Human papillomavirus DNA replication compartments in a transient DNA replication system

Many DNA viruses replicate their genomes at nuclear foci in infected cells. Using indirect immunofluorescence in combination with fluorescence in situ hybridization, we colocalized the human papillomavirus (HPV) replicating proteins E1 and E2 and the replicating origin-containing plasmid to nuclear foci in transiently transfected cells. The host replication protein A (RP-A) was also colocalized to these foci. These nuclear structures were identified as active sites of viral DNA synthesis by bromodeoxyuridine (BrdU) pulse-labeling. Unexpectedly, the great majority of RP-A and BrdU incorporation was found in these HPV replication domains. Furthermore, E1, E2, and RP-A were also colocalized to nuclear foci in the absence of an origin-containing plasmid. These observations suggest a spatial reorganization of the host DNA replication machinery upon HPV DNA replication or E1 and E2 expression. Alternatively, viral DNA replication might be targeted to host nuclear domains that are active during the late S phase, when such domains are limited in number. In a fraction of cells expressing E1 and E2, the promyelocytic leukemia protein, a component of nuclear domain 10 (ND10), was either partially or completely colocalized with E1 and E2. Since ND10 structures were recently hypothesized to be sites of bovine papillomavirus virion assembly, our observation suggests that HPV DNA amplification might be partially coupled to virion assembly.     

p53 status does not determine outcome of E1B 55-kilodalton mutant adenovirus lytic infection

The ability of the adenovirus type 5 E1B 55-kDa mutants dl1520 and dl338 to replicate efficiently and independently of the cell cycle, to synthesis viral DNA, and to lyse infected cells did not correlate with the status of p53 in seven cell lines examined. Rather, cell cycle-independent replication and virus-induced cell killing correlated with permissivity to viral replication. This correlation extended to S-phase HeLa cells, which were more susceptible to virus-induced cell killing by the E1B 55-kDa mutant virus than HeLa cells infected during G1. Wild-type p53 had only a modest effect on E1B mutant virus yields in H1299 cells expressing a temperature-sensitive p53 allele. The defect in E1B 55-kDa mutant virus replication resulting from reduced temperature was as much as 10-fold greater than the defect due to p53 function. At 39 degreesC, the E1B 55-kDa mutant viruses produced wild-type yields of virus and replicated independently of the cell cycle. In addition, the E1B 55-kDa mutant viruses directed the synthesis of late viral proteins to levels equivalent to the wild-type virus level at 39 degreesC. We have previously shown that the defect in mutant virus replication can also be overcome by infecting HeLa cells during S phase. Taken together, these results indicate that the capacity of the E1B 55-kDa mutant virus to replicate independently of the cell cycle does not correlate with the status of p53 but is determined by yet unidentified mechanisms. The cold-sensitive nature of the defect of the E1B 55-kDa mutant virus in both late gene expression and cell cycle-independent replication leads us to speculate that these functions of the E1B 55-kDa protein may be linked.     

DNA replication of human papillomavirus type 31 is modulated by elements of the upstream regulatory region that lie 5' of the minimal origin

The viral replication factors E1 and E2 of papillomaviruses are necessary and sufficient to replicate plasmids containing the minimal origin of DNA replication in transient assays. Under physiological conditions, the upstream regulatory region (URR) governs expression of the early viral genes. To determine the effect of URR elements on E1 and E2 expression specifically, and on the regulation of DNA replication during the various phases of the viral life cycle, we carried out a systematic replication study with entire genomes of human papillomavirus type 31 (HPV31), a high-risk oncogenic type. We constructed a series of URR deletions, spacer replacements, and point mutations to analyze the role of the keratinocyte enhancer (KE) element, the auxiliary enhancer (AE) domain, and the L1-proximal end of the URR (5'-URR domain) in DNA replication during establishment, maintenance, and vegetative viral DNA amplification. Using transient and stable replication assays, we demonstrate that the KE and AE are necessary for efficient E1 and E2 gene expression and that the KE can also directly modulate viral replication. KE-mediated activation of replication is dependent on the position and orientation of the element. Mutation of either one of the four Ap1 sites, the single Sp1 site, or the binding site for the uncharacterized footprint factor 1 reduced replication efficiency through decreased expression of E1 and E2. Furthermore, the 5'-URR domain and the Oct1 DNA binding site are dispensable for viral replication, since such HPV31 mutants are able to replicate efficiently in a transient assay, maintain a stable copy number over several cell generations, and amplify viral DNA under vegetative conditions. Interestingly, deletion of the 5'-URR domain leads to increased transient and stable replication levels. These findings suggest that elements in the HPV31 URR outside the minimal origin modulate viral replication through both direct and indirect mechanisms.     

