Solid phase synthesis of the proteinase of bovine leukemia virus. Comparison of its specificity to that of HIV-2 proteinase.

The 126-residue proteinase (PR) of bovine leukemia virus (BLV) was synthesized by solid-phase peptide synthesis and its activity was shown using various oligopeptide substrates representing cleavage sites in BLV, human T-cell leukemia virus type 1 (HTLV-1), murine leukemia virus (MuLV) and human immunodeficiency virus type 1 (HIV-1). The specificity of the BLV PR was also compared to that of chemically synthesized human immunodeficiency virus type 2 (HIV-2) PR. Many of the peptides were cleaved at the expected site, however, 6 out of 15 were hydrolyzed only by one of the PRs. Furthermore, one BLV peptide was processed differently by the two enzymes. These results, together with the relative activities and the lack of inhibition of BLV PR by two HIV-1 PR inhibitors, suggest that the BLV PR specificity is substantially different from that of HIV PRs.     

Identification,purification, and characterization of pathogenesis-related proteins from virus-infected Samsun NN tobacco leaves

Ten pH-3 soluble, low-molecular-weight pathogenesis-related proteins (PRs) were found to accumulate in leaves of tobacco cv. Samsun NN reacting hypersensitively to tobacco mosaic virus. Besides the previously characterized PRs 1a, 1b, 1c and 2, these proteins were provisionally designated N, O, P, Q, R, and S in order of decreasing electrophoretic mobility in native polyacrylamide gels. Two-dimensional gel electrophoresis indicated that the PRs consist of single polypeptides, except for R, which is composed of two components with slightly different molecular weights. Estimated molecular weights in SDS-containing gels were: PRs 1a and 1b 17 kD, 1c 16.5 kD, 2 31 kD, N 33 kD, O 35 kD, P 27 kD, Q 28 kD, R 13 and 15 kD, and S 25 kD. However, based on their elution from gel filtration columns and relative moblities in native gels of different acrylamide concentrations, P and Q appeared to have molecular weights similar to those of the PR 1 group. Upon chromatofocusing no additional components were resolved. The PRs were eluted between pH 7 and 4; except for R, their pIs, as judged from isoelectric focusing, appeared to lie in the range from pH 4 to 5.2. In the presence of 6 M urea PR 1a was split into two components, one of which was strongly retarded on gels, as were P and Q. None of the PRs was detected when gels were stained for glycoproteins.By combinations of gel filtration, DEAE-cellulose chromatography, and chromatofocusing, PRs 1a, 1b, 1c, 2 and N were purified, their amino acid compositions determined, and antisera raised against each of these components. By Western blotting, antisera against either PR 1a, 1b, or 1c reacted with each of the components of the PR 1 group, as well as with PR S. Similarly, the antisera against either PR 2 or N reacted with both 2 and N, as well as with O and R. On the basis of major similarities in molecular weight characteristics, amino acid compositions, and serological relationships, it is proposed to classify tobacco PRs into five groups: 1: PRs 1a, 1b, and 1c; 2: 2a (formerly 2), 2b (N), and 2c (O); 3: 3a (P), and 3b (Q); 4: 4a and 4b (the two components of R); and 5: PR 5 (S).     

Comparative analysis of the sequences and structures of HIV-1 and HIV-2 proteases

The different isolates available for HIV-1 and HIV-2 were compared for the region of the protease (PR) sequence, and the variations in amino acids were analyzed with respect to the crystal structure of HIV-1 PR with inhibitor. Based on the extensive homology (39 identical out of 99 residues), models were built of the HIV-2 PR complexed with two different aspartic protease inhibitors, acetylpepstatin and a renin inhibitor, H-261. Comparison of the HIV-1 PR crystal structure and the HIV-2 PR model structure and the analysis of the changes found in different isolates showed that correlated substitutions occur in the hydrophobic interior of the molecule and at surface residues involved in ionic or hydrogen bond interactions. The substrate binding residues of HIV-1 and HIV-2 PRs show conservative substitutions of four residues. The difference in affinity of HIV-1 and HIV-2 PRs for the two inhibitors appears to be due in part to the change of Val 32 in HIV-1 PR to Ile in HIV-2 PR.     

