Propagation of infectious human papillomavirus type 16 by using an adenovirus and Cre/LoxP mechanism

Human papillomavirus type 16 (HPV16) infection is a major risk factor for the development of squamous cell cancers of the cervix and of the head and neck. A major barrier to understanding the progression from initial infection to cancer has been the lack of in vitro models that allow infection, replication, and persistence of the viral genome as an episome in differentiated epithelial cells. To overcome this barrier, we designed an adenoviral delivery vector that contained a full HPV16 genome flanked by LoxP homologous recombination sites and a fluorescent reporter that was expressed only after the HPV genome was excised by Cre recombinase. This system delivered circular HPV16 genomes to cervical epithelial cells and well differentiated human airway epithelia. After delivery, the HPV16 genome replicated and persisted as an episome in cervical keratinocytes. These cells developed an immortalized phenotype and a dysplastic epithelial appearance. Moreover, induction of differentiation led to the expression of late genes and production of infectious HPV16 virions. This work provides a means of introducing biologically active HPV genomes into epithelial cells, which are normally difficult to transfect. These methods allow the study of HPV genome replication and gene expression in the earliest stages of HPV genome establishment, and they may provide a means to study nononcogenic HPV viral types.     

Host range transforming gene of polyoma virus plays a role in virus assembly.

Polyoma virus host range transforming (hr-t) mutants are blocked in virion assembly. In normal 3T3 cells, a nonpermissive host, these mutants synthesize 30-40% as much viral DNA and 80-100% as much capsid proteins as does wild-type virus and yet produce only 1-2% as much infectious virus. Intermediates in virion assembly have been followed by [3H]thymidine incorporation. hr-t mutants synthesize 95S replicating minichromosomes, which accumulate as 75S forms. However, the latter fail to undergo efficient transition to 240S virion structures. This block in encapsidation is overcome in permissive hosts such as primary baby mouse kidney (BMK) epithelial cells. The block in assembly of 240S particles is accompanied by a failure to induce a series of acidic isoelectric forms of the major capsid protein, VP1. Multiple species of post-translationally modified VP1 are seen by two-dimensional gel electrophoresis in wild-type virus-infected cells. These acidic VP1 subspecies are decreased 6- to 10-fold in hr-t mutant-infected 3T3 cells but are produced in normal amounts when the same mutants infect BMK cells. When 3T3 cells are coinfected with hr-t mutant and wild-type viruses, normal amounts of the VP1 subspecies are present, and hr-t mutant viral DNA is efficiently packaged into virions. These studies demonstrate an important role of the hr-t gene of polyoma virus in virus assembly. Specifically, we propose that VP1 is a target for hr-t gene-controlled modification and that modified forms of VP1 are essential for encapsidation of viral minichromosomes.     

Predicted poxvirus FEN1-like nuclease required for homologous recombination,double-strand break repair and full-size genome formation

Poxviruses encode many if not all of the proteins required for viral genome replication in the cytoplasm of the host cell. In this context, we investigated the function of the vaccinia virus G5 protein because it belongs to the FEN1-like family of nucleases and is conserved in all poxviruses. A vaccinia virus G5 deletion mutant was severely impaired, as the yield of infectious virus was reduced by approximately two orders of magnitude. The mutant virions contained an apparently normal complement of proteins but appeared spherical rather than brick-shaped and contained no detectable DNA. The inability of G5 with substitutions of the predicted catalytic aspartates to complement the deletion mutant suggested that G5 functions as a nuclease during viral DNA replication. Although the amount of viral DNA produced in the absence of G5 was similar to that made by wild-type virus, the mean size was approximately one-fourth of the genome length. Experiments with transfected plasmids showed that G5 was required for double-strand break repair by homologous recombination, suggesting a similar role during vaccinia virus genome replication.     

Papillomavirus L1 major capsid protein self-assembles into virus-like particles that are highly immunogenic.

Infection by certain human papillomavirus types is regarded as the major risk factor in the development of cervical cancer, one of the most common cancers of women worldwide. Analysis of the immunogenic and structural features of papillomavirus virions has been hampered by the inability to efficiently propagate the viruses in cultured cells. For instance, it has not been established whether the major capsid protein L1 alone is sufficient for virus particle assembly. In addition, it is not known whether L1, L2 (the minor capsid protein), or both present the immunodominant epitopes required for induction of high-titer neutralizing antibodies. We have expressed the L1 major capsid proteins of bovine papillomavirus type 1 and human papillomavirus type 16 in insect cells via a baculovirus vector and analyzed their conformation and immunogenicity. The L1 proteins were expressed at high levels and assembled into structures that closely resembled papillomavirus virions. The self-assembled bovine papillomavirus L1, in contrast to L1 extracted from recombinant bacteria or denatured virions, also mimicked intact bovine papillomavirus virions in being able to induce high-titer neutralizing rabbit antisera. These results indicate that L1 protein has the intrinsic capacity to assemble into empty capsid-like structures whose immunogenicity is similar to infectious virions. This type of L1 preparation might be considered as a candidate for a serological test to measure antibodies to conformational virion epitopes and for a vaccine to prevent papillomavirus infection.     

