HIV gp120 V(1)/V(2) and C(2)-V(3) domains glycoprotein compatibility is required for viral replication.

The envelope gene, especially the V(3) region, of HIV-1 has been shown to be a principal determinant of cell tropism, replication and cytopathogenicity of the virus. In addition, the V(1)/V(2) region of the envelope gene has been found to be an important factor in cell tropism. We examined the compatibility between the V(1)/V(2) and C(2)-V(3) domains of HIV-1 gp120 in different combinations on viral replication by using envelope recombinants between ME1 and ME46, two infectious molecular clones with diverse biologic activity longitudinally isolated from one seropositive subject. Our data demonstrate that a proper interaction between the regions of V(1)/V(2)and C(2) is essential for viral infection and hence replication. Sequence analysis and subsequent site directed mutagenesis study indicate that the pattern of potential envelope N-glycosylation in the V(1)/V(2) and C(2)-V(3) regions may be the determining factor in such interaction between these two regions. It is possible that improper N-glycosylation sites while not affecting virus assembly, can influence through steric hindrance the conformational change of the V(3) region that is required for the co-receptor attachment and hence the viral infectivity.

Development of a fluorescence polarization-based diagnostic assay for equine infectious anemia virus.

The control of equine infectious anemia virus (EIAV) infections of horses has been over the past 20 years based primarily on the identification and elimination of seropositive horses, predominantly by a standardized agar gel immunodiffusion (AGID) assay in centralized reference laboratories. This screening for EIAV-seropositive horses has been to date hindered by the lack of a rapid diagnostic format that can be easily employed in the field. We describe here the development of a rapid solution-phase assay for the presence of serum antibodies to EIAV based on fluorescence polarization (FP) (patent pending). Peptides derived from antigenic regions of EIAV core and envelope proteins were initially screened for their utility as probes in an FP assay to select the best peptide antigen candidates. The FP assay was optimized to detect the presence of EIAV-specific antibodies by a change in the FP of a fluorescein-labeled immunoreactive peptide diagnostic antigen. The most sensitive and specific peptide probe was a peptide corresponding to the immunodominant region of the EIAV transmembrane protein, gp45. This probe was tested for its reactivity in the optimized FP assay with 151 AGID-positive horse sera and 106 AGID-negative serum samples. The results of these studies demonstrated that the FP assay reactivity correlated with reported AGID results in 106 of 106 negative serum samples (100% specificity) and in 135 of 151 positive serum samples (89.4% sensitivity). The FP assay was also found to have a very low background reactivity and to readily detect antibodies produced early in infection (

Concentration-dependent differential induction of necrosis or apoptosis by HIV-1 lytic peptide 1.

The mechanism by which human immunodeficiency virus type 1 induces depletion of CD4+ T-lymphocytes remains controversial, but may involve cytotoxic viral proteins. Synthetic peptides (lentivirus lytic peptide type 1) corresponding to the carboxyl terminus of the human immunodeficiency virus type 1 transmembrane glycoprotein induce cytopathology at concentrations of 100 nM and above. At these concentrations lentivirus lytic peptide type 1 disrupts mitochondrial integrity of CD4+ T-lymphoblastoid cells and induces other changes characteristic of necrosis. In contrast, at concentrations of 20 nM, lentivirus lytic peptide type 1 potently induces apoptosis. Thus, the mechanism by which human immunodeficiency virus type 1 mediates cell death, necrosis or apoptosis, may depend, in part, on the tissue concentration of transmembrane glycoprotein.

Lentivirus-derived antimicrobial peptides: increased potency by sequence engineering and dimerization.

We have previously described a family of cationic amphipathic peptides derived from lentivirus envelope proteins that have properties similar to those of naturally occurring antimicrobial peptides. Here, we explored the effects of amino acid truncations and substitutions on the antimicrobial potency and selectivity of the prototype peptide, LLP1. Removal of seven residues from the C-terminus of LLP1 had little effect on potency, but abrogated haemolytic activity. Replacement of the two glutamic acid residues of LLP1 with arginine resulted in a peptide with greater bactericidal activity. We discovered that the cysteine-containing peptides spontaneously formed disulphide-linked dimers, which were 16-fold more bactericidal to Staphylococcus aureus. Monomeric and dimeric LLP1 possessed similar alpha helical contents, indicating that disulphide formation did not alter the peptide's secondary structure. The dimerization strategy was applied to magainin 2, enhancing its bactericidal activity eight-fold. By optimizing all three properties of LLP1, a highly potent and selective peptide, named TL-1, was produced. This peptide is significantly more potent than LLP1 against gram-positive bacteria while maintaining high activity against gram-negative organisms and low activity against eukaryotic cells. In addition to new antimicrobial peptides, these studies contribute useful information on which further peptide engineering efforts can be based.