Expression of sphingosine 1-phosphate receptors in the rat dorsal root ganglia and defined single isolated sensory neurons

Previously, we demonstrated that sphingosine 1-phosphate (S1P) increased the excitability of small-diameter sensory neurons, in part, through activation of S1P receptor 1 (S1PR(1)), suggesting that other S1PRs can modulate neuronal excitability. Therefore, studies were undertaken to establish the expression profiles of S1PRs in the intact dorsal root ganglion (DRG) and in defined single isolated sensory neurons. To determine mRNA expression of S1PRs in the DRG, SYBR green quantitative PCR (qPCR) was used. To determine the expression of S1PR mRNAs in single neurons of defined diameters, a preamplification protocol utilizing Taqman primer and probes was used to enhance the sensitivity of detection. The preamplification protocol also permitted detection of mRNA for two hallmark neuronal receptor/ion channels, TRPV1 and P(2)X(3). Expression profiles of S1PR mRNA isolated from lung and brain were used as positive control tissues. In the intact DRG, the order of expression of S1PRs was S1PR(3)>R(1)≈R(2)>R(5)≈R(4). In the single neurons, the expression of S1PRs was quite variable with some neurons expressing all five subtypes, whereas some expressing only one subtype. In contrast to the DRG, S1PR(1) was the highest expressing subtype in 10 of the 18 small-, medium-, and large-diameter sensory neurons. S1PR(1) was the second highest expressor in ～50% of those remaining neurons. Overall, in the single neurons, the order of expression was S1PR(1)>R(3)≈R(5)>R(4)>R(2). The results obtained from the single defined neurons are consistent with our previous findings wherein S1PR(1) plays a prominent but not exclusive role in the enhancement of neuronal excitability.     

Structural studies of FIV and HIV-1 proteases complexed with an efficient inhibitor of FIV protease

Three forms of feline immunodeficiency virus protease (FIV PR), the wild type (wt) and two single point mutants, V59I and Q99V, as well as human immunodeficiency virus type 1 protease (HIV-1 PR), were cocrystallized with the C2-symmetric inhibitor, TL-3. The mutants of FIV PR were designed to replace residues involved in enzyme-ligand interactions by the corresponding HIV-1 PR residues at the structurally equivalent position. TL-3 shows decreased (improved) inhibition constants with these FIV PR mutants relative to wt FIV PR. Despite similar modes of binding of the inhibitor to all PRs (from P3 to P3'), small differences are evident in the conformation of the Phe side chains of TL-3 at the P1 and P1' positions in the complexes with the mutated FIV PRs. The differences mimick the observed binding of TL-3 in HIV-1 PR and correlate with a significant improvement in the inhibition constants of TL-3 with the two mutant FIV PRs. Large differences between the HIV-1 and FIV PR complexes are evident in the binding modes of the carboxybenzyl groups of TL-3 at P4 and P4'. In HIV-1 PR:TL-3, these groups bind over the flap region, whereas in the FIV PR complexes, the rings are located along the major axis of the active site. A significant difference in the location of the flaps in this region of the HIV-1 and FIV PRs correlates with the observed conformational changes in the binding mode of the peptidomimetic inhibitor at the P4 and P4' positions. These findings provide a structural explanation of the observed Ki values for TL-3 with the different PRs and will further assist in the development of improved inhibitors.     

Synthetic "interface" peptides alter dimeric assembly of the HIV 1 and 2 proteases.

