Louise T. Chow

Complex splicing patterns of RNAs from the early regions of adenovirus-2

Abstract The RNA species encoded by the five early regions of adenovirus serotype 2 (Ad2) were isolated from the cytoplasm of HeLa cells at various times after infection either in the absence or in the presence of cycloheximide or cytosine arabinoside. The transcripts were characterized by electron microscopy of heteroduplexes formed with single-stranded Ad2 DNA. Each early region gave rise to a family of composite RNA species with up to four conserved segments spliced together after the deletion of intervening sequences. Transcripts from each family contained some sequences in common but differed in the lengths and positions of the conserved segments and thus had alternative splicing patterns. They were mapped on the adenovirus genome and the relative abundance of each species was determined. The map co-ordinates of the early region promoters were refined, based upon the indication that the 5′ ends of mature cytoplasmic RNAs are derived from promoter-proximal DNA sequences. The 5′ leader segment of the region 2 message for the single-stranded DNA binding protein changed during the course of infection, suggesting the utilization of different promoters at early and late times. The transition was prevented by blocking protein synthesis with cycloheximide. Transcription of region 3 was under the control of its own promoter at early times but could also be directed by the major r -strand late promoter at intermediate to late times. The internal splicing patterns of the region 3 transcripts seen at early times persisted at late times, but some molecules had the 5′ tripartite leaders common to late r -strand mRNAs. One of the major early region 3 transcripts contained three RNA segments which are also present in a presumptive processing intermediate for the fiber RNA, a late message which is separated from its promoter by early region 3. These extra segments in some fiber RNAs apparently reflect the recognition of early RNA splicing signals at late times. From the large, complex arrays of composite RNA structures, numerous insights into the RNA splicing mechanisms were inferred.

A map of cytoplasmic RNA transcripts from lytic adenovirus type 2, determined by electron microscopy of RNA:DNA hybrids

Abstract We have used electron microscopic RNA loop mapping to determine, to within 200 nucleotides, the chromosome coordinates of the 5′ and 3′ ends of adenovirus type 2 transcripts isolated from the cytoplasm of productively infected human KB cells. The major blocks of transcription and many of the individual transcripts have discrete lengths and well defined 5′ and 3′ termini. Early cytoplasmic RNA is transcribed from four separate regions of the adenovirus genome, in map intervals 1.3–11.1, 62.4–67.9, 78.6–86.1 and 91.5–96.8. The most frequent late RNA loop occurred between coordinates 51.9 and 62.2. A late transcript complementary to interval 86.3–91.5 formed a characteristic R loop with a long branch of displaced RNA from coordinate 89.8–91.5, possibly a result of secondary structure near coordinate 89.8. Other late cytoplasmic RNA loops were located between coordinates 11.1 and 14.9, and in the intervals from 30–52 and 68–83. Transcripts from several regions of the chromosome had variable lengths but common 5′ or 3′ termini. Few R loops were seen between map coordinates 15 and 30. Switches between template strands were observed at map positions 11.2, 62.4 and 91.5. Many of the R loops have been correlated with gene products mapped previously by translation of RNA complementary to different portions of the chromosome.

Identification of the gene and mRNA for the adenovirus terminal protein precursor

The precursor of the 55K adenovirus terminal protein is an 87K protein that is covalently linked to viral DNA. This protein is likely to be identical to the 80,000 dalton protein described by Challberg et al. (1980). The mRNA for the 87K terminal protein precursor, like that for the E2-72K DNA binding protein, is detectable at both early and late times of infection, and its production is sensitive to protein synthesis inhibition (Lewis and Mathews, 1980). The 87K protein, together with proteins of 105,000 and 75,000 daltons, are translated from leftward transcribed (1-strand) messenger RNAs that are complementary to the viral genome between positions 11.2 and 31.5. Additional hybridization to the region between coordinates 37.3 and 41 suggests that the RNA body is spliced to sequences mapping farther right in the genome. Electron microscopic heteroduplex analysis has revealed a family of 1-strand RNAs that probably encode these proteins. The RNA bodies extend from coordinated 30, 26 and 23 to 11.1, with leaders at 39, 68.5 and 75 map units, defining a new adenovirus early region. These RNAs and region E2 RNAs share the first leader and presumably the same promoter, and may be coordinately expressed. Virions of the protease-deficient adenovirus 2 mutant ts1 grown at the restrictive temperature contain only the 87K form; when grown at the permissive temperature they contain both the 87K and 55K forms, and an additional 62K form; wild-type virions contain only the 55K form. Peptide analysis shows all these proteins to be related. The DNA-protein complex containing the 87K form is active as a template for viral DNA replication in vitro. This data supports a model of adenovirus DNA replication in which the 87K terminal protein precursor is the primary translation product and primes DNA synthesis. The 87K precursor is processed curing virus maturation to the 55K terminal protein, possibly via a 62K intermediate form, by the virus-specified Ad2ts1 protease.

