Human papillomaviruses (HPVs) infect keratinocytes and induce proliferative lesions. In infected cells, viral gene products alter the activities of cellular proteins, such as Rb and p53, resulting in altered cell cycle response. It is likely that HPV gene products also alter expression of cellular genes. In this study we used microarray analysis to examine the global changes in gene expression induced by high-risk HPV type 31 (HPV31). Among 7,075 known genes and ESTs (expressed sequence tags) tested, we found that 178 were upregulated and 150 were downregulated twofold or more in HPV31 cells compared to normal human keratinocytes. While no specific pattern could be deduced from the list of genes that were upregulated, downregulated genes could be classified to three groups: genes that are involved in the regulation of cell growth, genes that are specifically expressed in keratinocytes, and genes whose expression is increased in response to interferon stimulation. The basal level of expression of several interferon-responsive genes was found to be downregulated in HPV31 cells by both microarray analysis and Northern blot analysis in different HPV31 cell lines. When cells were treated with alpha or gamma interferon, expression of interferon-inducible genes was impaired. At high doses of interferon, the effects were less pronounced. Among the genes repressed by HPV31 was the signal transducer and activator of transcription (Stat-1), which plays a major role in mediating the interferon response. Suppression of Stat-1 expression may contribute to a suppressed response to interferon as well as immune evasion.

No abstract

Earlier studies have shown that the U L 31 protein is homogeneously distributed throughout the nucleus and cofractionates with nuclear matrix. We report the construction from an appropriate cosmid library a deletion mutant which replicates in rabbit skin cells carrying the U L 31 gene under a late (␥ 1 ) viral promoter. The mutant virus exhibits cytopathic effects and yields 0.01 to 0.1% of the yield of wild-type parent virus in noncomplementing cells but amounts of virus 10-to 1,000-fold higher than those recovered from the same cells 3 h after infection. Electron microscopic studies indicate the presence of small numbers of full capsids but a lack of enveloped virions. Viral DNA extracted from the cytoplasm of infected cells exhibits free termini indicating cleavage/packaging of viral DNA from concatemers for packaging into virions, but analyses of viral DNAs by pulsed-field electrophoresis indicate that at 16 h after infection, both the yields of viral DNA and cleavage of viral DNA for packaging are decreased. The repaired virus cannot be differentiated from the wild-type parent. These results suggest the possibility that U L 31 protein forms a network to enable the anchorage of viral products for the synthesis and/or packaging of viral DNA into virions. MATERIALS AND METHODS Cells and viruses. Rabbit skin cells and human 143TKϪ cells were obtained from John McLaren and Carlo Croce, respectively; Vero cells were obtained from American Type Culture Collection. Human lung fibroblast (HLF) cells were obtained from George Kemble (Aviron, Mountain View, Calif.). Cells were maintained in Dulbecco's modified Eagle medium supplemented with either 5% newborn calf serum (rabbit skin cells and Vero cells), 5% fetal bovine serum (143TK Ϫ cells), or 10% fetal bovine serum (HLF cells). Infected cells were maintained in medium consisting of mixture 199 supplemented with 1% calf serum (199V). Virus titer was measured on infected cells maintained in 199V plus 0.2% human immunoglobulin. RD14 and RD17 U L 31-expressing cell lines were two independent clones derived from rabbit skin cells and maintained in 10% fetal bovine serum. Herpes simplex virus 1 strain F [HSV-1(F)], a limitedpassage virus, serves as the prototype HSV-1 strain in this laboratory (16).Generation of U L 31 polyclonal antibody. A DNA fragment encoding U L 31 codons 1 to 163 was cloned into an Escherichia coli anthranilate synthetase (TrpE) expression vector (22). The fusion protein was induced after 2-h logphase growth of transformed E. coli with 20 g of indoleacrylic acid per ml. After 4 h of additional incubation, the bacteria were resuspended in lysis buffer (50 mM Tris-HCl [pH 7.5], 5 mM EDTA, 3 mg of lysozyme per ml) and stored on ice for 2 h. The lysate was then adjusted to 0.3 M NaCl and 0.65% Nonidet P-40 and centrifuged in a Sorvall SS34 rotor at 10,000 rpm for 10 min. The pellet was solubilized in disruption buffer (50 mM Tris-HCl [pH 7.0], 2% sodium dodecyl sulfate [SDS], 0.7 M ␤-mercaptoethanol, 2.75% sucrose) and subjected to electrophoresis in a ...