Retroviral proteases are obligate homodimers and play an essential role in the viral life cycle. Dissociation of dimers or prevention of their assembly may inactivate these enzymes and prevent viral maturation. A salient structural feature of these enzymes is an extended interface composed of interdigitating N- and C-terminal residues of both monomers, which form a four-stranded beta-sheet. Peptides mimicking one beta-strand (residues 95-99), or two beta-strands (residues 1-5 plus 95-99 or 95-99 plus 95-99) from the human immunodeficiency virus 1 (HIV1) interface were shown to inhibit the HIV1 and 2 proteases (PRs) with IC50's in the low micromolar range. These interface peptides show cognate enzyme preference and do not inhibit pepsin, renin, or the Rous sarcoma virus PR, indicating a degree of specificity for the HIV PRs. A tethered HIV1 PR dimer was not inhibited to the same extent as the wild-type enzymes by any of the interface peptides, suggesting that these peptides can only interact effectively with the interface of the two-subunit HIV PR. Measurements of relative dissociation constants by limit dilution of the enzyme show that the one-strand peptide causes a shift in the observed Kd for the HIV1 PR. Both one- and two-strand peptides alter the monomer/dimer equilibrium of both HIV1 and HIV2 PRs. This was shown by the reduced cross-linking of the HIV2 PR by disuccinimidyl suberate in the presence of the interface peptides. Refolding of the HIV1 and HIV2 PRs with the interface peptides shows that only the two-strand peptides prevent the assembly of active PR dimers. Although both one- and two-strand peptides seem to affect dimer dissociation, only the two-strand peptides appear to block assembly. The latter may prove to be more effective backbones for the design of inhibitors directed toward retroviral PR dimerization in vivo.     

Molecular basis for the relative substrate specificity of human immunodeficiency virus type 1 and feline immunodeficiency virus proteases

We have used a random hexamer phage library to delineate similarities and differences between the substrate specificities of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) proteases (PRs). Peptide sequences were identified that were specifically cleaved by each protease, as well as sequences cleaved equally well by both enzymes. Based on amino acid distinctions within the P3-P3' region of substrates that appeared to correlate with these cleavage specificities, we prepared a series of synthetic peptides within the framework of a peptide sequence cleaved with essentially the same efficiency by both HIV-1 and FIV PRs, Ac-KSGVF/VVNGLVK-NH(2) (arrow denotes cleavage site). We used the resultant peptide set to assess the influence of specific amino acid substitutions on the cleavage characteristics of the two proteases. The findings show that when Asn is substituted for Val at the P2 position, HIV-1 PR cleaves the substrate at a much greater rate than does FIV PR. Likewise, Glu or Gln substituted for Val at the P2' position also yields peptides specifically susceptible to HIV-1 PR. In contrast, when Ser is substituted for Val at P1', FIV PR cleaves the substrate at a much higher rate than does HIV-1 PR. In addition, Asn or Gln at the P1 position, in combination with an appropriate P3 amino acid, Arg, also strongly favors cleavage by FIV PR over HIV PR. Structural analysis identified several protease residues likely to dictate the observed specificity differences. Interestingly, HIV PR Asp30 (Ile-35 in FIV PR), which influences specificity at the S2 and S2' subsites, and HIV-1 PR Pro-81 and Val-82 (Ile-98 and Gln-99 in FIV PR), which influence specificity at the S1 and S1' subsites, are residues which are often involved in development of drug resistance in HIV-1 protease. The peptide substrate KSGVF/VVNGK, cleaved by both PRs, was used as a template for the design of a reduced amide inhibitor, Ac-GSGVF Psi(CH(2)NH)VVNGL-NH(2.) This compound inhibited both FIV and HIV-1 PRs with approximately equal efficiency. These findings establish a molecular basis for distinctions in substrate specificity between human and feline lentivirus PRs and offer a framework for development of efficient broad-based inhibitors.     

