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L A Laimins

Publications and source records attributed to L A Laimins.

At least 37 records · Page 2Linked to original sources
PubMed1997

The 5' region of the human papillomavirus type 31 upstream regulatory region acts as an enhancer which augments viral early expression through the action of YY1.

Cis-elements which control human papillomavirus early gene expression have previously been localized to sequences in the upstream regulatory region (URR) which are proximal to the E6 open reading frame. These elements include an enhancer element which functions preferentially in keratinocytes as well as promoter elements. The function of the remaining approximate 500-bp region of the URR in regulating viral expression in the high risk papillomaviruses has been largely uncharacterized. In HPV 6, a negative regulator of early expression, or silencer, has been identified in this 5' region of the URR. In this study, we have investigated the role of the 5' portion of the HPV 31 URR in regulating viral expression. Sequences in this region were found to exert a minimal negative effect on homologous or heterologous promoters. In contrast, a 128-bp sequence within this region was found to exhibit enhancer activity on heterologous and homologous promoters. This enhancer also augmented the activity of the previously identified minimum keratinocyte enhancer. The cellular factors, YY1 and TEF-1, were determined by DNase I footprint analysis and electrophoretic mobility shift assays (EMSAs) to bind the 128-bp enhancer. The binding of YY1 to two of these sites was found to be important for enhancer activity.

Base Sequence↗
PubMed1997

Abrogation of a mitotic checkpoint by E2 proteins from oncogenic human papillomaviruses correlates with increased turnover of the p53 tumor suppressor protein.

Human papillomavirus (HPV) E2 and E1 proteins are required for the replication of viral genomes in vivo. We have examined the effects of increasing the level of E2 on viral and cellular replication using recombinant adenoviruses. Infection of cells which maintain HPV 31 DNA episomally with E2 recombinant adenoviruses resulted in a 5-fold increase in genome copy number as well as an S phase arrest allowing for the continued replication of cellular DNA. Similar effects on cell cycle progression were seen following infection of normal human foreskin keratinocytes, the natural host cell. The DNA content of these cells increased beyond 4N indicating that multiple rounds of replication had occurred without an intervening mitotic event. In addition, increased cyclin A and E associated kinase activity was observed, while no change was detected in cyclin B associated kinase activity or in the activation state of cdc2 kinase. Interestingly, the levels of the p53 tumor suppresser protein were dramatically reduced through a post-transcriptional mechanism following infection. These data suggest a role for E2 in regulating viral and cellular replication by abrogation of a mitotic checkpoint, which is, at least in part, controlled by p53.

Adenoviridae↗
PubMed1997

Expression of AP1 during cellular differentiation determines human papillomavirus E6/E7 expression in stratified epithelial cells.

E6 and E7 oncoproteins of human papillomavirus (HPV) play significant roles in the pathogenesis of cervical cancer. However, the pattern of E6/E7 expression during the productive virus life cycle in differentiating epithelia of the uterine cervix remains unclear. In addition, little is known about the cellular factors regulating E6/E7 expression in differentiating epithelia. In the present study, using transient expression assays and DNA binding assays, we demonstrated that E6/E7 transcription is critically regulated by the cellular factor AP1, a Jun/Fos heterodimer complex. Immunohistochemical analyses of various uterine cervical lesions showed AP1 expression in lower cell layers of normal cervix and low-grade cervical intraepithelial neoplasia (CIN), while it was detected throughout all layers in high-grade CIN and invasive cancer. In situ RNA-RNA hybridization analyses of organotypic raft culture specimens of an HPV-31-containing cell line revealed that E6/E7 transcripts were expressed in most cell layers, with reduced expression in differentiated cells. This pattern of HPV expression correlated with the pattern of AP1 expression detected by immunohistochemical analyses. These findings suggest that E6/E7 expression in differentiating epithelia is dependent on AP1, which appears to be associated with proliferative activity of the cells. Since E6/E7 expression induces cell proliferation, co-expression of AP1 and E6/E7 in undifferentiated cell layers might create a positive regulatory loop, probably contributing to maintenance of initial HPV infection and subsequent activation in basal and suprabasal cell layers.

Binding Sites↗
PubMed1997

Differential effects of the splice acceptor at nucleotide 3295 of human papillomavirus type 31 on stable and transient viral replication.

