Differential requirements for conserved E2 binding sites in the life cycle of oncogenic human papillomavirus type 31

Abstract

Human papillomavirus (HPV) E2 proteins regulate viral replication by binding to sites in the upstream regulatory region (URR) and by complex formation with the E1 origin recognition protein. In the genital HPV types, the distribution and location of four E2 binding sites (BS1 to BS4) which flank a single E1 binding site are highly conserved. We have examined the roles of these four E2 sites in the viral life cycle of HPV type 31 (HPV31) by using recently developed methods for the biosynthesis of papillomaviruses from transfected DNA templates (M. G. Frattini et al., Proc. Natl. Acad. Sci. USA 93:3062-3067, 1996). In transient assays, no single site was found to be necessary for replication, and mutation of the early promoter-proximal site (BS4) led to a fourfold increase in replication. Cotransfection of the HPV31 wild-type (HPV-wt) and mutant genomes with expression vectors revealed that E1 stimulated replication of HPV31-wt as well as the HPV31-BS1, -BS2, and -BS3 mutants. In contrast, increased expression of E2 decreased replication of these genomes. Replication of the HPV31-BS4 mutant genome was not further increased by cotransfection of E1 expression vectors but was stimulated by E2 coexpression. In stably transfected normal human keratinocytes, mutation of either BS1, BS3, or BS4 resulted in integration of viral genomes into host chromosomes. In contrast, mutation of BS2 had no effect on stable maintenance of episomes or copy number. Following growth of stably transfected lines in organotypic raft cultures, the differentiation-dependent induction of late gene expression and amplification of viral DNA of the BS2 mutant was found to be similar to that of HPV31-wt. We were unable to find a role for BS2 in our assays for viral functions. We conclude that at least three of the four E2 binding sites in the URRs of HPVs are essential for the productive viral life cycle. The specific arrangement of E2 binding sites within the URR appears to be more important for viral replication than merely the number of sites.

FIG. 1

Schematic depiction of the linearized HPV31 genome. (Top) Upstream regulatory region located between the late and the early regions. Numbered boxes represent BS1 through -4, and the binding site for E1 is shown as an oval. The start sites for the early promoter P97 and the late P742 promoter are indicated by arrows. Open reading frames (ORFs) are shown in black. (Bottom) Mutated E2BS in mutant genomes are shown as crossed squares. The positions of translational stop codons introduced into the E1 and E2 genes are indicated by arrows.

FIG. 2

Representative Southern analysis of transiently replicating HPV31 in SCC-13 cells. SCC-13 cells were transfected with religated HPV31-wt, -BS1, -BS2, -BS3, or -BS4 or genomes that contained a disrupted E1 (E1N-TTL) or E2 (E2N-TTL) gene. Low-molecular weight DNA was isolated 120 h posttransfection, digested with BanII and DpnI, and analyzed by Southern analysis using a full-length HPV31 probe. The marker lane (M) contains 100 pg of EcoRI-linearized HPV31 DNA.

FIG. 3

Representative Southern analysis of transiently replicating HPV31 and E2BS mutant DNAs in the presence of expression vectors for E1 or E2. SCC-13 cells were cotransfected with 2 μg of religated HPV31 or HPV31-BS1, -BS2, -BS3, or -BS4 and 0.5 μg of pSG5 (−), pSG31:E1 (E1), or pSG31:E2 (E2). Low-molecular-weight DNA was isolated 120 h after transfection of SCC-13 cells and analyzed as described for Fig. 2. The marker lane (M) contains 100 pg of linearized HPV31 genome. The arrow indicates the position of the replicated DNA.

FIG. 4

Southern analysis of DNA from keratinocyte cell lines established after transfection of HPV31 or HPV31 genomes containing E2BS mutants. Ten micrograms of total cellular DNA was digested with BamHI (lanes N), which does not cut the HPV31 genome, or with EcoRV (lanes S), which cuts the HPV31 genome once. Genome copy number control lanes representing 50 and 5 copies per cell are shown on the left. Marker sizes are indicated in kilobases to the right. The migration of supercoiled (a), linearized (b), and open circle, concatemeric, or integrated (c) DNA forms is indicated to the right.

FIG. 5

RNase protection analysis of RNA isolated from keratinocytes containing HPV31-wt (wt; LKP cell line [15]), HPV31-BS2 (BS2), or HPV31-BS3 (BS3) grown in monolayer cultures (M) or differentiated in raft cultures (R). Total cellular RNA (10 μg) was analyzed by RNase protection using the antisense RNA probe transcribed from linearized pRP742. Lane P contains undigested antisense probe. Lane S contains ³²P-end-labeled 1-kb ladder (Gibco BRL), and the sizes are indicated in nucleotides to the right. Transcripts initiated at P97 or P742 are indicated by arrows to the left. The structure of the antisense RNA probe is shown below the autoradiograph. The major initiation sites for the late promoter P742 and the initiation site for the early promoter P97 are indicated by arrowheads, and parts of the E7 and E1 genes are shown below. The dashed line indicates that the P97 start site is not covered by the probe. The splice donor (SD) site at nt 877 is shown as an arrow.

FIG. 6

RNase protection analysis of total RNA (20 μg) isolated from keratinocytes containing HPV31-wt (WT; LKP cell line [15]), HPV31-BS2 (BS2), or HPV31-BS3 (BS3) grown in monolayer cultures (M) or differentiated in raft cultures (R). Lane P contains undigested antisense probe which was transcribed from pRPA31L1. Lane S contains ³²P-end-labeled 1-kb ladder (Gibco BRL), and the sizes are indicated in nucleotides to the right. The structure of the antisense RNA probe is shown below. Parts of the L2 and L1 open reading frames are indicated. The splice acceptor site at nt 5552 is depicted by a dashed line. Spliced L1 and unspliced L2/L1 transcripts are indicated by arrows on the left of the autoradiogram.

FIG. 7

DNA in situ hybridization analysis of HPV31-wt (WT) and HPV31-BS2 (BS2) cell lines differentiated in raft cultures. Tissue cross sections were hybridized to an HPV31/33/51-specific probe. Cells which have amplified viral DNA are darkly stained. A representative cell in each panel is indicated by a white arrow.