Robust production and passaging of infectious HPV in squamous epithelium of primary human keratinocytes

Using Cre-loxP-mediated recombination, we established a highly efficient and reproducible system that generates autonomous HPV-18 genomes in primary human keratinocytes (PHKs), the organotypic raft cultures of which recapitulated a robust productive program. While E7 promoted S-phase re-entry in numerous suprabasal differentiated cells, HPV DNA unexpectedly amplified following a prolonged G2 arrest in mid- and upper spinous cells. As viral DNA levels intensified, E7 activity diminished and then extinguished. These cells then exited the cell cycle to undergo virion morphogenesis. High titers of progeny virus generated an indistinguishable productive infection in naïve PHK raft cultures as before, never before achieved until now. An immortalization-defective HPV-18 E6 mutant genome was also characterized for the first time. Numerous cells accumulated p53 protein, without inducing apoptosis, but the productive program was severely curtailed. Complementation of mutant genomes by E6-expressing retrovirus restored proper degradation of p53 as well as viral DNA amplification and L1 production. This system will be invaluable for HPV genetic dissection and serves as a faithful ex vivo model for investigating infections and interventions.

Clonal Selection for Transcriptionally Active Viral Oncogenes during Progression to Cancer

ABSTRACT Primary keratinocytes immortalized by human papillomaviruses (HPVs), along with HPV-induced cervical carcinoma cell lines, are excellent models for investigating neoplastic progression to cancer. By simultaneously visualizing viral DNA and nascent viral transcripts in interphase nuclei, we demonstrated for the first time a selection for a single dominant papillomavirus transcription center or domain (PVTD) independent of integrated viral DNA copy numbers or loci. The PVTD did not associate with several known subnuclear addresses but was almost always perinucleolar. Silent copies of the viral genome were activated by growth in the DNA methylation inhibitor 5-azacytidine. HPV-immortalized keratinocytes supertransduced with HPV oncogenes and selected for marker gene coexpression underwent crisis, and the surviving cells transcribed only the newly introduced genes. Thus, transcriptional selection in response to environmental changes is a dynamic process to achieve optimal gene expression for cell survival. This phenomenon may be critical in clonal selection during carcinogenesis. Examination of HPV-associated cancers supports this hypothesis.

Cyclin/CDK Regulates the Nucleocytoplasmic Localization of the Human Papillomavirus E1 DNA Helicase

ABSTRACT Cyclin-dependent kinases (CDKs) play key roles in eukaryotic DNA replication and cell cycle progression. Phosphorylation of components of the preinitiation complex activates replication and prevents reinitiation. One mechanism is mediated by nuclear export of critical proteins. Human papillomavirus (HPV) DNA replication requires cellular machinery in addition to the viral replicative DNA helicase E1 and origin recognition protein E2. E1 phosphorylation by cyclin/CDK is critical for efficient viral DNA replication. We now show that E1 is phosphorylated by CDKs in vivo and that phosphorylation regulates its nucleocytoplasmic localization. We identified a conserved regulatory region for localization which contains a dominant leucine-rich nuclear export sequence (NES), the previously defined cyclin binding motif, three serine residues that are CDK substrates, and a putative bipartite nuclear localization sequence. We show that E1 is exported from the nucleus by a CRM1-dependent mechanism unless the NES is inactivated by CDK phosphorylation. Replication activities of E1 phosphorylation site mutations are reduced and correlate inversely with their increased cytoplasmic localization. Nuclear localization and replication activities of most of these mutations are enhanced or restored by mutations in the NES. Collectively, our data demonstrate that CDK phosphorylation controls E1 nuclear localization to support viral DNA amplification. Thus, HPV adopts and adapts the cellular regulatory mechanism to complete its reproductive program.

Electron microscopic characterization of DNAs of non-defective deletion mutants of bacteriophage Mu

We have discovered that the Mu prophage in Escherichia coli strain DK445, a λcIts857 Sam7 lac5: :Mu cI+ dilysogen (in which Mu is integrated in the lacZ gene of the plac5 prophage in the E. coli chromosome) contains a 2600 base-pair long insertion of unknown nature. The DNAs of seven independent phage Mu isolates recovered from induced DK445 have been studied by electron microscopic heteroduplex methods. All were found to contain either a deletion or a substitution in the G and/or beta regions of the chromosome. All seven mutants have growth rate, burst size and plaque morphology indistinguishable from wild-type Mu. Thus, they are the first non-defective deletion mutants of Mu. The regions deleted in these mutants are non-essential. They span the right-hand 1.3 kilobases of the G segment in the lytic orientation and the entire beta region except the rightmost 0.15 kilobase on the S (right) end. The mechanism which generates these Mu deletion mutants with high efficiency (77) is not known. The lengths of the bacterial DNA on the S end vary according to the amount of DNA inserted into or deleted from the Mu genome. Therefore, the model of headful encapsidation of Mu DNA from the c (left) end (Bukhari & Taylor, 1975) is substantiated. None of the mutants has lost the right-hand end of beta, as would have been anticipated by straight-forward headful packaging from the c end of prophage with a sizeable insertion. Therefore, the right-hand end of beta, one of its normal attachment sites, is probably essential for Mu viability. The identification of a Mu mutant with a substitution in beta which is incapable of promoting inversion of the G segment suggests that part of the G segment inversion system might be located on the left portion of the beta region. The G segment inverts normally in the Mu prophage in DK445. Yet, all seven deletion mutants have the G segment arranged in the lytic orientation. These observations suggest that the orientation of Mu G DNA may affect the phage viability and that there may be essential functions encoded within the left-hand 1.7 kilobases of the G segment in the lytic orientation.

