Human Papillomavirus Type 16 Capsid Proteins Produced from Recombinant Semliki Forest Virus Assemble into Virus-like Particles

Heino, Pirkko; Dillner, Joakim; Schwartz, Stefan

doi:10.1006/viro.1995.0044

Cited by 46 publications

(16 citation statements)

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“…However, the forward process of capsid maturation has not previously been examined in detail (36). It is possible that the maturation of papillomavirus capsids has been largely overlooked, because L1 is usually expressed using lytic systems, such as baculovirus, vaccinia virus, or Semliki Forest virus vectors (13,15,20,39). In such systems, viral cytopathic effects probably induce an altered redox potential (31) in a fraction of the cells, resulting in a mixture of immature and mature capsids.…”

Section: Discussionmentioning

confidence: 99%

Maturation of Papillomavirus Capsids

Buck¹,

Thompson

Pang

et al. 2005

J Virol

265

342

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The papillomavirus capsid is a nonenveloped icosahedral shell formed by the viral major structural protein, L1. It is known that disulfide bonds between neighboring L1 molecules help to stabilize the capsid. However, the kinetics of inter-L1 disulfide bond formation during particle morphogenesis have not previously been examined. We have recently described a system for producing high-titer papillomavirus-based gene transfer vectors (also known as pseudoviruses) in mammalian cells. Here we show that papillomavirus capsids produced using this system undergo a maturation process in which the formation of inter-L1 disulfide bonds drives condensation and stabilization of the capsid. Fully mature capsids exhibit improved regularity and resistance to proteolytic digestion. Although capsid maturation for other virus types has been reported to occur in seconds or minutes, papillomavirus capsid maturation requires overnight incubation. Maturation of the capsids of human papillomavirus types 16 and 18 proceeds through an ordered accumulation of dimeric and trimeric L1 species, whereas the capsid of bovine papillomavirus type 1 matures into more extensively cross-linked forms. The presence of encapsidated DNA or the minor capsid protein, L2, did not have major effects on the kinetics or extent of capsid maturation. Immature capsids and capsids formed from L1 mutants with impaired disulfide bond formation are infectious but physically fragile. Consequently, capsid maturation is essential for efficient purification of papillomavirus-based gene transfer vectors. Despite their obvious morphological differences, mature and immature capsids are similarly neutralizable by various L1-and L2-specific antibodies.Papillomaviruses, which are etiologically implicated in the development of warts, cervical cancer, and other epithelial tumors, replicate in the stratified squamous epithelium of the skin or mucous membranes (reviewed in reference 18). The papillomavirus life cycle is closely tied to the epithelial differentiation program, which takes many days to complete. The eventual release of the nonenveloped virion is thought to depend primarily on the normal cellular disintegration typically seen near the surface of squamous epithelia.The complexity of mimicking stratified epithelial differentiation in culture has made it challenging to study the papillomaviral life cycle in vitro. As a result, much of what is known about papillomavirus morphogenesis has been learned by studying recombinant versions of the two viral structural proteins, L1 and L2. The major capsid protein, L1, can spontaneously self-assemble into 72-pentamer virus-like particles (VLPs) that closely resemble native papillomavirus virions (20). The minor capsid protein, L2, is important for papillomavirus infectivity (38), but its arrangement in the virion and its role in encapsidation of the 8-kb double-stranded circular viral genomic DNA remain unclear.We have recently described a system for generating hightiter papillomavirus-based gene transfer vectors (also known as...

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Section: Discussionmentioning

confidence: 99%

Maturation of Papillomavirus Capsids

Buck¹,

Thompson

Pang

et al. 2005

J Virol

265

342

View full text Add to dashboard Cite

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“…However, even when placed under strong heterologous promoters, expression of the papillomavirus capsid genes proved to be difficult to achieve. Only by the use of virus vector systems, such as recombinant baculoviruses for insect cells or vaccinia viruses and Semliki Forest viruses for mammalian cells, is it possible to efficiently express papillomavirus capsid genes (12,41). While vaccinia virus and Semliki Forest virus generate their mRNAs in the cytoplasm, thus circumventing the nuclear export process, this is not the case with recombinant baculoviruses or in the yeast systems, which also allow efficient production of papillomavirus late proteins (25).…”

Section: Discussionmentioning

confidence: 99%

Enhancement of Capsid Gene Expression: Preparing the Human Papillomavirus Type 16 Major Structural Gene L1 for DNA Vaccination Purposes