Association of the human papillomavirus type 11 E1 protein with histone H1

The E1 and E2 proteins are the only virus-encoded factors required for human papillomavirus (HPV) DNA replication. The E1 protein is a DNA helicase responsible for initiation of DNA replication at the viral origin. Its recruitment to the origin is facilitated by binding to E2, for which specific recognition elements are located at the origin. The remaining replication functions for the virus, provided by the host cell's replication machinery, may be mediated by further interactions with E1 and E2. Histone H1 was identified as an HPV type 11 (HPV-11) E1-binding protein by far-Western blotting and by microsequence analyses of a 34-kDa protein purified by E1 affinity chromatography. E1 also bound in vitro to H1 isolated under native conditions in association with intact nucleosomes. In addition, E1 and H1 were coimmunoprecipitated by an E1 antiserum from a nuclear extract prepared from cells expressing recombinant E1. Bound H1 was displaced from HPV-11 DNA by the addition of E1, suggesting that E1 can promote replication initiation and elongation by alteration of viral chromatin structure and disruption of nucleosomes at the replication fork. Furthermore, a region of the HPV-11 genome containing the origin of replication was identified which had weaker affinity for H1 than that of the remaining genome. This result suggests that the presence of a DNA structure at or near the HPV origin facilitates initiation of DNA replication by exclusion of H1. These results are similar to those of studies of simian virus 40 DNA replication, in which a large T antigen-H1 interaction and an H1-resistant region at the origin of DNA replication have also been demonstrated.     

Differential requirements for conserved E2 binding sites in the life cycle of oncogenic human papillomavirus type 31

Human papillomavirus (HPV) E2 proteins regulate viral replication by binding to sites in the upstream regulatory region (URR) and by complex formation with the E1 origin recognition protein. In the genital HPV types, the distribution and location of four E2 binding sites (BS1 to BS4) which flank a single E1 binding site are highly conserved. We have examined the roles of these four E2 sites in the viral life cycle of HPV type 31 (HPV31) by using recently developed methods for the biosynthesis of papillomaviruses from transfected DNA templates (M. G. Frattini et al., Proc. Natl. Acad. Sci. USA 93:3062-3067, 1996). In transient assays, no single site was found to be necessary for replication, and mutation of the early promoter-proximal site (BS4) led to a fourfold increase in replication. Cotransfection of the HPV31 wild-type (HPV-wt) and mutant genomes with expression vectors revealed that E1 stimulated replication of HPV31-wt as well as the HPV31-BS1, -BS2, and -BS3 mutants. In contrast, increased expression of E2 decreased replication of these genomes. Replication of the HPV31-BS4 mutant genome was not further increased by cotransfection of E1 expression vectors but was stimulated by E2 coexpression. In stably transfected normal human keratinocytes, mutation of either BS1, BS3, or BS4 resulted in integration of viral genomes into host chromosomes. In contrast, mutation of BS2 had no effect on stable maintenance of episomes or copy number. Following growth of stably transfected lines in organotypic raft cultures, the differentiation-dependent induction of late gene expression and amplification of viral DNA of the BS2 mutant was found to be similar to that of HPV31-wt. We were unable to find a role for BS2 in our assays for viral functions. We conclude that at least three of the four E2 binding sites in the URRs of HPVs are essential for the productive viral life cycle. The specific arrangement of E2 binding sites within the URR appears to be more important for viral replication than merely the number of sites.     

