The phytopathogenic fungi Phytophthora cryptogea and Phytophthora capsici cause systemic leaf necrosis on their non‐host tobacco; in culture they release proteins, called cryptogein and capsicein, which elicit similar necrosis. In addition, both proteins protect tobacco against invasion by the pathogen Phytophthora nicotianae, the agent of the tobacco black shank, that is unable to produce such an elicitor. Cryptogein causes visible leaf necrosis starting at about 1 μg/plant, whereas 50‐fold as much capsicein is required for the same reaction. Capsicein induces protection even in near absence of leaf necrosis. The activities of both elicitors are eliminated upon pronase digestion. They are proteins of similar Mr (respectively 10323 and 10155) and their complete amino acid sequences were determined. They consist of 98 residues, with some internal repetitions of hexapeptides and heptapeptides. 85% identity was observed between both sequences: only two short terminal regions are heterologous, while the central core is entirely conserved. Secondary structure predictions, hydropathy and flexibility profiles differ only around position 15 and at the C‐terminus; these modifications could play a role in the modulation of their biological activities. After a search of the sequence data bases, they appear to be novel proteins.

This paper describes the identification of a new class of extracellular bacterial proteins, typified by PopA1 and its derivative PopA3, which act as specific hypersensitive response (HR) elicitors. These two heat‐stable proteins, with HR‐like elicitor activities on tobacco (non‐host plant) but without activity on tomato (host plant), have been characterized from the supernatant of the plant pathogenic bacterium Pseudomonas solanacearum strain GMI1000. These two proteins induced the same pattern of response on Petunia, as a function of the genotypes tested. popA, the structural gene for PopA1, maps outside of the hrp gene cluster but belongs to the hrp regulon. The amino acid sequence of PopA1 does not show homology to any characterized proteins. Its secretion is dependent on hrp genes and is followed by stepwise removal of the 93 amino‐terminal amino acids, producing the protein PopA3. Petunia lines responsive to PopA3 and its precursors were resistant to infection by strain GMI1000, whereas non‐responsive lines were sensitive, suggesting that popA could be an avirulence gene. A popA mutant remained fully pathogenic on sensitive plants, indicating that this gene is not essential for pathogenicity. While lacking PopA1, this mutant, which remained avirulent on tobacco and on resistant Petunia lines, still produced additional extracellular necrogenic compounds. On the basis of both their structural features and the biological properties of the popA mutant, PopA1 and PopA3 clearly differ from hairpins characterized in other plant pathogenic bacteria.

Odorant-binding proteins (OBPs) are small abundant extracellular proteins belonging to the lipocalin superfamily. They are thought to participate in perireceptor events of odor detection by carrying, deactivating, and/or selecting odorant molecules. Putative human OBP genes (hOBP) have recently been described [Lacazette et al. (2000) Hum. Mol. Genet. 9, 289-301], but the presence of the corresponding proteins remained to be established in the human olfactory mucus. This paper reports the first evidence of such expression in the mucus covering the olfactory cleft, where the sensory olfactory epithelium is located. On the contrary, hOBPs were not observed in the nasal mucus covering the septum and the lower turbinate. To demonstrate the odorant binding activity of these proteins, a corresponding recombinant protein variant, hOBP(IIa)(alpha), was secreted by the yeast Pichia pastoris and thoroughly characterized. It appears as a monomer with one disulfide bond located between C59 and C151, a conservative feature of all other vertebrate OBPs. By measuring the displacement of several fluorescent probes, we show that hOBP(IIa)(alpha) is able to bind numerous odorants of diverse chemical structures, with a higher affinity for aldehydes and large fatty acids. A computed 3D model of hOBP(IIa)(alpha) is proposed and reveals that two lysyl residues of the binding pocket may account for the increased affinity for aldehydes. The relatively limited specificity of hOBP(IIa)(alpha) suggests that other human OBPs are expected to take into account the large diversity of odorant molecules.

