0000000000332333
AUTHOR
Christoph Volpers
Development of type-specific and cross-reactive serological probes for the minor capsid protein of human papillomavirus type 33.
Human papillomavirus type 33 (HPV33) is associated with malignant tumors of the cervix. In an attempt to develop immunological probes for HPV33 infections, antisera against various bacterial fusion proteins carrying sequences of the minor capsid protein encoded by L2 were raised in animals. Antigenic determinants on the HPV33 L2 protein were identified by using truncated fusion proteins and were classified as type specific or cross-reactive with respect to HPV1, -8, -11, -16, and -18. Cross-reactive epitopes map to amino acids 98 to 107 or to amino acids 102 to 112 and 107 to 117, respectively, depending on the fusion protein used for immunization. Antibodies directed toward these epitopes …
Conformational and linear epitopes on virus-like particles of human papillomavirus type 33 identified by monoclonal antibodies to the minor capsid protein L2
The organization of epitopes on the minor capsid protein L2 of human papillomavirus (HPV) type 33 has been analysed using three monoclonal antibodies (MAbs) generated against a large fragment of the L2 protein (amino acids 82-259) expressed as a glutathione S-transferase fusion protein. The topology of the L2 epitopes has been investigated with respect to the structure of HPV-33 virus-like particles (VLPs). Two of the MAbs reacted with linear epitopes which were mapped to amino acids 153-160 and 163-170, respectively. These epitopes were accessible in denatured but not in native VLPs consisting of L1 and L2, suggesting an internal location. The third antibody was unable to detect denatured …
Organization of the major and minor capsid proteins in human papillomavirus type 33 virus-like particles.
The organization of the major (L1) and minor (L2) proteins in the human papillomavirus capsid is still largely unknown. In this study we analysed the disulphide bonding between L1 proteins and the association of L2 proteins with capsomers using virus-like particles obtained in insect cells by co-expression of the L1 and L2 genes of human papillomavirus type 33. About 50% of the L1 protein molecules in these particles (1.29 g/cm3) formed disulphide-bonded trimers. Reduction of the intermolecular disulphide bonds by dithiothreitol (DTT) treatment caused disassembly of virus-like particles into capsomers. This indicates that disulphide bonds between capsomers at the threefold symmetry position…
Analysis of type-restricted and cross-reactive epitopes on virus-like particles of human papillomavirus type 33 and in infected tissues using monoclonal antibodies to the major capsid protein
A panel of six monoclonal antibodies recognizing at least three different antigenic regions has been raised against the L1 major capsid protein of human papillo-mavirus type 33 (HPV-33), which is associated with cervical carcinoma. The antigenic sites defined by these antibodies have been mapped and classified as type-restricted or broadly cross-reactive using bacterially expressed L1 fusion proteins of a variety of HPV types. Conformational and linear epitopes have been distinguished using native and denatured virus-like particles. HPV infection of genital lesions has been analysed using both monoclonal antibodies and DNA amplification by PCR. The antibodies obtained should be useful to pr…
Genome organization and nucleotide sequence of human papillomavirus type 39
The 7833-bp nucleotide sequence of human papillomavirus type 39 (HPV39), which is associated with genital intraepithelial neoplasias and invasive carcinomas, has been determined. The genome organization deduced from the sequence shares characteristic features with other genital papillomaviruses. According to sequence comparisons, HPV39 most closely resembles HPV18 and may be a member of a subgroup of genital papillomaviruses distinct from the HPV16/31/33 group. As a novel feature, we report a 1.3-kb open reading frame on the DNA strand which lacks major open reading frames in the other sequenced HPV genomes.
Assembly of the Major and the Minor Capsid Protein of Human Papillomavirus Type 33 into Virus-like Particles and Tubular Structures in Insect Cells
Native virions of human papillomaviruses (HPV) can be isolated from genital lesions only in very limited amounts. Recent studies have shown that virus-like particles can be obtained by expression of the capsid proteins using vaccinia virus recombinants or the baculovirus system. We now present the first detailed characterization of virus-like particles of a human papillomavirus associated with malignant genital lesions, HPV-33, produced in high yield using the baculovirus expression system. Assembly of the major capsid protein L1 alone or together with the minor capsid protein L2 has been obtained. Both spherical virus-like particles of 50-60 nm diameter and tubular structures of either 25-…
Binding and internalization of human papillomavirus type 33 virus-like particles by eukaryotic cells
Infection of cells by human papillomaviruses (HPVs) associated with malignant genital lesions has not been studied because of the lack of an in vitro system and the unavailability of virions. We have now used virus-like particles (VLPs) of HPV type 33 to analyze the initial events in the interaction of the HPV capsid with cell lines. Binding of VLPs to HeLa cells was observed in biochemical assays and by immunofluorescence. VLP binding was inhibited by antisera raised against VLPs but not by monoclonal antibodies recognizing either L1 or L2 epitopes accessible on VLPs. Under saturating conditions, approximately 2 x 10(4) VLPs were bound per cell, with a dissociation constant of about 100 pM…