A negative regulatory element in the human papillomavirus type 16 genome acts at the level of late mRNA stability.

A negative regulatory element present in the human papillomavirus type 16 genome has been characterized. Deletion analysis has localized the 5' end of the element to the late region of the genome at the extreme 3' end of the coding region of the L1 open reading frame, around the L1 stop codon, with the element extending into the L1 3' untranslated region. For the cell lines used, the element's function was independent of cell type, tissue, or species of origin, unlike papillomavirus infection, which is very dependent on such factors. By using an mRNA decay assay, we have determined that polyadenylated RNA containing the element is much less stable than polyadenylated RNA lacking the element. This indicates that the element acts as an mRNA instability element. The significance of A-rich, GU-rich, and AUG-rich sequences for the functioning of this human papillomavirus type 16 instability element is discussed.     

In vitro generation and type-specific neutralization of a human papillomavirus type 16 virion pseudotype.

We report a system for generating infectious papillomaviruses in vitro that facilitates the analysis of papillomavirus assembly, infectivity, and serologic relatedness. Cultured hamster BPHE-1 cells harboring autonomously replicating bovine papillomavirus type 1 (BPV1) genomes were infected with recombinant Semliki Forest viruses that express the structural proteins of BPV1. When plated on C127 cells, extracts from cells expressing L1 and L2 together induced numerous transformed foci that could be specifically prevented by BPV neutralizing antibodies, demonstrating that BPV infection was responsible for the focal transformation. Extracts from BPHE-1 cells expressing L1 or L2 separately were not infectious. Although Semliki Forest virus-expressed L1 self-assembled into virus-like particles (VLPs), viral DNA was detected in particles only when L2 was coexpressed with L1, indicating that genome encapsidation requires L2. Expression of human papillomavirus type 16 (HPV16) L1 and L2 together in BPHE-1 cells also yielded infectious virus. These pseudotyped virions were neutralized by antiserum to HPV16 VLPs derived from European (114/K) or African (Z-1194) HPV16 variants but not by antisera to BPV VLPs, to a poorly assembling mutant HPV16 L1 protein, or to VLPs of closely related genital HPV types. Extracts from BPHE-1 cells coexpressing BPV L1 and HPV16 L2 or HPV16 L1 and BPV L2 were not infectious. We conclude that (i) mouse C127 cells express the cell surface receptor for HPV16 and are able to uncoat HPV16 capsids; (ii) if a papillomavirus DNA packaging signal exists, then it is conserved between the BPV and HPV16 genomes; (iii) functional L1-L2 interaction exhibits type specificity; and (iv) protection by HPV virus-like particle vaccines is likely to be type specific.     

Laboratory production in vivo of infectious human papillomavirus type 11.

Human papillomaviruses (HPV) induce among patients natural lesions which produce small amounts of virus. Infection of human cell cultures does not lead to the multiplication of virus, which also does not replicate in experimental animals. We have developed a unique system for the laboratory production of HPV type 11 (HPV-11). Fragments of human neonatal foreskin were infected with an extract of naturally occurring human vulvar condylomata and grafted beneath the renal capsule of athymic mice. Later (3 to 5 months), condylomatous cysts developed from those grafts. Nuclei of koilocytotic cells contained large amounts of capsid antigen and intranuclear virions. The experimentally induced condylomata were homogenized, and the virions were extracted and used to infect another generation of human foreskin grafts in athymic mice. The HPV-11 DNA content and infectivity of the natural and experimental condylomata were similar. Extracts of experimental condylomata were subjected to differential ultracentrifugation and sedimentation in CsCl density gradients. A single, opalescent band was visible at a density of 1.34 g/ml. It contained HPV virions with HPV-11 DNA. This report is the first demonstration of the laboratory production of an HPV.     

Identification of a transforming gene of human papillomavirus type 16.