Gag-Pol Transframe Domain p6* Is Essential for HIV-1 Protease-Mediated Virus Maturation

HIV-1 protease (PR) is encoded by pol, which is initially translated as a Pr160^gag-pol polyprotein by a ribosomal frameshift event. Within Gag-Pol, truncated p6gag is replaced by a transframe domain (referred to as p6* or p6pol) located directly upstream of PR. p6* has been proposed as playing a role in modulating PR activation. Overlapping reading frames between p6* and p6gag present a challenge to researchers using genetic approaches to studying p6* biological functions. To determine the role of p6* in PR activation without affecting the gag reading frame, we constructed a series of Gag/Gag-Pol expression vectors by duplicating PR with or without p6* between PR pairs, and observed that PR duplication eliminated virus production due to significant Gag cleavage enhancement. This effect was mitigated when p6* was placed between the two PRs. Further, Gag cleavage enhancement was markedly reduced when either one of the two PRs was mutationally inactivated. Additional reduction in Gag cleavage efficiency was noted following the removal of p6* from between the two PRs. The insertion of a NC domain (wild-type or mutant) directly upstream of PR or p6*PR did not significantly improve Gag processing efficiency. With the exception of those containing p6* directly upstream of an active PR, all constructs were either noninfectious or weakly infectious. Our results suggest that (a) p6* is essential for triggering PR activation, (b) p6* has a role in preventing premature virus processing, and (c) the NC domain within Gag-Pol is not a major determinant of PR activation.     

Altered gag polyprotein cleavage specificity of feline immunodeficiency virus/human immunodeficiency virus mutant proteases as demonstrated in a cell-based expression system

We have used feline immunodeficiency virus (FIV) protease (PR) as a mutational system to study the molecular basis of substrate-inhibitor specificity for lentivirus PRs, with a focus on human immunodeficiency virus type 1 (HIV-1) PR. Our previous mutagenesis studies demonstrated that discrete substitutions in the active site of FIV PR with structurally equivalent residues of HIV-1 PR dramatically altered the specificity of the mutant PRs in in vitro analyses. Here, we have expanded these studies to analyze the specificity changes in each mutant FIV PR expressed in the context of the natural Gag-Pol polyprotein ex vivo. Expression mutants were prepared in which 4 to 12 HIV-1-equivalent substitutions were made in FIV PR, and cleavage of each Gag-Pol polyprotein was then assessed in pseudovirions from transduced cells. The findings demonstrated that, as with in vitro analyses, inhibitor specificities of the mutants showed increased HIV-1 PR character when analyzed against the natural substrate. In addition, all of the mutant PRs still processed the FIV polyprotein but the apparent order of processing was altered relative to that observed with wild-type FIV PR. Given the importance of the order in which Gag-Pol is processed, these findings likely explain the failure to produce infectious FIVs bearing these mutations.     

Evolutionary analysis of the segment from helix 3 through helix 5 in vertebrate progesterone receptors

The interaction between helix 3 and helix 5 in the human mineralocorticoid receptor [MR], progesterone receptor [PR] and glucocorticoid receptor [GR] influences their response to steroids. For the human PR, mutations at Gly-722 on helix 3 and Met-759 on helix 5 alter responses to progesterone. We analyzed the evolution of these two sites and the rest of a 59 residue segment containing helices 3, 4 and 5 in vertebrate PRs and found that a glycine corresponding to Gly-722 on helix 3 in human PR first appears in platypus, a monotreme. In lamprey, skates, fish, amphibians and birds, cysteine is found at this position in helix 3. This suggests that the cysteine to glycine replacement in helix 3 in the PR was important in the evolution of mammals. Interestingly, our analysis of the rest of the 59 residue segment finds 100% sequence conservation in almost all mammal PRs, substantial conservation in reptile and amphibian PRs and divergence of land vertebrate PR sequences from the fish PR sequences. The differences between fish and land vertebrate PRs may be important in the evolution of different biological progestins in fish and mammalian PR, as well as differences in susceptibility to environmental chemicals that disrupt PR-mediated physiology.     