Human Papillomavirus E6E7-Mediated Adenovirus Cell Killing: Selectivity of Mutant Adenovirus Replication in Organotypic Cultures of Human Keratinocytes

ABSTRACT Replication-competent adenoviruses are being investigated as potential anticancer agents. Exclusive virus replication in cancer cells has been proposed as a safety trait to be considered in the design of oncolytic adenoviruses. From this perspective, we have investigated several adenovirus mutants for their potential to conditionally replicate and promote the killing of cells expressing human papillomavirus (HPV) E6 and E7 oncoproteins, which are present in a high percentage of anogenital cancers. For this purpose, we have employed an organotypic model of human stratified squamous epithelium derived from primary keratinocytes that have been engineered to express HPV-18 oncoproteins stably. We show that, whereas wild-type adenovirus promotes a widespread cytopathic effect in all infected cells, E1A- and E1A/E1B-deleted adenoviruses cause no deleterious effect regardless of the coexpression of HPV18 E6E7. An adenovirus deleted in the CR2 domain of E1A, necessary for binding to the pRB family of pocket proteins, shows no selectivity of replication as it efficiently kills all normal and E6E7-expressing keratinocytes. Finally, an adenovirus mutant deleted in the CR1 and CR2 domains of E1A exhibits preferential replication and cell killing in HPV E6E7-expressing cultures. We conclude that the organotypic keratinocyte culture represents a distinct model to evaluate adenovirus selectivity and that, based on this model, further modifications of the adenovirus genome are required to restrict adenovirus replication to tumor cells.

The Hinge of the Human Papillomavirus Type 11 E2 Protein Contains Major Determinants for Nuclear Localization and Nuclear Matrix Association

ABSTRACT The E2 protein of papillomaviruses is a site-specific DNA binding nuclear protein. It functions as the primary replication origin recognition protein and assists in the assembly of the preinitiation complex. It also helps regulate transcription from the native viral promoter. The E2 protein consists of an amino-terminal (N)trans-acting domain, a central hinge (H) domain, and a carboxyl-terminal (C) protein dimerization and DNA binding domain. The hinge is highly divergent among papillomaviruses, and little is known about its functions. We fused the enhanced green fluorescent protein (GFP) with the full-length human papillomavirus type 11 (HPV-11) E2 protein and showed that the resultant fusion, called gfpE2, maintained transcription and replication functions of the wild-type protein and formed similar subnuclear foci. Using a series of GFP fusion proteins, we showed that the hinge conferred strong nuclear localization, whereas the N or C domain was present in both cytoplasm and nucleus. Biochemical fractionation demonstrated that the N domain and hinge, but not the C domain, independently associated with the nuclear matrix. Mutational analyses showed that a cluster of basic amino acid residues, which is conserved among many mucosotropic papillomaviruses, was required for efficient nuclear localization and nuclear matrix association. This mutation no longer repressed the HPV-11 upstream regulatory region-controlled reporter expression. However, a very small fraction of this mutant colocalized with E1 in the nucleus, perhaps by a piggyback mechanism, and was able to support transient replication. We propose that the hinge is critical for the diverse regulatory functions of the HPV-11 E2 protein during mRNA transcription and viral DNA replication.

Post-transcriptional induction of p21cip1 protein by human papillomavirus E7 inhibits unscheduled DNA synthesis reactivated in differentiated keratinocytes.

Productive infection by human papillomaviruses (HPVs) occurs only in differentiated squamous epithelial cells in papillomas, condylomata, and low grade intraepithelial neoplasias. Host DNA replication is reactivated in a fraction of terminally differentiated keratinocytes in benign human lesions and in organotypic raft cultures of primary human keratinocytes (PHKs) transduced with retroviruses expressing HPV-18 E7 oncogene from its native upstream regulatory region (URR). Thus the natural function of E7 protein, which inactivates pRB family proteins, is to induce host genes essential to support viral DNA replication in post-mitotic cells. Using this raft culture model system, we show that HPV-18 URR-E7 induces the universal cyclin-dependent kinase inhibitor p21cip1 protein in a fraction of differentiated PHKs. Induction is mediated by post-transcriptional mechanisms independent of p53. Double immunofluorescence studies demonstrate that, in raft cultures and in laryngeal papillomas, p21cip1 induction and reactivated host DNA synthesis take place in a mutually exclusive manner in PCNA-positive, differentiated keratinocytes. We suggest that p21cip1 induction effectively blocks unscheduled DNA synthesis reactivated by E7. These results begin to explain the inverse relationship between p21cip1 induction and HPV activities previously observed in a spectrum of benign lesions regardless of HPV types present.