In human papillomavirus type 31 (HPV-31), the E1--E4 and E5 open reading frames are expressed from polycistronic mRNAs. The major polycistronic mRNAs which encode E1--E4 and E5 are spliced messages which utilize a splice acceptor at nucleotide (nt) 3295 (SPA3295). Our laboratory recently developed a recombinant system for the synthesis of HPVs following immortalization of primary keratinocytes with cloned HPV-31 genomes (M. G. Frattini et al., Proc. Natl. Acad. Sci. USA 93:3062-3067, 1996). These immortalized cell lines are capable of maintaining HPV-31 DNA as episomes and induce the synthesis of virions in organotypic raft culture. In this study, we used these methods to begin an analysis of the roles of E1--E4 and E5 in HPV pathogenesis by mutating the major splice at nt 3295. Mutation of SPA3295 did not significantly alter the ability of HPV-31 genomes to replicate transiently in keratinocytes, nor did the mutation affect the immortalization potential of HPV-31. However, genomes carrying the SPA3295 mutation were not stably maintained as viral episomes, and the resulting immortalized keratinocyte cell line contained multiple, integrated copies of the mutated HPV-31 DNA. Northern analysis indicated that cell lines immortalized with the mutant HPV-31 expressed transcripts which were similar in size and abundance to wild-type messages, including those transcripts which rely on utilization of SPA3295. RNase protection and reverse transcription-PCR revealed that mutation of SPA3295 resulted in the utilization of a cryptic splice acceptor at nt 3298. These data suggest that the requirements for stable maintenance of HPV genomes are more stringent than those for transient replication and that factors which define these requirement rely on the major splice acceptor at nt 3295.

Alternative Splicing↗
PubMed1997

Initiation of DNA synthesis by human papillomavirus E7 oncoproteins is resistant to p21-mediated inhibition of cyclin E-cdk2 activity.

The E6 and E7 proteins from the high-risk human papillomaviruses (HPVs) bind and inactivate the tumor suppressor proteins p53 and Rb, respectively. In HPV-positive cells, expression of E6 proteins from high-risk types results in increased turnover of p53, which leads to an abrogation of p21-mediated G1/S arrest in response to DNA-damaging agents. In contrast, keratinocytes which express E7 alone have increased levels of p53 but, interestingly, also fail to undergo a G1/S arrest. We investigated the mechanism by which E7 bypasses this p21 arrest by using both keratinocytes which stably express E7 as well as U20S cells which stably or transiently express E7. We observed that E7 does not affect the induction of p21 synthesis by p53. While glutathione S-transferase (GST)-E7 bound a low level of in vitro-translated p21, we were unable to detect E7 and p21 in the same complex by GST-E7 binding assays or immunoprecipitations from cell extracts. Furthermore, E7 did not prevent p21-mediated inhibition of cyclin E kinase activity. In keratinocytes expressing E7, increased levels of p53, p21, and cyclin E, as well as increased cyclin E kinase activity, were observed. To determine if this increase in cyclin E activity was necessary for E7's ability to overcome p21-mediated G1/S arrest, we examined U20S cells in which cyclin E levels are not increased in response to E7 expression. U20S cells which stably express E7 were found to initiate DNA synthesis in the presence of DNA-damaging agents despite the inhibition of cyclin E activity by p21. In transient assays, cotransfection of E7 or E2F-1 along with p21 into U20S cells rescued G1 arrest and resulted in S-phase entry, as measured by the ability to incorporate bromodeoxyuridine. These data indicate that E7 is able to overcome G1/S arrest without directly affecting p21 function and likely acts through deregulation of E2F activity.

CDC2-CDC28 Kinases↗
PubMed1997

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.

Capsid↗
PubMed1996

In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation-dependent late expression.

Human papillomavirus (HPV) types 16, 18, 31, and 51 are the etiologic agents of many anogenital cancers including those of the cervix. These "high risk" HPVs specifically target genital squamous epithelia, and their lytic life cycle is closely linked to epithelial differentiation. We have developed a genetic assay for HPV functions during pathogenesis using recircularized cloned HPV 31 genomes that were transfected together with a drug resistance marker into monolayer cultures of normal human foreskin keratinocytes, the natural host cell. After drug selection, cell lines were isolated that stably maintained HPV 31 DNA as episomes and underwent terminal differentiation when grown in organotypic raft cultures. In differentiated rafts, the expression of late viral genes, amplification of viral DNA, and production of viral particles were detected in suprabasal cells. This demonstrated the ability to synthesize HPV 31 virions from transfected DNA templates and allowed an examination of HPV functions during the vegetative viral life cycle. We then used this system to investigate whether an episomal genome was required for the induction of late viral gene expression. When an HPV 31 genome (31E1*) containing a missense mutation in the E1 open reading frame was transfected into normal human keratinocytes, the mutant viral sequences were found to integrate into the host cell chromosomal DNA with both early and late regions intact. While high levels of early viral gene transcription were observed, no late gene expression was detected in rafts of cell lines containing the mutant viral genome despite evidence of terminal differentiation. Therefore, the induction of late viral gene expression required that the viral genomes be maintained as extrachromosomal elements, and terminal differentiation alone was not sufficient. These studies provide the basis for a detailed examination of HPV functions during viral pathogenesis.