Previously, we observed sequential two-step alteration, growth stimulation, and progression to a more malignant state in NIH 3T3 cells transfected by human papillomavirus type 16 (HPV-16) DNA. In this study, we prepared a cDNA library from RNA extracted from cells transfected with the HPV-16 DNA and isolated cDNA clones which had growth-stimulating activity. Analysis of these cDNA clones indicated that the E7 open reading frame alone is responsible for inducing both steps of this cell transformation.     

Mutational analysis of human papillomavirus type 16 E7 functions.

The human papillomavirus type 16 E7 gene encodes a nuclear oncoprotein (98 amino acids [AAs] long) consisting of three regions: regions 1 (AAs 1 to 20) and 2 (AAs 21 to 40), which show high homology to the sequences of conserved domains 1 and 2, respectively, of adenovirus E1A; and region 3 (AAs 41 to 98) containing two metal-binding motifs Cys-X-X-Cys (AAs 58 and 91 to 94). We constructed AA deletion (substitution) mutants and single-AA substitution mutants of E7 placed under the control of the simian virus 40 promoter and examined their biological functions. Stable expression of E7 protein in monkey COS-1 cells required almost the entire length of E7 and was markedly lowered by the mutations in region 3. Transactivation of the adenovirus E2 promoter in monkey CV-1 cells was lowered by the mutations. It was abolished by changing Cys-24 to Gly and markedly decreased by a mutation at His-2 or at the metal-binding motifs in region 3. Focal transformation of rat 3Y1 cells by E7 was eliminated by changing His-2 to Asp or Cys-24 to Gly and was greatly impaired by changing Cys-61 or Cys-94 to Gly. The transforming function survived mutations at Leu-13 and Cys-68 and deletion of Asp-Ser-Ser (AAs 30 to 32). The data suggest that regions 1 to 3 are required for its functions and that the meta-binding motifs in region 3 are required to maintain a stable or functional structure of the E7 protein.     

Characterization of a human papillomavirus type 16 variant-dependent neutralizing epitope.

We have determined that three type-specific and conformationally dependent monoclonal antibodies, H16.E70, H16.U4, and H16.V5, neutralize pseudotype human papillomavirus type 16 (HPV16) virions in vitro. H16.U4 and H16.V5 neutralized pseudotype virions derived from the German HPV16 variant 114K and the Zairian variant Z-1194 with equal efficiency. In contrast, neutralization of Z-1194 pseudotype virions by H16.E70 was two orders of magnitude weaker than neutralization of 114K pseudotype virions. This difference correlated with enzyme-linked immunosorbent assay reactivity of H16.E70 to L1 virus-like particles of the two variants. A substitution at residue 282 of L1 was responsible for this differential reactivity, suggesting that this residue constitutes part of the H16.E70 epitope.     

Analysis of the Saccharomyces cerevisiae mitochondrial COX3 mRNA 5' untranslated leader: translational activation and mRNA processing.

We used transformation of yeast mitochondria and homologous gene replacement to study features of the 613-base COX3 mRNA 5' untranslated leader (5'-UTL) required for translational activation by the protein products of the nuclear genes PET54, PET122, and PET494 in vivo. Elimination of the single AUG triplet in the 5'-UTL had no detectable effect on expression, indicating that activator proteins do not work by allowing ribosomes to bypass that AUG. Deletion of the entire 5'-UTL completely prevented translation, suggesting that the activator proteins do not function by antagonizing any other negative element in the 5'-UTL. Removal of the 15 terminal bases from the 5' end of the 5'-UTL did not block activator-dependent translation. The largest internal deletion that did not interfere with translation removed 125 bases from the upstream portion of the leader. However, two large deletions that blocked translation could be reverted to activator-dependent expression by secondary changes in the remaining 5'-UTL sequences, indicating that the original deletions had not removed the translational activator target but only deformed it. Taken together, the deletion mutations and revertants define a region of 151 bases (between positions -480 and -330 relative to the start codon) containing sequences that are sufficient for translational activation when modified slightly. Suppression of the respiratory phenotypes of two 5'-UTL mutations by overexpression of PET54, PET122, and PET494 indicated functional interactions between the leader and the three activator proteins. The mature COX3 mRNA is cleaved from a precursor immediately downstream of the preceding tRNAVal in a fashion resembling mRNA processing in vertebrate mitochondria. Our results indicate that the site of this cleavage in Saccharomyces cerevisiae is determined solely by the position of the tRNA.     