Cleavage of vimentin by different retroviral proteases

Proteases (PRs) of retroviruses cleave viral polyproteins into their mature structural proteins and replication enzymes. Besides this essential role in the replication cycle of retroviruses, PRs also cleave a variety of host cell proteins. We have analyzed the in vitro cleavage of mouse vimentin by proteases of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2), bovine leukemia virus (BLV), Mason-Pfizer monkey virus (M-PMV), myeloblastosis-associated virus (MAV), and two active-site mutants of MAV PR. Retroviral proteases display significant differences in specificity requirements. Here, we show a comparison of substrate specificities of several retroviral proteases on vimentin as a substrate. Vimentin was cleaved by all the proteases at different sites and with different rates. The results show that the physiologically important cellular protein vimentin can be degraded by different retroviral proteases.     

Essential roles of sphingosine 1-phosphate receptor types 1 and 3 in human hepatic stellate cells motility and activation

The biological roles of sphingosine 1-phosphate (S1P) and S1P receptors (S1PRs) have been broadly investigated. However, at present pathophysiological roles of S1P/S1PRs axis in liver fibrosis are not well defined. Here, we investigated the functions of S1P/S1PRs axis in human hepatic stellate cells (HSC) line, LX-2 cells. We found that S1PR types 1, 2 and 3 (S1PR1-3) are clearly detected in LX-2 cells, as determined by RT-PCR, Western blot and immunocytochemistry analysis. S1P exerted a powerful migratory action on LX-2 cells, as determined in Boyden chambers, and stimulated fibrogenic activity of LX-2 cells, as demonstrated by increase of expression of smooth muscle α-actin, procollagen α1(I) and α1(III) and total hydroxyproline content. Moreover, the effects of S1P were mimicked by S1PR1 agonist SEW2871, and abrogated by W146 (S1PR1 antagonist) and/or silencing S1PR1, three expression with small interfering RNA, suggesting the main roles of S1PR1 and 3. However, studies with S1PR2 antagonist JTE-013 and silencing S1PR2 expression indicated that S1PR2 negatively regulated S1P-induced cell migration. Interestingly, exogenously added S1P induced significant up-regulation of sphingosine kinase-1 and the synthesis of additional S1P, and expression of S1PR1,3, but not S1PR2. In conclusion, our data have identified an additional function regulated by S1P/S1PR1,3 axis involving migration and fibrogenic activation of HSCs. These results suggest that selective modulation of S1PR activity may represent a new antifibrotic strategy.     

The Drosophila larval visual system: high-resolution analysis of a simple visual neuropil

The task of the visual system is to translate light into neuronal encoded information. This translation of photons into neuronal signals is achieved by photoreceptor neurons (PRs), specialized sensory neurons, located in the eye. Upon perception of light the PRs will send a signal to target neurons, which represent a first station of visual processing. Increasing complexity of visual processing stems from the number of distinct PR subtypes and their various types of target neurons that are contacted. The visual system of the fruit fly larva represents a simple visual system (larval optic neuropil, LON) that consists of 12 PRs falling into two classes: blue-senstive PRs expressing Rhodopsin 5 (Rh5) and green-sensitive PRs expressing Rhodopsin 6 (Rh6). These afferents contact a small number of target neurons, including optic lobe pioneers (OLPs) and lateral clock neurons (LNs). We combine the use of genetic markers to label both PR subtypes and the distinct, identifiable sets of target neurons with a serial EM reconstruction to generate a high-resolution map of the larval optic neuropil. We find that the larval optic neuropil shows a clear bipartite organization consisting of one domain innervated by PRs and one devoid of PR axons. The topology of PR projections, in particular the relationship between Rh5 and Rh6 afferents, is maintained from the nerve entering the brain to the axon terminals. The target neurons can be subdivided according to neurotransmitter or neuropeptide they use as well as the location within the brain. We further track the larval optic neuropil through development from first larval instar to its location in the adult brain as the accessory medulla.     