Blotting, Northern↗
PubMed1996

Human papillomavirus E7 oncoproteins bind a single form of cyclin E in a complex with cdk2 and p107.

The E6 and E7 proteins of the high-risk human papillomaviruses (HPVs) act coordinately to immortalize human keratinocytes. These viral oncoproteins function by binding and altering the activity of cellular proteins which regulate cell cycle progression. Among the proteins bound by E7 are the retinoblastoma protein, Rb, as well as the related p107 and p130 proteins. In addition, E7 binds cyclin A, which regulates transit through the S and G2/M phases of the cell cycle. In this study, we demonstrate that HPV 18 E7 also associates with cyclin E which controls the G1/S transition. E7/cyclin E complexes were immunoprecipitated from E7-expressing cells as well as from cell extracts using GST-E7 fusion proteins. E7 was found to complex with a single form of cyclin E, and the binding was mediated through p107. Both E7/cyclin E and E7/cyclin A complexes exhibit kinase activity through associated cdk2 proteins which can contribute to phosphorylation of p107. The association of E7 with proteins which regulate transit through the cell cycle may provide an additional mechanism by which infection with human papillomaviruses results in cellular hyperproliferation.

CDC2-CDC28 Kinases↗
PubMed1995

Transcriptional activity of human papillomavirus type 31b enhancer is regulated through synergistic interaction of AP1 with two novel cellular factors.

Transcription of human papillomaviruses (HPV) is regulated by enhancer sequences located in the upstream regulatory region. The factors regulating expression of one of the high risk genital HPV types, HPV 31b, were investigated using transient expression and protein binding assays. A region of 262 base pairs in length was identified as the minimal functional enhancer and a series of five protected binding sites were observed by footprint analyses. Electrophoretic mobility shift assays demonstrated that AP1, Oct-1, as well as three novel factors bound these sequences. Mutational analyses indicated that AP1 synergistically activated the HPV31b enhancer together with either of two novel factors. One of these novel factors bound a sequence similar to an NF1 site but was distinct from NF-1. The second factor bound sequences bearing similarity to KRF-1 binding sites which have previously been characterized in HPV 18. Competition binding assays demonstrated that this factor was not identical to KRF-1. Additional studies implicated Oct-1 as a negative regulator of HPV 31b expression as mutation of Oct-1 binding sequences resulted in an increase in viral expression. None of the factors observed to be important for HPV 31b enhancer activity was found exclusively in epithelial cells and instead were detected in a variety of cell types. Of these factors, AP1 binding correlated most strongly with enhancer function in a variety of cell types, implicating it as a principal regulator of HPV expression. Variations in the constituents of the AP1 complex that bind the HPV 31b enhancer were also observed in different cell types, suggesting that changes in the distribution of jun proteins may play a significant role in determining the tropism of HPV. These results indicate that AP1 may be a common regulator for various HPV types and that it contributes to enhancer specificity. In addition, a set of novel factors, which may be specific for each HPV type, act synergistically with AP1 for full activation of the enhancer.

Base Sequence↗
PubMed1995

E6 and E7 expression from the HPV 18 LCR: development of genital hyperplasia and neoplasia in transgenic mice.

Human papillomavirus type 18 infection is highly associated with malignant tumors of the genital tract. To investigate the tissue specificity of the HPV long control region (LCR) and the transforming ability of the E6-E7 oncoproteins, an HPV-18 transgene containing the viral LCR and E6 and E7 genes was introduced into mice. Three founder males exhibited enlarged seminal vesicles and preputial glands by 50 weeks of age. A line of transgenic mice was established by in vitro fertilization, and subsequent generations of transgenic males and females were monitored for lesions. Approximately 80% of hemizygous transgenic males exhibited enlarged seminal vesicles and preputial glands as early as 12 weeks of age. Histological examination indicated that this enlargement was due to distension by fluid, along with polyploid hyperplasia of the lining secretory epithelium. E6 and E7 transcripts were limited to affected organs and kidney. Approximately 41% of transgenic females developed cervical neoplasms between 1-2 years of age. Histologically, tumors were mesenchymal rather than epithelial in origin. E6 and E7 transcripts were restricted to cervical tumor tissue and kidney. These findings suggest that the HPV-18 LCR has an element(s) which directs expression specifically to the urogenital tract in transgenic mice.