The long-term effects of interferon treatment on cell lines that maintain human papillomavirus type 31 (HPV-31) episomes have been examined. High doses and prolonged interferon treatment resulted in growth arrest of HPV-positive cells, with a high percentage of cells undergoing apoptosis. These effects were not seen with interferon treatment of either normal human keratinocytes or cells derived from HPV-negative squamous carcinomas, which exhibited only slight decreases in their rates of growth. Within 2 weeks of the initiation of treatment, a population of HPV-31-positive cells that were resistant to interferon appeared consistently and reproducibly. The resistant cells had growth and morphological characteristics similar to those of untreated cells. Long-term interferon treatment of HPV-positive cells also resulted in a reduction in HPV episome levels but did not significantly decrease the number of integrated copies of HPV. Cells that maintained HPV genomes lacking E5 were sensitive to interferon, while cells expressing only the E6/E7 genes were resistant. In contrast, cells that expressed E2 from a tetracycline-inducible promoter were found to be significantly more sensitive to interferon treatment than parental cells. This suggests that at least a portion of the sensitivity to interferon could be mediated through the E2 protein. These studies indicate that cells maintaining HPV episomes are highly sensitive to interferon treatment but that resistant populations arise quickly.Human papillomaviruses (HPV) are small double-stranded DNA viruses that infect epithelial tissues. More than 85 subtypes have been identified, and each of these types exhibits strict tissue specificity (13). About one-third of HPV types infect the anogenital epithelia and induce the most common form of sexually transmitted disease (71). HPV infect cells in the basal layer of epithelia and establish a latent infection in these cells. Production of HPV virions, however, requires infected cells to migrate away from the basal layer and undergo differentiation (28,31,71). The HPV that infect the anogenital region can be divided into high-risk and low-risk HPV types depending on their association with malignancy. The low-risk HPVs, such as HPV type 6 (HPV-6) and HPV-11, cause hyperproliferative lesions of external genitalia and are rarely associated with malignancies. In contrast, the high-risk HPVs, , are the etiological agents of cervical cancer (28,31,35,71). The difference in clinical outcome between low-and high-risk HPV infections has been an area of major research interest.The genomes of all genital HPV types encode 8 to 10 proteins. In the high-risk HPVs, E6 and E7 function as oncogenes. E6 binds to the cellular ubiquitin ligase, E6AP, which then targets p53 for degradation (29,53,54,67). In addition, E6 activates the expression of htert, the catalytic subunit of telomerase. E7 binds to and inhibits the activity of retinoblastoma protein, pRB, and promotes the constitutive activation of E2F family members (9,16,38,44). The E1 and E2 g...

The nucleotide sequence of the UL31 open reading frame is predicted to encode a basic protein with a hydrophilic amino terminus and a nuclear localization signal. To identify its gene product, we constructed a viral genome in which the thymidine kinase gene was inserted between the UL31 and UL32 open reading frames. The thymidine kinase gene was then deleted, and in the process, the 5' terminus of the UL31 open reading frame was replaced with a 64-bp sequence in frame with the complete, authentic sequence of the UL31 open reading frame. The inserted sequence encoded a hydrophilic epitope derived from glycoprotein B of human cytomegalovirus and for which a monoclonal antibody is available. We report that in infected cells, the tagged protein localized in and was dispersed throughout the nucleus. Nuclear fractionation studies revealed that the UL31 protein partitions with the nuclear matrix. The protein is phosphorylated in infected cells maintained in medium containing 32pi. According to current counts, the herpes simplex virus 1 (HSV-1) genome encodes 77 open reading frames. Of this number, 58 are in the unique sequences of the long component (UL) (2, 19, 22), 13 are in the unique sequences of the short component (Us) (12, 23), and 3 map in the inverted repeats ab and ca, which flank UL and US sequences (1, 11), respectively. Approximately half of the genes encoded by the virus are dispensable for growth in cells in culture, and the functions of many genes, whether dispensable for growth in cells in culture or essential, are not known (36). The focus of this report is on one of a stretch of essential genes mapping between 0.4 and 0.5 map units (Fig. 1). At the left terminus of this stretch are a small portion of the UL29 gene, which encodes the singlestranded-DNA-binding protein ICP8, and the UL30 gene, which encodes the viral DNA polymerase. Near the right terminus of this stretch are three genes encoding virion proteins; one of these, encoded by U134, is one of the few nonglycosylated, essential membrane phosphoproteins (33, 34). Little is known about the products of the other genes. This report concerns the protein product of the UL31 open reading frame, which is predicted to contain 306 amino acids. The U,L31 transcript has been mapped by Holland et al. (17), and its synthesis has been shown to be restricted in the presence of 1-,3-D-arabinofuranosylthymine, an inhibitor of viral DNA synthesis. Attempts to delete the UL31 open reading frame have not been successful (la, 9a). In this report, we provide the first characterization of the gene product. The results presented here indicate that the gene product is a nuclear phosphoprotein which shares properties with proteins known to form the nuclear matrix. MATERIALS AND METHODS Cells and viruses. HSV-1 strain F [HSV-1(F)] is the prototype HSV-1 strain used in this laboratory. HSV-1(F)A305 was derived from HSV-1(F) by deleting approximately 500 bp from the thymidine kinase (tk) gene (30) and is the parent strain for * Corresponding author. all of the viral recomb...