Adaptations in chloroplast membrane lipid synthesis from synthesis in ancestral cyanobacterial endosymbionts.

Cyanobacteria and chloroplasts are believed to share a common ancestor, but synthetic pathways for membrane lipids are different. Lyso-phosphatidic acid (lyso-PA) is the precursor for the synthesis of all membrane lipids and synthesized by an acyl-ACP dependent glycerol-3-phosphate acyltransferase (GPAT) in chloroplasts. In cyanobacteria, GPAT genes are not found and, instead, genes coding for enzymes in the acyl-phosphate dependent lyso-PA synthetic pathway (plsX and plsY) are conserved. We report that the PlsX/Y dependent lyso-PA synthetic pathway is essential in cyanobacteria, but can be replaced by acyl-ACP dependent GPAT from Escherichia coli (plsB) and Arabidopsis thaliana (ATS1). Cyanobacteria thus display the capacity to accept enzymes from other organisms to synthesize essential components. This ability may have enabled them to evolve into current chloroplasts from their ancestral origins.

Thiol-reactive reagents inhibits intracellular trafficking of human papillomavirus type 16 pseudovirions by binding to cysteine residues of major capsid protein L1.

BACKGROUND: A human papillomavirus (HPV) virion is composed of capsid proteins L1 and L2. Several cysteine residues are located on L1 of various HPVs at markedly similar relative positions, suggesting their important functions. Although the authentic virions cannot be studied with cultured cells, surrogate pseudovirions consisting of capsid and reporter plasmid are available for studies dealing with infectivity. RESULTS: HPV type16-pseudovirions (16PVs) were found to lose their infectivity after incubation with thiol-reactive reagents [biotin polyethyleneoxide iodoacetamide (BPEOIA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), N-ethylmaleimide (NEM), 4-(N-maleimido)benzyl-trimethylammonium iodide (MBTA), and [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET)]. A labelled streptavidin was detected to bind to the complex of BPEOIA and L1 of the 16PVs incubated with BPEOIA. The analysis of molecular mass of trypsin-fragments derived from the complex of the BPEOIA and L1 indicated that BPEOIA bound to at least C146, C225, and C229. No appreciable change of the 16PVs carrying DTNB or NEM was detected by sedimentation analysis or electron microscopy. The 16PVs carrying DTNB or NEM were able to bind to and enter HeLa cells but degraded before they reached the perinuclear region. CONCLUSION: HPV16 L1 C146, C225, and C229 have free thiol, which are accessible to BPEOIA, DTNB, NEM, MBTA, and MTSET. Binding of DTNB or NEM to the thiols may cause conformational changes that result in the inhibition of the entry and trafficking of the 16PVs.

Modification of human papillomavirus-like particle vaccine by insertion of the cross-reactive L2-epitopes.

Infection with human papillomavirus 16 (HPV16), which is one of the 15 types of HPV causally associated with cervical cancer and accounts for 50% of the cases, can be prevented in a type-specific manner by an HPV16 virus-like particle (VLP) vaccine comprised of particles of the L1 protein alone. We attempted to modify the VLP vaccine by inserting the HPV16 L2-peptides including cross-neutralization epitopes into the L1 polypeptide. The chimeric L1 had, between L1 amino acids (aa) 430 and 433, the L2 sequence of aa 18-38, 56-75, or 96-115 (with the replacements of S at aa 101 and T at aa 112 with L and S, respectively). The three chimeric L1s were each expressed from the recombinant baculovirus in insect Sf9 cells, and the resultant VLPs were characterized. The chimeric VLPs were shown to present the L2-peptides on their surface. By immunizing rabbits with the VLPs, it was shown that they retained capability to induce the antibody neutralizing HPV16 and acquired capability to elicit antibodies cross-neutralizing the infectious HPV18, 31, 52, and 58 pseudovirions. Although the cross-neutralizing titers were lower than the type-specific neutralizing titer, the results suggest that the chimeric VLPs have potential to serve as a vaccine candidate for a broad spectrum of high-risk HPVs.

Neutralization of HPV16, 18, 31, and 58 pseudovirions with antisera induced by immunizing rabbits with synthetic peptides representing segments of the HPV16 minor capsid protein L2 surface region.

Neutralizing antibody against human papillomavirus (HPV) minor capsid protein L2 can cross-neutralize different HPV genotypes in vitro. To identify the segments containing the cross-neutralization epitopes of HPV16 L2, we characterized antisera obtained by immunizing two rabbits with each of the ten synthetic peptides of 14 to 20 amino acids (aa) long, which represents a part of the HPV16 L2 sequence from aa 14 to 144. The antisera against the peptides within the region from aa 18 to 144 efficiently bound to HPV16 L1/L2-capsids and neutralized HPV16 pseudovirions, indicating that the region is displayed on the surface of the capsids and contains several neutralization epitopes. Antiserum against the peptide from aa 18 to 38 (anti-P18/38) cross-neutralized HPV18. Anti-P56/75 cross-neutralized HPV18, 31, and 58. Anti-P61/75 and anti-P64/81 cross-neutralized HPV18 and 58. Anti-P96/115 and the antiserum induced by a mutant P96/115 (S and T at aa 101 and 112 were replaced with L and S, respectively) cross-neutralized HPV31 and 58. The mixture of equal volumes of three antisera, anti-P18/38, anti-P56/75, and anti-mutant P96/115, neutralized HPV16, 18, 31, and 58 more efficiently than anti-P56/75 alone, suggesting that there is a synergistic effect of antibodies on the cross-neutralization. The cross-neutralization appears to be correlated with conserved aa sequences among HPV types. The data in this study provide a basis for designing vaccine antigens effective against a broader spectrum of the high-risk HPVs.