Animals↗
PubMed1995

Differentiation-dependent expression of E1--E4 proteins in cell lines maintaining episomes of human papillomavirus type 31b.

The life cycle of human papillomaviruses (HPVs) is dependent on epithelial differentiation. Among the viral proteins expressed in differentiated epithelial cells are the viral capsid proteins, L1 and L2, as well as the E1E4 fusion proteins. In this study, the expression and intracellular localization of the E1E4 proteins of HPV type 31b were examined in both monolayer and raft cultures of the CIN-612 cell line which maintains episomal copies of HPV-31b. In this cell line, a high level of E1E4 protein expression was observed in the cytoplasm of a small percentage of cells in monolayer culture. A large increase in E1E4 protein levels was observed upon stratification of the CIN-612 cell line in raft cultures, with E1E4 protein expression limited to the uppermost layers of the epithelium. A diffuse, slightly grainy cytoplasmic localization of E1E4 protein was observed in both monolayer and raft culture systems. Although virion synthesis is entirely dependent upon phorbol ester or synthetic diacylglycerol treatment of raft cultures, E1E4 expression was observed in both treated and untreated monolayer and raft cultures of the CIN-612 cell line. In monolayer cultures of two simian virus 40-transformed cell lines, cos-7 and MK-6, transiently transfected with an E1E4 expression vector, the distribution of E1E4 protein was found to differ substantially from that in the CIN-612 cells. In these cell lines E1E4 protein was found to exhibit a total collapse into either cytoplasmic inclusion granules in the cos-7 cells or a perinuclear halo-like structure in the MK-6 cell line. The host cell, its differentiation state, and the amount of expression can therefore significantly affect the distribution of the E1E4 proteins.

Animals↗
PubMed1995

Human papillomavirus type 31b late gene expression is regulated through protein kinase C-mediated changes in RNA processing.

Expression of the human papillomavirus (HPV) capsid genes, L1 and L2, as well as amplification of viral DNA and virion assembly occur in the terminally differentiated layers of infected stratified squamous epithelium in vivo. These processes can be duplicated in the laboratory through the use of organotypic or raft cultures. When CIN612 cells, which contain episomal copies of the high-risk HPV type 31b, are allowed to differentiate in raft cultures, the expression of transcripts encoding the early genes E1--E4 and E5 is induced. These transcripts are initiated at the differentiation-dependent P742 promoter located in the middle of the E7 open reading frame. Exposure of raft cultures to activators of protein kinase C, such as phorbol esters, results in the further induction of late gene expression as well as virion assembly. In this study, we have investigated the mechanism by which activators of protein kinase C induce late gene expression. The major L1 transcript was found to be encoded by a bicistronic E1--E4, L1 RNA which initiated at the differentiation-dependent promoter P742. Additional low-level expression of L1-containing RNAs was also observed from the early-region promoter, P97. The major L2 transcripts were found to be encoded by E1--E4, E5, L2, L1 RNAs which were also initiated in the early region, probably at the differentiation-specific promoter P742. While early and late RNAs were found to be expressed from the same promoter, they differed in utilization of splicing and polyadenylation sites. Raft cultures treated with activators of protein kinase C induced expression of late genes, but no change in the abundance of early RNAs initiated at the P742 promoter was observed. Thus, the increase in late gene expression was likely due to changes in RNA processing or stabilization rather than an increase in the rate of transcription from P742. Regulation of HPV late gene expression therefore occurs at two levels: differentiation-dependent induction of the P742 promoter, which can be mimicked in vitro by growth in raft cultures, and posttranscriptional changes that can be induced by activation of protein kinase C. These posttranscriptional changes may occur through inactivation or down-regulation of splicing factors which inhibit use of the late region polyadenylation site, resulting in increased stability of late region transcripts.

Cells, Cultured↗
PubMed1994

Binding of the human papillomavirus E1 origin-recognition protein is regulated through complex formation with the E2 enhancer-binding protein.