The early region 1B 55-kilodalton oncoprotein of adenovirus relieves growth restrictions imposed on viral replication by the cell cycle

The E1B 55-kDa oncoprotein of adenovirus enables the virus to overcome restrictions imposed on viral replication by the cell cycle. Approximately 20% of HeLa cells infected with an E1B 55-kDa mutant adenovirus produced virus when evaluated by electron microscopy or by assays for infectious centers. By contrast, all HeLa cells infected with a wild-type adenovirus produced virus. The yield of E1B mutant virus from randomly cycling HeLa cells correlated with the fraction of cells in S phase at the time of infection. In synchronously growing HeLa cells, approximately 75% of the cells infected during S phase with the E1B mutant virus produced virus, whereas only 10% of the cells infected during G1 produced virus. The yield of E1B mutant virus from HeLa cells infected during S phase was sevenfold greater than that of cells infected during G1 and threefold greater than that of cells infected during asynchronous growth. Cells infected during S phase with the E1B mutant virus exhibited severe cytopathic effects, whereas cells infected with the E1B mutant virus during G1 exhibited a mild cytopathic effect. Viral DNA synthesis appeared independent of the cell cycle because equivalent amounts of viral DNA were synthesized in cells infected with either wild-type or E1B mutant virus. The inability of the E1B mutant virus to replicate was not mediated by the status of p53. These results define a novel property of the large tumor antigen of adenovirus in relieving growth restrictions imposed on viral replication by the cell cycle.     

A C-terminal helicase domain of the human papillomavirus E1 protein binds E2 and the DNA polymerase alpha-primase p68 subunit

The human papillomavirus (HPV) E1 and E2 proteins bind cooperatively to the viral origin of replication (ori), forming an E1-E2-ori complex that is essential for initiation of DNA replication. All other replication proteins, including DNA polymerase alpha-primase (polalpha-primase), are derived from the host cell. We have carried out a detailed analysis of the interactions of HPV type 16 (HPV-16) E1 with E2, ori, and the four polalpha-primase subunits. Deletion analysis showed that a C-terminal region of E1 (amino acids [aa] 432 to 583 or 617) is required for E2 binding. HPV-16 E1 was unable to bind the ori in the absence of E2, but the same C-terminal domain of E1 was sufficient to tether E1 to the ori via E2. Of the polalpha-primase subunits, only p68 bound E1, and binding was competitive with E2. The E1 region required (aa 397 to 583) was the same as that required for E2 binding but additionally contained 34 N-terminal residues. In confirmation of these differences, we found that a monoclonal antibody, mapping adjacent to the N-terminal junction of the p68-binding region, blocked E1-p68 but not E1-E2 binding. Sequence alignments and secondary-structure prediction for HPV-16 E1 and other superfamily 3 (SF3) viral helicases closely parallel the mapping data in suggesting that aa 439 to 623 constitute a discrete helicase domain. Assuming a common nucleoside triphosphate-binding fold, we have generated a structural model of this domain based on the X-ray structures of the hepatitis C virus and Bacillus stearothermophilus (SF2) helicases. The modelling closely matches the deletion analysis in suggesting that this region of E1 is indeed a structural domain, and our results suggest that it is multifunctional and critical to several stages of HPV DNA replication.     

Different HPV16 E6/E7 oncogene expression patterns in epithelia reconstructed from HPV16-immortalized human endocervical cells and genital keratinocytes

Human papillomavirus type 16 (HPV16) E6/E7 oncogenes immortalize two types of human genital epithelial cells in vitro, endocervical cells and ectocervical or foreskin keratinocytes. Epithelia reconstructed in in vivo nude mouse implants or in vitro organotypic raft cultures from immortalized endocervical cells form higher grade dysplasia than those from keratinocytes. Here, we compared viral E6/E7 mRNA expression in immortalized cell lines of the three cell types using implants, rafts and in situ hybridization assays. Endocervical cells expressed E6/E7 throughout their reconstructed epithelia. In contrast, oncogenes were limited to basal cells for keratinocyte lower grade dysplasias. To study the role of the HPV16 promoter/enhancer in this repression in the upper layers of keratinocyte epithelia, new cell lines were established by immortalization with E6/E7 controlled by the SV40 promoter. The oncogenes were shown to be controlled from the SV40 elements after immortalization. Nevertheless, E6/E7 in the two cell types had the same cell-specific expression pattern as that controlled from the homologous HPV16 promoter. In addition, naturally occurring premalignant lesions having integrated HPV16 DNA expressed E6/E7 extensively in the high-grade dysplastic region of undifferentiated metaplasia. On the other hand, oncogene expression was restricted to lower layers in the lower grade dysplastic region of more mature differentiation. Our data suggest that keratinocytes have an inherent HPV16 promoter-nonspecific mechanism of repression. Apparently this mechanism, which can be acquired during maturation, is initially nonfunctional in in vitro and in vivo epithelia derived from metaplastic endocervical cells.     