Early evolution of histone mRNA 3' end processing

The replication-dependent histone mRNAs in metazoa are not polyadenylated, in contrast to the bulk of mRNA. Instead, they contain an RNA stem-loop (SL) structure close to the 3' end of the mature RNA, and this 3' end is generated by cleavage using a machinery involving the U7 snRNP and protein factors such as the stem-loop binding protein (SLBP). This machinery of 3' end processing is related to that of polyadenylation as protein components are shared between the systems. It is commonly believed that histone 3' end processing is restricted to metazoa and green algae. In contrast, polyadenylation is ubiquitous in Eukarya. However, using computational approaches, we have now identified components of histone 3' end processing in a number of protozoa. Thus, the histone mRNA stem-loop structure as well as the SLBP protein are present in many different protozoa, including Dictyostelium, alveolates, Trypanosoma, and Trichomonas. These results show that the histone 3' end processing machinery is more ancient than previously anticipated and can be traced to the root of the eukaryotic phylogenetic tree. We also identified histone mRNAs from both metazoa and protozoa that are polyadenylated but also contain the signals characteristic of histone 3' end processing. These results provide further evidence that some histone genes are regulated at the level of 3' end processing to produce either polyadenylated RNAs or RNAs with the 3' end characteristic of replication-dependent histone mRNAs.     

Optimization of a weak 3' splice site counteracts the function of a bovine papillomavirus type 1 exonic splicing suppressor in vitro and in vivo

Alternative splicing is a critical component of the early to late switch in papillomavirus gene expression. In bovine papillomavirus type 1 (BPV-1), a switch in 3' splice site utilization from an early 3' splice site at nucleotide (nt) 3225 to a late-specific 3' splice site at nt 3605 is essential for expression of the major capsid (L1) mRNA. Three viral splicing elements have recently been identified between the two alternative 3' splice sites and have been shown to play an important role in this regulation. A bipartite element lies approximately 30 nt downstream of the nt 3225 3' splice site and consists of an exonic splicing enhancer (ESE), SE1, followed immediately by a pyrimidine-rich exonic splicing suppressor (ESS). A second ESE (SE2) is located approximately 125 nt downstream of the ESS. We have previously demonstrated that the ESS inhibits use of the suboptimal nt 3225 3' splice site in vitro through binding of cellular splicing factors. However, these in vitro studies did not address the role of the ESS in the regulation of alternative splicing. In the present study, we have analyzed the role of the ESS in the alternative splicing of a BPV-1 late pre-mRNA in vivo. Mutation or deletion of just the ESS did not significantly change the normal splicing pattern where the nt 3225 3' splice site is already used predominantly. However, a pre-mRNA containing mutations in SE2 is spliced predominantly using the nt 3605 3' splice site. In this context, mutation of the ESS restored preferential use of the nt 3225 3' splice site, indicating that the ESS also functions as a splicing suppressor in vivo. Moreover, optimization of the suboptimal nt 3225 3' splice site counteracted the in vivo function of the ESS and led to preferential selection of the nt 3225 3' splice site even in pre-mRNAs with SE2 mutations. In vitro splicing assays also showed that the ESS is unable to suppress splicing of a pre-mRNA with an optimized nt 3225 3' splice site. These data confirm that the function of the ESS requires a suboptimal upstream 3' splice site. A surprising finding of our study is the observation that SE1 can stimulate both the first and the second steps of splicing.     

Abrogation of growth arrest signals by human papillomavirus type 16 E7 is mediated by sequences required for transformation.