The papillomavirus E1 and E2 proteins form heteromeric complexes and individually bind specific sequences within the viral origin of replication. The mechanism by which these proteins are recruited to the origin and the role of the E1/E2 complex in replication remain undefined. To examine the interplay of these replication proteins, we have analyzed the binding of human papillomavirus (HPV) type 31b E1 and E2 proteins to the origin of replication. Binding of E1 to the origin was increased by E2 proteins and required the presence of E2 binding sites. This increase was due to the formation of E1/E2 complexes which preferentially bound E2-responsive sequences, and the magnitude was determined by the relative affinity of the E2 binding sites. While the E1 protein alone bound an A/T-rich sequence at the HPV-31b origin with low affinity, complexes of E1 and E2 bound instead to E2 binding sites with high affinity. The E1/E2 complex bound a similar sequence as E2 homodimers, but only E2 homodimer binding induced a significant increase in hypersensitivity as indicated by DNase I footprinting. In the presence of excess E1, E1 proteins bound both the A/T-rich sequence and E2 binding sites through complex formation with E2. In E2 excess, E1/E2 complexes preferentially formed, and binding was specific for E2 sites. Therefore, changes in the relative amounts of E1 and E2 proteins can dramatically alter the pattern of binding of viral replication factors to the origin. These observations suggest a model whereby modulation of the relative levels of E1 and E2 during the viral life cycle may alter the pattern of origin binding and possibly episomal copy number.

Base Sequence↗
PubMed1994

The role of the E1 and E2 proteins in the replication of human papillomavirus type 31b.

Using transient assays, the cis and trans requirements for human papillomavirus type 31b (HPV-31b) replication in transformed and primary keratinocytes have been determined. We demonstrate that expression of both the E1 and E2 open reading frames are necessary and sufficient for replication of a plasmid construct containing a genomic 291-bp fragment in both cell types. The roles of the E1 and E2 proteins in replication were further examined by overexpressing the gene products in a glutathione-S-transferase bacterial expression system. In addition to a full-length E1 protein, a truncated E1 protein (E1*) which consisted of the amino-terminal 268 aa was synthesized. Both E1 and E1* were found to complex efficiently with the E2 protein in vivo and in vitro. In addition, site-specific DNA binding by both the E2 and E1 proteins to HPV-31b origin containing DNA was observed. The E1* protein, however, failed to bind HPV-31b DNA specifically and could not cooperate with E2 for replication in transient assays. DNase I footprinting demonstrated that the E1 protein of HPV-31b bound to an A/T-rich region (nucleotides 7905-24) which shares similarities with the binding site for the bovine papillomavirus type 1 (BPV-1) E1 protein. These studies describe both similarities and differences in the requirements for replication of HPV-31b and BPV-1.

Base Sequence↗
PubMed1994

Inhibition of p53 DNA binding by human papillomavirus E6 proteins.

Transformation by the human papillomavirus (HPV) early gene products, E6 and E7, involves their interaction with cellular proteins p53 and Rb. Using glutathione S-transferase (GST) fusion proteins, we found that HPV E6 bound human p53 and that the relative efficiency of binding varied such that the GST-HPV type 16 E6 (16E6) protein bound p53 with highest affinity, followed by GST-31E6, GST-18E6, and GST-11E6. The GST-E6 fusion proteins were sufficient for binding p53 purified from a baculovirus expression system as well as in vitro translation sources, while no association was observed with GST-18E7 or a GST-16E6 mutant bearing a five-amino-acid deletion in E6. When the site-specific DNA binding activity of p53 was examined in the presence of GST-E6 proteins, an inhibition of DNA binding was observed. The degree of inhibition correlated with the relative affinity of different E6 proteins for p53; thus, GST-16E6 was the most potent inhibitor of p53 DNA binding activity, and GST-11E6 was the least effective. Prevention of p53 DNA binding is likely to play a role in the abrogation of the transcriptional activity of p53 by HPV E6 and provides a further mechanism for E6 disruption of p53 growth suppressor function in addition to its role in directing specific degradation of p53 through the ubiquitin-mediated pathway. The variation in inhibition of DNA binding seen with the various E6 proteins may thus contribute to the differences in oncogenic potential seen among the HPV types.

Amino Acid Sequence↗
PubMed1993

Specific repression of TATA-mediated but not initiator-mediated transcription by wild-type p53.

The p53 protein is apparently central to the development of human cancers because both alleles are often found to be mutated in different tumour types. In addition, wild-type p53 can inhibit transformation by viral and cellular oncogenes in vitro, so p53 has been classified as a tumour suppressor. Investigations of the normal function of p53 have indicated that at least one of its functions could involve the activation of gene expression through the binding of specific DNA-regulatory sequences. Also, overexpression of p53 can mediate growth arrest and repress transcription from a variety of promoters. We demonstrate here both in vivo and in vitro that expression of wild-type p53 specifically represses the activity of promoters whose initiation is dependent on the presence of a TATA box. Promoters whose accurate transcription is directed by a pyrimidine-rich initiator element, however, are immune to the effects of p53. Furthermore, we observe that repression is mediated by an interaction of p53 with basal transcription factor(s). Thus, p53 appears to repress the activity of certain promoters through direct communication with TATA box-dependent basal transcription machinery.

3T3 Cells↗