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. 1998 Oct;72(10):8115-23.
doi: 10.1128/JVI.72.10.8115-8123.1998.

Transactivation by the E2 protein of oncogenic human papillomavirus type 31 is not essential for early and late viral functions

F Stubenrauch  1 , A M Colbert, L A Laimins
Affiliations

Transactivation by the E2 protein of oncogenic human papillomavirus type 31 is not essential for early and late viral functions

F Stubenrauch et al. J Virol. 1998 Oct.

Abstract

The activation of transcription and of DNA replication are, in some cases, mediated by the same proteins. A prime example is the E2 protein of human papillomaviruses (HPVs), which binds ACCN6GGT sequences and activates heterologous promoters from multimerized binding sites. The E2 protein also has functions in replication, where it complexes with the virally encoded origin recognition protein, E1. Much of the information on these activities is based on transient-transfection assays as well as biochemical analyses; however, their importance in the productive life cycle of oncogenic HPVs remains unclear. To determine the contributions of these E2 functions to the HPV life cycle, a genetic analysis was performed by using an organotypic tissue culture model. HPV type 31 (HPV31) genomes that contained mutations in the N terminus of E2 (amino acid 73) were constructed; these mutants retained replication activities but were transactivation defective. Following transfection of normal human keratinocytes, these mutant genomes were established as stable episomes and expressed early viral transcripts at levels similar to those of wild-type HPV31. Upon differentiation in organotypic raft cultures, the induction of late gene expression and amplification of viral DNA were detected in cell lines harboring mutant genomes. Interestingly, only a modest reduction in late gene expression was observed in the mutant lines. We conclude that the transactivation function of E2 is not essential for the viral life cycle of oncogenic HPVs, although it may act to moderately augment late expression. Our studies suggest that the primary positive role of E2 in the viral life cycle is as a replication factor.