Persistent Human Papillomavirus Infection

Simple SummaryThe success of HPV as an infectious agent lies not within its ability to cause disease, but rather in the adeptness of the virus to establish long-term persistent infection. The ability of HPV to replicate and maintain its genome in a stratified epithelium is contingent on the manipulation of many host pathways. HPVs must abrogate host anti-viral defense programs, perturb the balance of cellular proliferation and differentiation, and hijack DNA damage signaling and repair pathways to replicate viral DNA in a stratified epithelium. Together, these characteristics contribute to the ability of HPV to achieve long-term and persistent infection and to its evolutionary success as an infectious agent.AbstractPersistent infection with oncogenic human papillomavirus (HPV) types is responsible for ~5% of human cancers. The HPV infectious cycle can sustain long-term infection in stratified epithelia because viral DNA is maintained as low copy number extrachromosomal plasmids in the dividing basal cells of a lesion, while progeny viral genomes are amplified to large numbers in differentiated superficial cells. The viral E1 and E2 proteins initiate viral DNA replication and maintain and partition viral genomes, in concert with the cellular replication machinery. Additionally, the E5, E6, and E7 proteins are required to evade host immune responses and to produce a cellular environment that supports viral DNA replication. An unfortunate consequence of the manipulation of cellular proliferation and differentiation is that cells become at high risk for carcinogenesis.     

The large form of hepatitis delta antigen is crucial for assembly of hepatitis delta virus.

The virions of hepatitis delta virus (HDV) contain two species of HDV-specific protein, a large and a small form of hepatitis delta antigen (HDAg). We examined the role of individual HDAgs in virion assembly in cotransfection experiments. First, we constructed a replication-competent HDV mutant expressing only the small HDAg. When cotransfected with a plasmid expressing hepatitis B virus surface antigens to the HuH-7 cells, the mutant did not produce HDV virions, whereas the wild-type HDV clone did. Therefore, though the small HDAg is important for viral replication and is incorporated into the virus, the small-form delta antigen by itself is insufficient for virion formation. When the system was co-transfected with an additional plasmid providing the large HDAg, the HDV virion was then recovered. There was also evidence suggesting that the large HDAg could be copackaged into the HBsAg particles, without the presence of the HDV genome and the small HDAg. The results indicate a crucial role of the large HDAg in HDV assembly.     

Human papillomavirus type 16 alters human epithelial cell differentiation in vitro.

Human papillomavirus (HPV) types 16, 18, 31, and 33 have been implicated as etiologic agents of cervical and penile cancer. Using a cell culture system for keratinocytes which allows stratification and production of differentiation-specific keratins, we have examined the effects of one of these viruses, HPV-16, on the differentiation capabilities of human epithelial cells. A plasmid containing the HPV-16 genome and a neomycin-selectable marker was transfected into primary human epidermal cells and SCC-13 cells, an immortalized squamous cell carcinoma cell line. Cloned neomycin-resistant cell lines were isolated and examined by cell culture on raised collagen rafts. Cell lines containing HPV-16 DNA retained the ability to stratify and express differentiation-specific keratins in the raft system but otherwise failed to differentiate normally. The histological abnormalities induced by HPV-16 closely resembled those seen in genital intraepithelial neoplasia in vivo. Hence, our results support the role of HPV-16 as an etiologic agent in the development of genital neoplasias and suggest a specific system for the study of HPV-16-induced epithelial cancers.     

Genetic consequences of packaging two RNA genomes in one retroviral particle: pseudodiploidy and high rate of genetic recombination.

Retroviruses contain two complete viral genomic RNAs in each virion. A system to study in a single round of replication the products of virions with two different genomic RNAs was established. A spleen necrosis virus-based splicing vector containing both the neomycin-resistance gene (neo) and the hygromycin B phosphotransferase gene (hygro) was used. Two frameshift mutants were derived from this vector such that the neo and the hygro genes were inactivated in separate vectors. Thus, each vector confers resistance to only one selection. The vectors with frameshift mutations were separately propagated and were pooled to infect DSDh helper cells. Doubly resistant cell clones were isolated, and viruses produced from these clones were used to infect D17 cells. This protocol allowed virions containing two different genomic RNAs (heterozygotes) to complete one round of retroviral replication. The molecular nature of progeny that conferred resistance to single or double selection and their ratio were determined. Our data demonstrate that each infectious heterozygous virion produces only one provirus. The rate of retroviral recombination is approximately 2% per kilobase per replication cycle. Recombinant proviruses are progeny of heterozygous virions.     