The RNA-dependent RNA polymerase complex of respiratory syncytial virus (RSV) is composed of the large polymerase (L), the phosphoprotein (P), the nucleocapsid protein (N) and the co-factors M2-1 and M2-2. The P protein plays a central role within the replicase-transcriptase machinery, forming homo-oligomers and complexes with N and L. In order to study P-P and N-P complexes, and the role of P phosphorylation in these interactions, the human RSV P and N proteins were expressed in E. coli as His-tagged or GST-fusion proteins. The non-phosphorylated status of recombinant P protein was established by mass spectrometry. GST-P and GST-N fusion proteins were able to interact with RSV proteins extracted from infected cells in a GST pull-down assay. When co-expressed in bacteria, GST-P and His-P were co-purified by glutathione-Sepharose affinity, showing that the RSV P protein can form oligomers within bacteria. This result was confirmed by chemical cross-linking experiments and gel filtration studies. The P oligomerization domain was investigated by a GST pull-down assay using a series of P deletion constructs. This domain was mapped to a small region situated in the central part of P (aa 120-150), which localized in a computer-predicted coiled-coil domain. When co-expressed in bacteria, RSV N and P proteins formed a soluble complex that prevented non-specific binding of N to bacterial RNA. Therefore, RSV P protein phosphorylation is not required for the formation of P-P and N-P complexes, and P controls the RNA binding activity of N.

Chemosensory proteins (CSPs) are ubiquitous soluble small proteins isolated from sensory organs of a wide range of insect species, which are believed to be involved in chemical communication. We report the cloning of a honeybee CSP gene called ASP3c, as well as the structural and functional characterization of the encoded protein. The protein was heterologously secreted by the yeast Pichia pastoris using the native signal peptide. ASP3c disulfide bonds were assigned after trypsinolysis followed by chromatography and mass spectrometry combined with microsequencing. The pairing (Cys(I)–Cys(II), Cys(III)–Cys(IV)) was found to be identical to that of Schistocerca gregaria CSPs, suggesting that this pattern occurs commonly throughout the insect CSPs. CD measurements revealed that ASP3c mainly consists of α‐helices, like other insect CSPs. Gel filtration analysis showed that ASP3c is monomeric at neutral pH. Using ASA, a fluorescent fatty acid anthroyloxy analogue as a probe, ASP3c was shown to bind specifically to large fatty acids and ester derivatives, which are brood pheromone components, in the micromolar range. It was unable to bind tested general odorants and other tested pheromones (sexual and nonsexual). This is the first report on a natural pheromonal ligand bound by a recombinant CSP with a measured affinity constant.

The polyprotein of infectious bursal disease virus (IBDV), an avian birnavirus, is processed by the viral protease, VP4. Previous data obtained on the VP4 of infectious pancreatic necrosis virus (IPNV), a fish birnavirus, and comparative sequence analysis between IBDV and IPNV suggest that VP4 is an unusual eukaryotic serine protease that shares properties with prokaryotic leader peptidases and other bacterial peptidases. IBDV VP4 is predicted to utilize a serine-lysine catalytic dyad. Replacement of the members of the predicted catalytic dyad (Ser-652 and Lys-692) confirmed their indispensability. The two cleavage sites at the pVP2-VP4 and VP4-VP3 junctions were identified by N-terminal sequencing and probed by site-directed mutagenesis. Several additional candidate cleavage sites were identified in the C-terminal domain of pVP2 and tested by cumulative site-directed mutagenesis and expression of the mutant polyproteins. The results suggest that VP4 cleaves multiple (Thr/Ala)-X-Ala Ala motifs. A trans activity of the VP4 protease of IBDV, and also IPNV VP4 protease, was demonstrated by co-expression of VP4 and a polypeptide substrate in Escherichia coli. For both proteases, cleavage specificity was identical in the cis-and trans-activity assays. An attempt was made to determine whether VP4 proteases of IBDV and IPNV were able to cleave heterologous substrates. In each case, no cleavage was observed with heterologous combinations. These results on the IBDV VP4 confirm and extend our previous characterization of the IPNV VP4, delineating the birnavirus protease as a new type of viral serine protease.