Leder

Kleinschmidt

Wiethe

et al. 2001

J Virol

148

114

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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.The human papillomaviruses (HPV) comprise a heterogeneous group of epitheliotropic DNA viruses. It is assumed that each of the more than 80 described HPV genotypes represents also a separate serotype (23, 24). The papillomavirus life cycle requires the infection of differentiating epithelia. In this environment, expression of the viral genes is controlled by the cell differentiation program (5, 17). Infections by human papillomaviruses are the major cause of uterine cancer in humans (22,43,44). It is estimated that worldwide half a million new cases of cervical cancer are caused by these viruses every year. The most important HPV type in this respect is HPV type 16 (HPV-16), accounting for approximately 50% of all cases of cervical cancer. Since the recognition of HPV infection as a major health burden, efforts have been undertaken to interrupt the cycle of papillomavirus infections in order to prevent virusinduced disease. Most promising for the prevention of papillomavirus-associated cancer seems to be the development of subviral vaccines that evoke protective immunity by the induction of neutralizing, capsid-directed antibodies. In fact, viruslike particles (VLP) based on the viral capsid protein L1 or L1 plus L2 are currently being developed for prophylactic and therapeutic vaccination against papillomavirus infections (19,26,27). Because they require costly production and purification protocols, it is predictable that it will require a long time for VLP-based vaccines to become affordable in the lessdeveloped countries, which suffer most from papillomaviruscaused cancer. For the same reasons, production and purification of VLP-based vaccines likely have to be restricted to a very limited number of HPV serotypes. As an alternative approach, capsid-specific neutralizing antibodies could be induced by simple DNA vaccination strategies. Since production of DNA vaccines are standardized, it is feasible to produce vaccines against ...

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“…A dominant antibody response in humans against HPV-16 capsids involves a type-specific epitope only present on assembled capsids and not on the individual pentavalent capsomeres (Heino et al, 1995;Wang et al, 1997). The L1 forms pentamers when its N terminus is fused to GST and this can allow detection of other L1-reactive antibodies.…”

Section: Discussionmentioning

confidence: 99%

Human papillomavirus type 16 E2- and L1-specific serological and T-cell responses in women with vulval intraepithelial neoplasia

Davidson

Sehr

Faulkner

et al. 2003

Journal of General Virology

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Human papillomavirus type 16 (HPV-16)-associated vulval intraepithelial neoplasia (VIN) is frequently a chronic, multifocal high-grade condition with an appreciable risk of progression to vulval cancer. The requirement to treat women with VIN has recently stimulated the use of immunotherapy with E6/E7 oncogene vaccines. Animal models have shown that E2 may also be a useful vaccine target for HPV-associated disease; however, little is known about E2 immunity in humans. This study investigated the prevalence of HPV-16 E2-specific serological and T-cell responses in 18 women with HPV-16-associated VIN and 17 healthy volunteers. E2 responses were determined by full-length E2-GST ELISA with ELISPOT and proliferation assays using E2 C-terminal protein. As positive controls, HPV-16 L1 responses were measured using virus-like particles (VLPs) and L1-GST ELISA with ELISPOT and proliferation using VLPs as antigen. The VIN patients all showed a strong serological response to L1 compared with the healthy volunteers by VLP (15/18 vs 1/17, P<0?001) and L1-GST ELISA (18/18 vs 1/17, P<0?001). In contrast, L1-specific cellular immune responses were detected in a significant proportion of controls but were more prevalent in the VIN patients by proliferation assay (9/17 vs 17/18, P<0?02) and interferon-c ELISPOT (9/17 vs 13/18, P=not significant). Similar and low numbers of patients and controls were seropositive for E2-specific Ig (2/18 vs 1/17). In spite of previous studies showing the immunogenicity of E2 in eliciting primary T-cell responses in vitro, there was a low prevalence of E2 responses in the VIN patients and controls (2/18 vs 0/17).

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Human Papillomavirus Type 16 Capsid Proteins Produced from Recombinant Semliki Forest Virus Assemble into Virus-like Particles

Cited by 46 publications

References 0 publications

Maturation of Papillomavirus Capsids

Maturation of Papillomavirus Capsids

Enhancement of Capsid Gene Expression: Preparing the Human Papillomavirus Type 16 Major Structural Gene L1 for DNA Vaccination Purposes

Human papillomavirus type 16 E2- and L1-specific serological and T-cell responses in women with vulval intraepithelial neoplasia

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