal de ester cianoacrílico.

Through a percutaneous intrarenal injection of 2 ml cyanoacrylic ester to each kidney, acute renal failure was induced in 10 experimental rabbits. Seven of the animals died as a consequence of uremia before a period of 64 hours. The other three remained alive for 96 hours although suffering from stable renal failure. Cyanoacrylic ester solidifies immediately after injection, leading to a hardened body which fills the renal pelvis, ureters and also the renal vessels. Each injection induces hydronephrosis and necrotic-hemorrhagic lesions, and infarcts in the case of vascular injection. With this simple technique immediate acute renal failure can be obtained experimentally without causing trauma or involving extrarenal structures.

Parenteral nutrition via the peritoneum with dextrose and amino acids.

We filled the peritoneal cavity of 9 rabbits through a silastic pediatric Tenckhoff-type catheter with a volume 10% of the animal's weight of an isotonic 'absorption mass' made up by a hydroelectrolytic solution to which 535 mg/dl glucose and 2.53 g/l L-amino acids were added. Then we slowly and continuously infused into it a mixture of a 50% glucose solution with a 13.3% L-amino acid solution to provide 10 +/- 0.2 g glucose and 2.18 +/- 0.24 g L-amino acid (0.334 +/- 0.039 g nitrogen) per kilogram weight in 24 h. So we mimicked the scheme and quantities of total parenteral nutrition. Osmolarity and volume of peritoneal fluid were constant throughout the experiment. In the first 4 h, glucose concentration rose to 1,407 +/- 431 mg/dl and amino acids decreased to nearly two thirds of their initial concentrations. Thereafter, both amino acid and glucose concentrations stabilized. Blood sugar rose slightly from basal 145 +/- 16 mg/dl to a maximum of 200 +/- 59 mg/dl. Plasma amino acids showed a moderate but irregular variation with increases in proline, glutamic acid, methionine, lysine and arginine concentrations, a decline of isoleucine and tryptophan concentrations and no changes for the other amino acids given. There were no variations in plasma values of sodium, potassium, chloride, urea, creatinine, osmolarity, total proteins and hematocrit. We achieved a glucose absorption of 8.72 +/- 0.94 g/kg weight (87.07 +/- 10.11% of the administered amount) and a nitrogen absorption of 303 +/- 47 mg/kg/day (91.4 +/- 14% of administered amount).(ABSTRACT TRUNCATED AT 250 WORDS)