Dataset Information

Enhancement of capsid gene expression: preparing the human papillomavirus type 16 major structural gene L1 for DNA vaccination purposes.

ABSTRACT: Expression of the structural proteins L1 and L2 of the human papillomaviruses (HPV) is tightly regulated. As a consequence, attempts to express these prime-candidate genes for prophylactic vaccination against papillomavirus-associated diseases in mammalian cells by means of simple DNA transfections result in insufficient production of the viral antigens. Similarly, in vivo DNA vaccination using HPV L1 or L2 expression constructs produces only weak immune responses. In this study we demonstrate that transient expression of the HPV type 16 L1 and L2 proteins can be highly improved by changing the RNA coding sequence, resulting in the accumulation of significant amounts of virus-like particles in the nuclei of transfected cells. Data presented indicate that, in the case of L1, adaptation for codon usage accounts for the vast majority of the improvement in protein expression, whereas translation-independent posttranscriptional events contribute only to a minor degree. Finally, the adapted L1 genes demonstrate strongly increased immunogenicity in vivo compared to that of unmodified L1 genes.

SUBMITTER: Leder C

PROVIDER: S-EPMC114488 | biostudies-literature | 2001 Oct

REPOSITORIES: biostudies-literature

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Enhancement of capsid gene expression: preparing the human papillomavirus type 16 major structural gene L1 for DNA vaccination purposes.

Leder C C Kleinschmidt J A JA Wiethe C C Müller M M

Journal of virology 20011001 19

Expression of the structural proteins L1 and L2 of the human papillomaviruses (HPV) is tightly regulated. As a consequence, attempts to express these prime-candidate genes for prophylactic vaccination against papillomavirus-associated diseases in mammalian cells by means of simple DNA transfections result in insufficient production of the viral antigens. Similarly, in vivo DNA vaccination using HPV L1 or L2 expression constructs produces only weak immune responses. In this study we demonstrate t ...[more]

PMID: 11533183

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Project description:Papillomaviruses are a family of nonenveloped DNA viruses that infect the skin or mucosa of their vertebrate hosts. The viral life cycle is closely tied to the differentiation of infected keratinocytes. Papillomavirus virions are released into the environment through a process known as desquamation, in which keratinocytes lose structural integrity prior to being shed from the surface of the skin. During this process, virions are exposed to an increasingly oxidative environment, leading to their stabilization through the formation of disulfide cross-links between neighboring molecules of the major capsid protein, L1. We used time-lapse cryo-electron microscopy and image analysis to study the maturation of HPV16 capsids assembled in mammalian cells and exposed to an oxidizing environment after cell lysis. Initially, the virion is a loosely connected procapsid that, under in vitro conditions, condenses over several hours into the more familiar 60-nm-diameter papillomavirus capsid. In this process, the procapsid shrinks by ~5% in diameter, its pentameric capsomers change in structure (most markedly in the axial region), and the interaction surfaces between adjacent capsomers are consolidated. A C175S mutant that cannot achieve normal inter-L1 disulfide cross-links shows maturation-related shrinkage but does not achieve the fully condensed 60-nm form. Pseudoatomic modeling based on a 9-Å resolution reconstruction of fully mature capsids revealed C-terminal disulfide-stabilized "suspended bridges" that form intercapsomeric cross-links. The data suggest a model in which procapsids exist in a range of dynamic intermediates that can be locked into increasingly mature configurations by disulfide cross-linking, possibly through a Brownian ratchet mechanism. Importance: Human papillomaviruses (HPVs) cause nearly all cases of cervical cancer, a major fraction of cancers of the penis, vagina/vulva, anus, and tonsils, and genital and nongenital warts. HPV types associated with a high risk of cancer, such as HPV16, are generally transmitted via sexual contact. The nonenveloped virion of HPVs shows a high degree of stability, allowing the virus to persist in an infectious form in environmental fomites. In this study, we used cryo-electron microscopy to elucidate the structure of the HPV16 capsid at different stages of maturation. The fully mature capsid adopts a rigid, highly regular structure stabilized by intermolecular disulfide bonds. The availability of a pseudoatomic model of the fully mature HPV16 virion should help guide understanding of antibody responses elicited by HPV capsid-based vaccines.

Dataset Information

Enhancement of capsid gene expression: preparing the human papillomavirus type 16 major structural gene L1 for DNA vaccination purposes.

Publications

Enhancement of capsid gene expression: preparing the human papillomavirus type 16 major structural gene L1 for DNA vaccination purposes.

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