Interruption of T-cell signal transduction by lentivirus lytic peptides from HIV-1 transmembrane protein.

Two peptide segments designated LLP1 (residues 828-855) and LLP2 (residues 768-788) of the HIV-1 transmembrane (TM) envelope protein display structural and functional properties of calmodulin (CaM) binding. These LLP segments may contribute to cytopathogenesis by binding cellular CaM and inhibiting normal CaM-regulated signal transduction pathways. To determine whether these peptides could interrupt signal transduction in vivo, a cellular assay which uses a reporter gene linked to the nuclear factor of activated T cells (NF-AT) was used. Signal transduction perturbation was tested by exogenous addition of LLPs, W-7 or ionomycin; the LLPs inhibited NF-AT-mediated signal transduction as measured by reduced reporter activity. The LLP inhibition profile of NF-AT-driven luciferase activity was similar to the CaM inhibitor W-7. This was in direct contrast to ionomycin, a mobile calcium ion carrier which caused a significant increase in luciferase activity. These findings are consistent with the hypothesis that the CaM-binding properties of TM may contribute to defects in signal transduction leading to the T-cell anergy observed in patients infected with HIV-1.

A synthetic peptide corresponding to the carboxy terminus of human immunodeficiency virus type 1 transmembrane glycoprotein induces alterations in the ionic permeability of Xenopus laevis oocytes.

The carboxy-terminal 29 amino acids of the human immunodeficiency virus type 1 transmembrane glycoprotein (HIV-1 TM) are referred to as lentivirus lytic peptide 1 (LLP-1). Synthetic peptides corresponding to LLP-1 have been shown to induce cytolysis and to alter the permeability of cultured cells to various small molecules. To address the mechanisms by which LLP-1 induces cytolysis and membrane permeability changes, various concentrations of LLP-1 were incubated with Xenopus laevis oocytes, and two-electrode, voltage-clamp recording measurements were performed. LLP-1 at concentrations of 75 nM and above induced dramatic alterations in the resting membrane potential and ionic permeability of Xenopus oocytes. These concentrations of LLP-1 appeared to induce a major disruption of plasma membrane electrophysiological integrity. In contrast, concentrations of LLP-1 of 20-50 nM induced changes in membrane ionic permeability that mimic changes induced by compounds, such as the bee venom peptide melittin, that are known to form channel-like structures in biological membranes at sublytic concentrations. An analog of LLP-1 with greatly reduced cytolytic activity failed to alter the electrophysiological properties of Xenopus oocytes. Thus, by altering plasma membrane ionic permeability, the carboxy terminus of TM may contribute to cytolysis of HIV-1-infected CD4+ cells.

Novel antimicrobial peptides derived from human immunodeficiency virus type 1 and other lentivirus transmembrane proteins.

We have previously described a conserved set of peptides derived from lentiviral envelope transmembrane proteins that are similar to the natural antimicrobial peptides cecropins and magainins in overall structure but bear no sequence homology to them or other members of their class. We describe here an evaluation of the antimicrobial properties of these virally derived peptides, designated lentivirus lytic peptides (LLPs). The results of this study demonstrate that they are potent and selective antibacterial peptides: the prototype sequence, LLP1, is bactericidal to both gram-positive and gram-negative organisms at micromolar concentrations in 10 mM phosphate buffer. Furthermore, LLP1 kills bacteria quite rapidly, causing a 1,000-fold reduction in viable organisms within 50 s. Peptides corresponding to sequences from three lentivirus envelope proteins were synthesized and characterized. Several of these peptides are selective, killing bacteria at concentrations 50- to 100-fold lower than those required to lyse erythrocytes. Development of antimicrobial agents based on these peptides may lead to improved therapeutics for the management of a variety of infectious diseases.

Metal-dependent conformers of the periplasmic ferric ion binding protein.