Production of infectious bovine papillomavirus from cloned viral DNA by using an organotypic raft/xenograft technique

Bovine papillomavirus type 1 (BPV-1) induces fibropapillomas in its natural host and can transform fibroblasts in culture. The viral genome is maintained as an episome within fibroblasts, which has allowed extensive genetic analyses of the viral functions required for DNA replication, gene expression, and transformation. Much less is known about BPV-1 gene expression and replication in bovine epithelial cells because the study of the complete viral life cycle requires an experimental system capable of generating a fully differentiated stratified bovine epithelium. Using a combination of organotypic raft cultures and xenografts on nude mice, we have developed a system in which BPV-1 can replicate and produce infectious viral particles. Organotypic cultures were established with bovine keratinocytes plated on a collagen raft containing BPV-1-transformed fibroblasts. These keratinocytes were infected with virus particles isolated from a bovine wart or were transfected with cloned BPV-1 DNA. Several days after the rafts were lifted to the air interface, they were grafted on nude mice. After 6-8 weeks, large xenografts were produced that exhibited a hyperplastic and hyperkeratotic epithelium overlying a large dermal fibroma. These lesions were strikingly similar to a fibropapilloma caused by BPV-1 in the natural host. Amplified viral DNA and capsid antigens were detected in the suprabasal cells of the epithelium. Moreover, infectious virus particles could be isolated from these lesions and quantitated by a focus formation assay on mouse cells in culture. Interestingly, analysis of grafts produced with infected and uninfected fibroblasts indicated that the fibroma component was not required for productive infection or morphological changes characteristic of papillomavirus-infected epithelium. This system will be a powerful tool for the genetic analysis of the roles of the viral gene products in the complete viral life cycle.     

Recovery of infectious proviral DNA from mammalian cells infected with respiratory syncytial virus.

The DNA fraction from a line of bovine embryonic kidney cells originally exposed as primary cultures several months earlier to a temperature-sensitive (ts) mutant of respiratory syncytial (RS) virus could be used to transfect human HEp-2 cells with the production of infectious RS virus. The DNA donor cells, designated BEK/RS ts, retained their healthy fibroblastic appearance during continuous cultivation at a temperature (39 degrees) restrictive for growth of the original infecting mutant and showed no evidence for RS virus replication or viral antigen synthesis when directly examined for these activities by conventional methods. The infectious property of the DNA from BEK/RS ts cells was abolished by exposure of the nucleic acid preparation to DNase (but not RNase) or by pretreatment of recipient HEp-2 cells with actinomycin D or mitomycin C. The latter drug treatments substantially enhanced the replication of infecting wild-type RS virus in HEp-2 cells. Viral isolates derived from the progeny of a DNA transfection included clones possessing several genetic markers of the RS ts mutant originally used to infect BEK/RS ts cells and other virus clones that appeared to be either hybrid or wild-type for phenotypic properties such as their temperature sensitivity. An infectious proviral DNA was also detected in a line of virogenic HEp-2 cells (HEp-2/RS) persistently infected with respiratory syncytial virus after exposure to the wild-type strain 2 years earlier.     

Point Mutations in the Glycoprotein Ectodomain of Field Rabies Viruses Mediate Cell Culture Adaptation through Improved Virus Release in a Host Cell Dependent and Independent Manner

Molecular details of field rabies virus (RABV) adaptation to cell culture replication are insufficiently understood. A better understanding of adaptation may not only reveal requirements for efficient RABV replication in cell lines, but may also provide novel insights into RABV biology and adaptation-related loss of virulence and pathogenicity. Using two recombinant field rabies virus clones (rRABV Dog and rRABV Fox), we performed virus passages in three different cell lines to identify cell culture adaptive mutations. Ten passages were sufficient for the acquisition of adaptive mutations in the glycoprotein G and in the C-terminus of phosphoprotein P. Apart from the insertion of a glycosylation sequon via the mutation D247N in either virus, both acquired additional and cell line-specific mutations after passages on BHK (K425N) and MDCK-II (R346S or R350G) cells. As determined by virus replication kinetics, complementation, and immunofluorescence analysis, the major bottleneck in cell culture replication was the intracellular accumulation of field virus G protein, which was overcome after the acquisition of the adaptive mutations. Our data indicate that limited release of extracellular infectious virus at the plasma membrane is a defined characteristic of highly virulent field rabies viruses and we hypothesize that the observed suboptimal release of infectious virions is due to the inverse correlation of virus release and virulence in vivo.     

Active human immunodeficiency virus protease is required for viral infectivity.

Retroviral proteins are synthesized as polyprotein precursors that undergo proteolytic cleavages to yield the mature viral proteins. The role of the human immunodeficiency virus (HIV) protease in the viral replication cycle was examined by use of a site-directed mutation in the protease gene. The HIV protease gene product was expressed in Escherichia coli and observed to cleave HIV gag p55 to gag p24 and gag p17 in vitro. Substitution of aspartic acid residue 25 (Asp-25) of this protein with an asparagine residue did not affect the expression of the protein, but it eliminated detectable in vitro proteolytic activity against HIV gag p55. A mutant HIV provirus was constructed that contained the Asn-25 mutation within the protease gene. SW480 human colon carcinoma cells transfected with the Asn-25 mutant proviral DNA produced virions that contained gag p55 but not gag p24, whereas virions from cells transfected with the wild-type DNA contained both gag p55 and gag p24. The mutant virions were not able to infect MT-4 lymphoid cells. In contrast, these cells were highly sensitive to infection by the wild-type virions. These results demonstrate that the HIV protease is an essential viral enzyme and, consequently, an attractive target for anti-HIV drugs.