In this study we have provided evidence data that a naturally occurred mutation of A338V in HPV-2 E2 increases E2 DNA-binding capacity and reverses its transcriptional regulation activity on the viral early promoter. The effect of this mutation on the biological functions of E2 seems to be very critical, since the other four amino acid exchanges locate at the transactivation domain and the hinge regions of E2 have little impact. The E2 protein has the typical structure of transcriptional regulator, which consists of a multiple-protein-binding transactivation domain, a DNA-binding/dimerization domain, and a flexible linker [14
]. Consistent with other previous studies, our data confirm that although the C-terminal segment of E2 alone has DNA-binding capacity, lacking its N-terminal portion makes the truncated E2 almost loss it's of promoter repressor activity. Our data indicate that the E2 N-terminal alone works as a transcriptional activator, inducing about 1.5-fold increased promoter activity. However, this positive effect on the promoter is totally abolished in the context of whole E2 protein. The substitution of L118S in the E2 transactivation domain shows no influence on either DNA-binding or promoter activity. The contribution of E2 hinge region to its transcriptional regulator is believed to be not essential [15
], as three naturally occurred mutations in this area together do not influence either the E2 DNA-binding or transcriptional activation.
The structural analysis of the C-terminal DBD from several PV E2 proteins, e.g. HPV-16, -18, -31 and bovine papillomavirus (BPV-1), either alone or together with TAD, suggest it to be a tight dimer upon DNA binding [16
]. The structure of E2 DNA-binding domain is conserved among HPV families [18
]. E2 DNA-binding domains of HPV-2 and HPV-18 have 52% identity and there is only one gap between the alignments (Figure ). With software Modeller9.5 and NAMD2.6, we have constructed 3D structures of DNA-binding domains of wild-type (338A) and mutant (338 V) E2 proteins using the published crystal structure of HPV-18 E2 DNA-binding domain as the template. The amino acid residue 338 locates in the region of beta barrel that is far away from the helix region that binds to DNA (Figure ), indicating that the influence of the mutation on DNA-binding is not due to the direct alteration in the helix region. However, in beta barrel structures the hydrophobic residues are oriented into the interior of the barrel to form a hydrophobic core and the stability of theβ-barrel depends largely on the interaction of the inner hydrophobic amino acid residues. The mutation from Ala to Val at aa 338 increases the hydrophobic property and subsequently stabilizes the dimeric structure of E2, which is possibly responsible for the enhanced DNA binding activities observed in the EMSA.
Figure 7 Molecular modeling of HPV-2 E2 dimeric DNA-binding domain (prototype and mutated forms). A: Sequence alignment between HPV-2 E2 DNA-Binding domain and HPV-18's (PDB: 1F9F chain: D). There is only one gap between the alignment. In HPV-2 sequence, the A338V (more ...)
Previous study has showed that binding of the full-length wild-type BPV-1 E2 protein to the LCR sequences leads to formation of DNA loops and the transcriptional activating domain of E2 is necessary for this loop [19
]. Such structure will result in the tissue-specific enhancers shifting closer to the core transcription complex for transcriptional activation [20
]. Meanwhile, some studies have indicated that binding of the intact E2 to the LCR sequences may spatially prevent the transcriptional machine to active the promoter, which are the main molecular mechanism for E2 transcriptional repression [22
E2 has also been shown to be able to interact with other cellular agents, e.g. Brd4, to regulate its transcriptional activity [24
]. The identification of Brd4 as a component in a dominant form of E2 complexes indicates that Brd4 may be the cofactor for HPV E2 repressor function [25
]. Apparently, Brd4 recruits E2 that in turn prevents the recruitment of TFIID and pol II to the HPV promoter [26
]. Amino acid substitutions within the E2 transactivation domain impaired both the transcriptional activity and binding to Brd4 [27
]. Furthermore, Brd4 is a host chromatin adaptor for papillomavirus. The dimerization of the E2 is required for efficient Brd4 binding [28
]. The mutation from Ala to Val at aa 338 of HPV-2 E2, which would change the hydrophobicity and/or tertiary structure of E2, will lead to a modification of its interaction with the chromatin, and thus, modulates its transcriptional regulation activity. Although our data highlight a close correlation between the increased activity for DNA-binding and the enhanced activity for viral early promoters of the mutated E2 protein, the exact mechanism remains unclear.
Our data indicate that the DNA-binding capacity of the C-terminal fragment of E2 is stronger than those with the hinge region, and much stronger than the full-length E2. Earlier study has found that besides the full-length E2, bovine papillomavirus (BPV) E2 ORF also encodes two other E2 peptides, E2-TR and E8/E2 proteins [29
]. These shorter E2 proteins contain the DNA binding and dimerization domains of the C-terminus and hinge region, but lack the transactivation domain. Relative abundances of the truncated E2 proteins have been observed in BPV transformed cells (the molar ratio of E2:E2-TR:E8/E2 is 1:10:3) [30
]. Expression of HPV-31 E8E2C protein has been reported to be able to inhibit HeLa cell growth [31
]. However, the transcriptional profiles of other HPV E2 ORFs, regardless in benign or malignant cells, are rarely addressed. The fact that the C-terminus E2 binds DNA stronger suggests that it is more competitive than the full-length E2 in the cells.
Our study provides the evidence that HPV-2 E2, regardless of wild-type or mutant (A338V), induces the similar biological effectiveness under the controls of the homologous and heterologous HPV LCRs. This suggests that E2 protein may induce same regulative activity on the viral early promoters from different HPVs with similar upstream components. Although there are more than 100 genotypes of HPVs involving in various human benign or malignant proliferating diseases, the sequences of viral genomes are relatively conservative. Hence, the effectiveness of HPV-2 E2 may represent a common property of HPVs' E2 proteins.
In addition to the role in regulating viral transcription, HPV E2 protein involves in enhancing E1-dependent viral DNA replication and genome maintenance. In HPV genomes the viral DNA replication initiation site co-localizes with the viral transcription region. However, the regulative function of E2 in viral DNA replication is far from understood compared with its role in transcriptional regulation. Although the point mutations in TAD and in hinge region within this E2 mutant do not affect DNA-binding and transcriptional regulation, their influence on viral genome replication cannot be excluded. Sequences analyses of this variant HPV-2 strain have also identified several point mutations in its E1 ORF. Further studies of viral genome replication will help explore the inconvenient reason of such huge verrucae vulgaris.