Induction of human papillomavirus type 18 late gene expression and genomic amplification in organotypic cultures from transfected DNA templates

The genetic analysis of human papillomavirus (HPV) functions during the vegetative viral life cycle is dependent upon the ability to generate human keratinocyte cell lines which maintain episomal copies of transfected viral genomes. We have previously demonstrated that lipofection of normal human foreskin keratinocytes with recircularized cloned HPV-31 genomic sequences resulted in a high frequency of cell lines which maintained viral genomes as extrachromosomal elements (M.G. Frattini, H. Lim, and L.A. Laimins, Proc. Natl. Acad. Sci. USA 93:3062-3067, 1996). Following the growth of these cell lines in organotypic (raft) cultures, the differentiation-dependent expression of viral late genes, the amplification of viral genomes, and virion biosynthesis were observed. In the present study, we demonstrate that these methodologies are not restricted to HPV-31 but are applicable to other HPV types, including the oncogenic HPV-18. HPV-18 genomes were purified from bacterial vector sequences, religated, and transfected into normal human foreskin keratinocytes together with a neomycin-selectable marker. Following drug selection, resistant cells were expanded and examined for the state of the viral DNA. All cell lines examined were found to contain approximately 100 to 200 episomal copies of HPV-18 DNA per cell. Growth of these cell lines in raft cultures resulted in the differentiation-dependent expression of the E1 [symbol: see text] E4 and L1 capsid genes. In addition, viral genome amplification was observed in suprabasal cells following DNA in situ hybridization analysis of differentiated raft cultures. The induction of these late viral functions has previously been shown to be directly associated with differentiation-dependent virion biosynthesis. Our studies indicate the ability to perform a detailed genetic analysis of the various phases of the viral life cycle, including control of the differentiation-dependent late viral functions, using a second oncogenic HPV type.     

Assembly of bacteriophage phi X174: identification of a virion capsid precursor and proposal of a model for the functions of bacteriophage gene products during morphogenesis

A capsomeric structure sedimenting with an S value of 108 in sucrose gradients was isolated from Escherichia coli infected with bacteriophage phi X174. The 108S material contained viral proteins F, G, H, and D, and the relative amounts of these proteins in the 108S material were similar to those in the infectious 132S particle, which has previously been described as a possible intermediate in the assembly of 114S phage particles. Electron micrographs indicated that the size and shape of the 108S material resemble those of the 132S particle. The 108S material contained no DNA, and its formation occurred independently of DNA synthesis. The 108S material accumulated in infected cells when viral DNA replication was prevented either by mutation in phage genes A or C or by removal of thymidine from a culture infected with wild-type phage or with a lysis gene E mutant. Upon restoration of thymidine to cells infected with the lysis gene E mutant and then starved of thymidine, the accumulated 108S material was converted to 132S particles and to 114S phage particles, implying that the 108S material is a precursor of phage particles. A model that proposes possible functions for the products of phi X174 genes A, B, C, D, F, and G during viral replication and phage maturation is described.     

Expression of polysialylated NCAM but not L1 or N-cadherin by regenerating adult mouse optic fibers in vitro

Persistent/latent viral infections of insect cells are a prominent though poorly understood phenomenon. In this study, the long-term association between the Hz-1 virus and insect host cells, conventionally referred to as persistent viral infection, is described. With the aid of a newly developed fluorescent cell-labeling system, we found that productive viral replication occurs by spontaneous viral reactivation in fewer than 0.2% of persistently infected cell lines over a 5-day period. Once viral reactivation takes place, the host cell dies. The persistently infected cells contain various amounts of viral DNA, and, in an extreme case, up to 16% of the total DNA isolated from infected cells could be of viral origin. Both pulsed-field gel electrophoresis and in situ hybridization experiments showed that some of these viral DNA molecules are inserted into the host chromosomes but that the rest of viral DNA copies are free from host chromosomes. Thus, Hz-1 virus is the first nonretroviral insect virus known to insert its genome into the host chromosome during the infection process. These data also suggest that the previously described persistent infection of Hz-1 virus in insect cells should be more accurately referred to as latent viral infection.