Laimonis A. Laimins

Microarray Analysis Identifies Interferon-Inducible Genes and Stat-1 as Major Transcriptional Targets of Human Papillomavirus Type 31

ABSTRACT 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.

Human papillomavirus E6 proteins bind p53 in vivo and abrogate p53-mediated repression of transcription.

The transforming proteins of DNA tumor viruses SV40, adenovirus and human papillomaviruses (HPV) bind the retinoblastoma and p53 cell cycle regulatory proteins. While the binding of SV40 large T antigen and the adenovirus E1B 55 kDa protein results in the stabilization of the p53 protein, the binding of HPV16 and 18 E6 results in enhanced degradation in vitro. To explore the effect of viral proteins on p53 stability in vivo, we have examined cell lines immortalized in tissue culture by HPV18 E6 and E7 or SV40 large T antigen, as well as cell lines derived from cervical neoplasias. The half‐life of the p53 protein in non‐transformed human foreskin keratinocytes in culture was found to be approximately 3 h while in cell lines immortalized by E6 and E7, p53 protein half‐lives ranged from 2.8 h to less than 1 h. Since equivalent levels of E6 were found in these cells, the range in p53 levels observed was not a result of variability in amounts of E6. In keratinocyte lines immortalized by E7 alone, the p53 half‐life was found to be similar to that in non‐transformed cells; however, it decreased to approximately 1 h following supertransfection of an E6 gene. These observations are consistent with an interaction of E6 and p53 in vivo resulting in reductions in the stability of p53 ranging between 2‐ and 4‐fold. We also observed that the expression of various TATA containing promoters was repressed in transient assays by co‐transfection with plasmids expressing the wild‐type p53 gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Telomerase Activation by Human Papillomavirus Type 16 E6 Protein: Induction of Human Telomerase Reverse Transcriptase Expression through Myc and GC-Rich Sp1 Binding Sites

ABSTRACT High-risk human papillomaviruses (HPVs) immortalize keratinocytes by disrupting the retinoblastoma protein (Rb)/p16 pathway and activating telomerase. The E7 oncoprotein targets Rb, while the E6 oncoprotein induces telomerase activity in human keratinocytes. This study has examined the mechanism by which E6 activates telomerase. Expression of human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, was found to be increased in keratinocytes stably expressing HPV type 16 E6, suggesting that E6 acts to increase hTERT transcription. hTERT expression and telomerase activity were activated to significantly higher levels in cells expressing both E6 and E7 than in cells expressing E6 alone. This indicates that E7 may augment E6-mediated activation of hTERT transcription. In transient-transfection assays using hTERT reporters, the induction of hTERT expression by E6 was found to be mediated by a 258-bp fragment of the hTERT promoter, proximal to the ATG initiation codon. Previous studies have demonstrated that overexpression of Myc can activate hTERT expression, suggesting that Myc may be a mediator of E6-mediated hTERT induction. However, in cells stably expressing E6, no strict correlation between the level of Myc and the activation of hTERT was found. Consistent with this observation, mutation of the two Myc binding sites in the hTERT promoter only modestly reduced responsiveness to E6 in transient reporter assays. This indicates that activation of Myc-dependent transcription is not essential for E6-mediated upregulation of hTERT expression. The hTERT promoter also contains five GC-rich elements that can bind Sp1. Mutation of these sites within the 258-bp fragment partially reduced hTERT induction by E6. However, when mutations in the Sp1 sites were combined with the mutated Myc binding sites, all activation by E6 was lost. This indicates that it is the combinatorial binding of factors to Myc and Sp1 cis elements that is responsible for hTERT induction by E6.

The E8E2C protein, a negative regulator of viral transcription and replication, is required for extrachromosomal maintenance of human papillomavirus type 31 in keratinocytes.

ABSTRACT The viral E2 protein is a major regulator of papillomavirus DNA replication. An important way to influence viral replication is through modulation of the activity of the E2 protein. This could occur through the action of truncated E2 proteins, called E2 repressors, whose role in the replication cycle of human papillomaviruses (HPVs) has not been determined. In this study, using cell lines that contain episomal copies of the “high-risk” HPV type 31 (HPV31), we have identified viral transcripts with a splice from nucleotide (nt) 1296 to 3295. These transcripts are similar to RNAs from other animal and human papillomaviruses and have the potential to fuse a small open reading frame (E8) to the C terminus of E2, resulting in an E8u2009̂E2C fusion protein. E8u2009̂E2C transcripts were present throughout the complete replication cycle of HPV31. A genetic analysis of E8u2009̂E2C in the context of the HPV31 genome revealed that mutation of the single ATG of the E8 gene, introduction of a stop codon downstream of the ATG, or disruption of the splice donor site at nt 1296 led to a dramatic 30- to 40-fold increase in the transient DNA replication levels in both normal and immortalized human keratinocytes. High-level expression of E8u2009̂E2C from heterologous vectors was found to inhibit E1-E2-dependent DNA replication of an HPV31 origin of replication construct as well as to interfere with E2s ability to transactivate reporter gene constructs. In addition, HPV31 E8u2009̂E2C strongly repressed the basal activity of the major viral early promoter P97 independent of E2. E8u2009̂E2C may therefore exert its negative effect on viral DNA replication through modulating E2s ability to enhance E1-dependent DNA replication as well as by regulating viral gene expression. Surprisingly, HPV31 genomes that were unable to express E8u2009̂E2C could not be maintained extrachromosomally in human keratinocytes in long-term assays despite high transient DNA replication levels. This suggests that the E8u2009̂E2C protein may play a role in copy number control as well as in the stable maintenance of HPV episomes.

In Vitro Systems for the Study and Propagation of Human Papillomaviruses

Papillomaviruses were first shown to be pathogenic agents by Shope and Hurst (1933) who identified them as the causative agent of infectious papillomatosis of cottontail rabbits. Later, Rous and Beard (1935) observed that benign papillomas of rabbits induced by this virus could progress to carcinomas. Attempts to develop systems for the propagation of papillomaviruses in vitro were unsuccessful and resulted in a dormancy of papillomavirus research until the 1970s. The late 1970s brought the beginning of molecular biology technology and the cloning of papillomavirus genomes. This allowed for the isolation of sufficient quantities of material to begin a systematic study of papillomaviruses. In the 1980s a correlation between papillomaviruses and human neoplastic lesions of the anogenital area led to a significant increase in interest in the study of these viruses.