Homothorax controls a binary Rhodopsin switch in Drosophila ocelli

Visual perception of the environment is mediated by specialized photoreceptor (PR) neurons of the eye. Each PR expresses photosensitive opsins, which are activated by a particular wavelength of light. In most insects, the visual system comprises a pair of compound eyes that are mainly associated with motion, color or polarized light detection, and a triplet of ocelli that are thought to be critical during flight to detect horizon and movements. It is widely believed that the evolutionary diversification of compound eye and ocelli in insects occurred from an ancestral visual organ around 500 million years ago. Concurrently, opsin genes were also duplicated to provide distinct spectral sensitivities to different PRs of compound eye and ocelli. In the fruit fly Drosophila melanogaster, Rhodopsin1 (Rh1) and Rh2 are closely related opsins that originated from the duplication of a single ancestral gene. However, in the visual organs, Rh2 is uniquely expressed in ocelli whereas Rh1 is uniquely expressed in outer PRs of the compound eye. It is currently unknown how this differential expression of Rh1 and Rh2 in the two visual organs is controlled to provide unique spectral sensitivities to ocelli and compound eyes. Here, we show that Homothorax (Hth) is expressed in ocelli and confers proper rhodopsin expression. We find that Hth controls a binary Rhodopsin switch in ocelli to promote Rh2 expression and repress Rh1 expression. Genetic and molecular analysis of rh1 and rh2 supports that Hth acts through their promoters to regulate Rhodopsin expression in the ocelli. Finally, we also show that when ectopically expressed in the retina, hth is sufficient to induce Rh2 expression only at the outer PRs in a cell autonomous manner. We therefore propose that the diversification of rhodpsins in the ocelli and retinal outer PRs occurred by duplication of an ancestral gene, which is under the control of Homothorax.     

Molecular investigations of pathogenesis-related Bet v 1 homologues in Passiflora (Passifloraceae)

The major birch pollen allergen, Bet v 1, responsible for allergic reactions in many areas of the world, is homologous to a large number of pathogenesis-related proteins (PRs), identified as PR10. As part of a long-range investigation of these types of proteins and of evolution in Passiflora,DNA sequences from eight Bet v 1 homologue isoforms were obtained from five species of this genus in Brazil, and their sequences compared among themselves and with 30 others from 8 different species, classified in different taxonomic units. The objective was a first characterization of these PRs in wild passionflowers, and their use for evolutionary and applied investigations. High interspecific, but low intraspecific variability was observed, as expected from multigenic families subjected to concerted evolution. The relationships obtained both within Passiflora and between it and seven other genera probably best reflect functional similarities than evolutionary history.     

A primary response with an initial negative wave

In the anterosuperior and posterosuperior portions of the ectosylvian gyrus of dogs small foci were found in which clicks evoked responses with parameters close to those of primary responses (PRs), but with an initial negative wave. An analysis of PRs with an initial wave of different polarity was carried out by studying the reproducibility of the response during an increase in the frequency of stimulation and during the action of various drugs. PRs with an initial negative ("negative" focus) and positive (auditory area AI) waves were found to be reproduced when the frequency of stimulation was 20–30 Hz, whereas the negative phase of the classical PR disappeared when the stimulation frequency reached 10–15 Hz. The polarity of the response in these foci was unchanged after injection of a lethal dose of nembutal, but the negative phase of the classical PR disappeared during moderately deep anesthesia. Strychnine, on the other hand, considerably increased the amplitude of the negative wave in the positive-negative complex, but the initial negative potential was only very slightly and temporarily increased, and it was lost in the subsequent strychnine spike. GABA inhibited both the PR with initial negative wave and also the negative phase of the classical PR. The results suggest that PRs with different polarities of their initial wave differ in origin. The results of experiments with GABA indicate that PRs with an initial negative wave arise through excitation of apical dendrites.A. A. Bogomolets' Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 2, No. 5, pp. 488–496, September–October, 1970.