The polyprotein of infectious pancreatic necrosis virus (IPNV), a birnavirus, is processed by the viral protease VP4 (also named NS) to generate three polypeptides: pVP2, VP4, and VP3. Site-directed mutagenesis at 42 positions of the IPNV VP4 protein was performed to determine the active site and the important residues for the protease activity. Two residues (serine 633 and lysine 674) were critical for cleavage activity at both the pVP2-VP4 and the VP4-VP3 junctions. Wild-type activity at the pVP2-VP4 junction and a partial block (with an alteration of the cleavage specificity) at the VP4-VP3 junction were observed when replacement occurred at histidines 547 and 679. A similar observation was made when aspartic acid 693 was replaced by leucine, but wild-type activity and specificity were found when substituted by glutamine or asparagine. Sequence comparison between IPNV and two birnavirus (infectious bursal disease virus and Drosophila X virus) VP4s revealed that serine 633 and lysine 674 are conserved in these viruses, in contrast to histidines 547 and 679. The importance of serine 633 and lysine 674 is reminiscent of the protease active site of bacterial leader peptidases and their mitochondrial homologs and of the bacterial LexA-like proteases. Self-cleavage sites of IPNV VP4 were determined at the pVP2-VP4 and VP4-VP3 junctions by N-terminal sequencing and mutagenesis. Two alternative cleavage sites were also identified in the carboxyl domain of pVP2 by cumulative mutagenesis. The results suggest that VP4 cleaves the (Ser/Thr)-X-Ala2(Ser/Ala)-Gly motif, a target sequence with similarities to bacterial leader peptidases and herpesvirus protease cleavage sites.

The capsid proteins VP2 and VP3 of infectious bursal disease virus, a birnavirus, are derived from the processing of a large polyprotein: NH2-pVP2-VP4-VP3-COOH. Although the primary cleavage sites at the pVP2-VP4 and VP4-VP3 junctions have been identified, the proteolytic cascade involved in the processing of this polyprotein is not yet fully understood, particularly the maturation of pVP2. By using different approaches, we showed that the processing of pVP2 (residues 1 to 512) generated VP2 and four small peptides (residues 442 to 487, 488 to 494, 495 to 501, and 502 to 512). We also showed that in addition to VP2, at least three of these peptides (residues 442 to 487, 488 to 494, and 502 to 512) were associated with the viral particles. The importance of the small peptides in the virus cycle was assessed by reverse genetics. Our results showed that the mutants lacking the two smaller peptides were viable, although the virus growth was affected. In contrast, The birnaviruses are a family of small icosahedral viruses infecting insects, fish, and birds (15). Only five proteins, generally referred to as VP1, VP2, VP3, VP4, and VP5, are encoded by the viral genome. The Tϭ13 icosahedral birnavirus capsids are made by the VP2 and VP3 proteins. They contain the two double-stranded RNA genomic segments (A and B) and the VP1 protein, a putative RNA-dependent RNA polymerase. Translation of genomic segment A yields a polyprotein, pVP2-VP4-VP3, and a small protein, VP5, of unknown function. The B segment encodes VP1. The polyprotein processing gives rise to VP4, the viral protease, and VP2 and VP3. VP2 carries all the neutralizing epitopes, suggesting that it is at least partly exposed at the outer surface of the capsid. VP3, which interacts with VP1 (16, 24), is thought to be located on the inner surface of the capsid (6). VP3 contains charged residues at its carboxy-terminal domain, a domain suggested to be involved in the genomic RNA interaction. As found for other virus families, the capsid assembly seems to be regulated by polyprotein processing.The infectious bursal disease virus (IBDV), an avian birnavirus, is of major importance to the poultry industry. It causes an immunosuppressive disease in young chickens. After infection, IBDV multiplies rapidly in the B lymphocytes of the bursa of Fabricius, leading to increased susceptibility to other diseases. Very virulent strains have resulted in high rates of mortality in many countries.The first step governing the IBDV capsid assembly is the autoproteolytic cleavage of the polyprotein (1,012 amino acids). This process generates pVP2, VP4, and VP3. The pVP2-to-VP2 conversion involves several proteolytic cleavages at the carboxy end of pVP2 (1,14,20). Based on mutagenesis studies, the putative cleavage site was proposed as defined by the (Thr/ Ala)-X-Ala2Ala motif; three potential sites are present in the C-terminal domain of pVP2 (14).In the present study, we further analyzed the maturation process of VP2. By using mass spectrometry and N-terminal sequence analysis, we s...