One of the better understood structural correlates of Fe3+ binding by the transferrins is the conformational shift demonstrated by both lobes. FbpA, a prokaryotic protein involved in periplasmic iron transport, has previously been shown to be structurally and functionally homologous to the transferrins. Similar to each individual lobe of the transferrins, it is hypothesized that FbpA exists in two distinct conformations depending on whether metal is bound. Evidence for these changes is provided by the differential susceptibility of FbpA to trypsin digestion. Binding of Fe3+ by FbpA significantly decreases the ability of trypsin to digest wild-type protein. Construction of a null binding mutant, Tyr195Ile, confirms that protein "locked" in the apo-conformation is similarly susceptible to trypsin. This mutant also marks the initial characterization of an FbpA molecule unable to bind iron, suggesting that the Tyr195 residue is directly involved in iron binding. Other FbpA mutants which do bind iron show moderate resistance to digestion which suggests that they remain in the holo-protein conformation when binding Fe3+. The conformational states of FbpA may have important implications in protein-protein recognition during transport of Fe3+ between membranes, and may explain how these proteins function in the context of periplasm-to-cytosol Fe3+ transport.

Calmodulin-binding function of LLP segments from the HIV type 1 transmembrane protein is conserved among natural sequence variants.

LLP1 is a peptide, derived from the cytoplasmic tail of HIV-1 TM glycoprotein, that binds and inhibits calmodulin; this region is generally conserved among isolates, but amino acid variation does exist both within clade B and among different clades, as well as SIV. In light of previous studies showing that selected single amino acid changes can have a qualitatively significant effect on the calmodulin-binding properties of this peptide, we sought to examine the properties of naturally occurring variant LLP1 sequences. Using a quantitative fluorescence-based method to measure dissociation constants of calmodulin-LLP1 complexes, a remarkable conservation of calmodulin-binding function among natural variants was revealed. In contrast, engineered nonconservative single amino acid changes altered the affinity of the peptide for calmodulin. The results show that the calmodulin-binding function is well preserved despite the sequence variation observed in nature, suggesting that this region of the TM protein is important to viral replication.

Effect of amino acid substitutions on calmodulin binding and cytolytic properties of the LLP-1 peptide segment of human immunodeficiency virus type 1 transmembrane protein.

Previous studies have identified two highly basic amphipathic helical regions in the human immunodeficiency virus type 1 transmembrane protein that, in vitro, display both cytolytic and calmodulin-binding and -inhibitory properties that could contribute to cellular dysfunctions and cytopathogenesis during a persistent viral infection. In the current study, the structural specificity of the cytolytic and calmodulin-binding activities of the human immunodeficiency virus type 1 lentivirus lytic peptide (LLP-1) are examined with synthetic peptide homologs and analogs. The results of these studies demonstrate that even minor changes in LLP-1 amino acid content can markedly affect these properties, suggesting that sequence variation in these highly conserved LLP sequences may correlate with alterations in viral cytopathic properties.

Coordination of iron by the ferric iron-binding protein of pathogenic Neisseria is homologous to the transferrins.

The ferric iron-binding protein (Fbp) functions as a periplasmic-binding protein in the high-affinity active transport of growth-essential iron by pathogenic Neisseria. Fbp reversibly binds a single ferric ion per molecule of protein with high affinity. Similarly, the transferrins are a highly conserved family of bilobed vertebrate proteins that reversibly bind a single molecule of iron on each of the N- and C-terminal lobes. While evolutionarily divergent, iron binding by all described transferrin lobes is accomplished by a remarkably similar repertoire of residues, including two Tyr, one His, and one Asp, as well as a synergestic bicarbonate anion. With a molecular mass of ca. 34 kDa, Fbp approximates the size of a transferrin lobe. Given the similarities in iron-binding properties, it was investigated whether Fbp bound iron by a similar molecular strategy as the transferrins. The studies reported here demonstrate that the spectral properties of purified Fbp and human transferrin are similar in the visible range. Chemical modification of purified Fbp in the presence and absence of iron using the Tyr-specific modifier tetranitromethane demonstrates that between two and three Tyr residues are implicated in iron binding. A similar experiment using the His-specific reagent diethyl pyrocarbonate indicates that one of the six Fbp-encoded His residues is protected by iron. In addition, like the transferrins, a bicarbonate anion is required for the efficient coordination of iron by Fbp. The range of metals bound by Fbp and human transferrin, including the luminescent lanthanide terbium, is identical. Finally, terbium derivatives of Fbp and human transferrin yield virtually identical luminescence excitation spectra, implying a highly similar binding site environment. These studies suggest that the prokaryotic Fbp is a mono-sited analog for iron binding by the eukaryotic transferrins.