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Figures

FIG. 1
FIG. 1
Linear representation of the HPV31 E2 protein. The highly conserved amino- and carboxy-terminal domains of E2 are presented as black and striped boxes, respectively. The less-conserved hinge domain is shown in white. The arrows indicate mutations resulting in amino acid exchanges of highly conserved residues (residues 20, 37, 39, and 73) among E2 proteins.
FIG. 2
FIG. 2
(A) Gel retardation analysis of HPV31 wt E2 and E2 mutant proteins EN20, RK37, EQ39, and IL73 expressed in SCC-13 cells. Control lanes received no protein extract (lane −) or extract from cells transfected with the parental pSG5 vector (lane SG5). Lanes E2, 20, 37, 39, and 73 received identical amounts of extracts from cells transfected with the respective expression vectors. The retarded band corresponding to the E2-DNA complex is indicated with an arrow labeled c. The free 32P-labeled oligonucleotide containing an E2 binding site is indicated by an arrow labeled f. (B) Transient luciferase expression assays. SCC-13 cells were transfected with expression vectors for HPV31 wt E2 or E2 mutant protein EN20, RK37, EQ39, or IL73 and the E2-responsive reporter plasmid p6XE2BS-luc and analyzed for luciferase activity. The reporter plasmid consists of six E2-binding sites (E2) upstream of the minimal SV40 early promoter (SV) that drives the expression of the luciferase gene (luc) and is diagrammed below the graph. The luciferase activity obtained by cotransfection of the E2 mutant expression plasmids is given relative to the activity of wt E2-transfected cells, which was set to 1. The standard deviations are indicated by error bars. (C) Transient replication assay. SCC-13 cells were transfected with plasmid pGL31URR alone (−) or together with an expression vector for HPV31 E1 or both vectors together with expression vectors for HPV31 wt E2 or E2 mutant proteins EN20, RK37, EQ39, and IL73. Transient replication of pGL31URR was analyzed by Southern hybridization. A representative autoradiograph is shown below the graph. Replication levels of pGL31URR were quantitated by phosphoimaging analysis and are represented relative to the replication levels induced by HPV31 wt E2, which was set to 1. The standard deviations are indicated by error bars.
FIG. 3
FIG. 3
Southern analysis of DNA from human keratinocyte cell lines obtained after stable transfection of HPV31 wt, E2:EN20, E2:RK37, E2:EQ39, and E2:IL73 genomes. Ten micrograms of total cellular DNA was digested with either restriction enzyme BamHI (lanes N), which does not cut HPV31 DNA, or EcoRV (lanes S), which recognizes one site in the HPV31 genome, and then subjected to Southern analysis. As size markers, EcoRI-linearized HPV31 DNA equivalent to 5 and 25 viral copies per cell was used (lanes 5 and 25). Specific DNAs were detected with 32P-labeled HPV31 genomic DNA. To the left of the autoradiograph, the positions of concatemeric or integrated (C/I), open-circle (III), linear (II), and supercoiled (I) forms of viral DNA are indicated. To the right, sizes of HindIII-digested phage lamba DNA are shown in kilobases. On the far right, the results of a longer exposure of the lanes containing DNA from E2:RK37 cells are presented.
FIG. 4
FIG. 4
RNase protection analysis of RNA from HPV31 wt (lane WT), E2:RK37 (lane 37), and E2:IL73 (lane 73) cell lines grown in monolayer culture. Total cellular RNA (10 μg) was hybridized to a 32P-labeled antisense probe transcribed from plasmid pRP742 and subjected to RNase protection analysis. The positions of P97 transcripts, which are unspliced or spliced at a donor site at nt 877 (SD 877), are indicated by arrows to the left of the autoradiograph. Lane P contains undigested probe. A 32P-end-labeled 1-kb ladder was used as a size marker (lane M), and the sizes are indicated in nucleotides to the right. The structure of the antisense probe is depicted below the autoradiograph. The start sites for the P97 and P742 promoters as well as the splice donor site at nt 877 (SD 877) are indicated by arrows. The dotted line indicates that the start site for P97 is not included in the probe. Parts of the E7 and E1 genes that are covered by the probe are shown below the structure diagram.
FIG. 5
FIG. 5
(A) DNA in situ hybridization analysis of cross sections of HPV31 wt, E2:RK37, and E2:IL73, raft cultures. HPV31 DNA was detected with an HPV31/33/35-specific probe, and positive cells display a blue stain. (B) Immunohistochemical analysis for the differentiation-dependent expression of the HPV31 E1∧E4 protein in raft cultures. Tissue cross sections (5-μm thick) of HPV31 wt, E2:RK37, and E2:IL73 cell lines grown in raft culture were incubated with a polyclonal antibody generated against the HPV31 E1∧E4 protein. Specifically bound secondary antibodies (fluorescein isothiocyanate-conjugated) were detected by immunoflourescence.
FIG. 6
FIG. 6
RNase protection analysis of RNA from HPV31 wt (lane WT), E2:RK37 (lane 37), and E2:IL73 (lane 73) cell lines grown in raft cultures. Total cellular RNA (10 μg) was hybridized to a 32P-labeled antisense probe as described in the legend to Fig. 4. The positions of P97 and P742 transcripts, which are unspliced or spliced at a donor site at nt 877 (SD 877), are indicated to the left of the autoradiograph. Lane P contains undigested probe. A 32P-end-labeled 1-kb ladder was used as a size marker (lane M), and the sizes are indicated in nucleotides on the right. See the legend to Fig. 4 for a description of the diagram at the bottom of the figure.
FIG. 7
FIG. 7
RNase protection analysis of RNA from HPV31 wt (lanes WT), E2:RK37 (lanes 37), and E2:IL73 (lanes 73) cell lines grown in monolayer (M) or raft (R) cultures. Total cellular RNA (20 μg) was hybridized to a 32P-labeled antisense probe transcribed from plasmid pRPA31L1. The positions of late gene transcripts, which are unspliced or spliced at an acceptor site at nt 5552 (SA 5552), are indicated to the left of the autoradiograph. The structure of the antisense probe is shown below the autoradiograph. The parts of the L2 and L1 genes that are covered by the probe and the splice acceptor site (SA 5552) are indicated.
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