Cells arrest in the G1 or G0 phase of the cell cycle in response to a variety of negative growth signals that induce arrest by different molecular pathways. The ability of human papillomavirus (HPV) oncogenes to bypass these signals and allow cells to progress into the S phase probably contributes to the neoplastic potential of the virus. The E7 protein of HPV-16 was able to disrupt the response of epithelial cells to three different negative growth arrest signals: quiescence imposed upon suprabasal epithelial cells, G1 arrest induced by DNA damage, and inhibition of DNA synthesis caused by treatment with transforming growth factor beta. The same set of mutated E7 proteins was able to abrogate all three growth arrest signals. Mutant proteins that failed to abrogate growth arrest signals were transformation deficient and included E7 proteins that bound retinoblastoma protein in vitro. In contrast, HPV-16 E6 was able to bypass only DNA damage-induced G1 arrest, not suprabasal quiescence or transforming growth factor beta-induced arrest. The E6 and E7 proteins from the low-risk virus HPV-6 were not able to bypass any of the growth arrest signals.     

Production of human papillomavirus type 16 virions in a keratinocyte cell line.

Human papillomavirus type 16 (HPV-16) is strongly associated with carcinoma of the cervix, but the complete life cycle of the virus cannot be studied because no experimental system is available in which HPV-16 progeny are produced, and there is currently no source of HPV-16 virus particles. Most cell lines that harbor HPV-16 DNA contain the viral genome as integrated or concatenated DNA in which open reading frames are disrupted or deleted, but a human cervical keratinocyte cell line has been described which maintains HPV-16 DNA in monomeric episomal form (M.A. Stanley, H.M. Brown, M.W. Appleby, and A.C. Minson, Int. J. Cancer 43:672-676, 1989). This cell line was induced to form a stratified differentiating epithelium by grafting onto nude mice. Long-term grafts displayed the histological features of a low-grade cervical dysplasia, and terminally differentiated cells contained amplified levels of HPV-16 DNA, virus capsid antigen, and virus particles. This experimental system appears to permit the completion of the HPV-16 life cycle in virus-containing keratinocytes.     

Papilloma formation in human foreskin xenografts after inoculation of human papillomavirus type 16 DNA.

A mouse model of high-risk human papillomavirus infection was developed in which human papillomavirus (HPV) type 16 DNA was inoculated into human foreskin grafted to the skin of severe combined immunodeficient (scid) mice. Grafted skin contained human epidermis and dermis and, like normal human skin, expressed involucrin in differentiating keratinocytes. HPV type 16 DNA, attached to gold particles, was delivered directly into human epidermal cells and induced exophytic papilloma with histologic features of papillomavirus infection, including koilocytosis and expression of papillomavirus capsid antigen. This model should be useful for determining in vivo the functions of viral genes and for developing strategies to prevent and treat HPV-associated disease. It may also be of value in developing animal models of other human skin diseases.     

Effect of human papillomavirus type 16 oncogenes on MAP kinase activity.

The mitogen-activated protein (MAP) kinase signal transduction pathway is an intracellular signaling cascade which mediates cellular responses to growth and differentiation factors. The MAP kinase pathway can be activated by a wide range of stimuli dependent on the cell types, and this is normally a transient response. Oncogenes such as ras, src, raf, and mos have been proposed to transform cells in part by prolonging the activated stage of components within this signaling pathway. The human papillomavirus (HPV) oncogenes E6 and E7 play an essential role in the in vitro transformation of primary human keratinocytes and rodent cells. The HPV type 16 E5 gene has also been shown to have weak transforming activity and may enhance the epidermal growth factor (EGF)-mediated signal transduction to the nucleus. In the present study, we have investigated the effects of the oncogenic HPV type 16 E5, E6, and E7 genes on the induction of the MAP kinase signaling pathway. The E5 gene induced an increase in the MAP kinase activity both in the absence and in the presence of EGF. In comparison, the E6 and E7 oncoproteins do not alter the MAP kinase activity or prolong the MAP kinase activity induced with EGF. These findings suggest that E5 may function, at least in part, to enhance the cell response through the MAP kinase pathway. However, the transforming activity of E6 and E7 is not associated with alterations in the MAP kinase pathway. These findings are consistent with E5 enhancing the response to growth factor stimulation.