- Volume 92, Issue 10, 2011

Two splice sites on the human papillomavirus type 16 (HPV-16) genome are used exclusively by the late capsid protein L1 mRNAs: SD3632 and SA5639. These splice sites are suppressed in mitotic cells. This study showed that serine/arginine-rich protein 30c (SRp30c), also named SFRS9, activated both SD3632 and SA5639 and induced production of L1 mRNA. Activation of HPV-16 L1 mRNA splicing by SRp30c required an intact arginine/serine-repeat (RS) domain of SRp30c. In addition to this effect, SRp30c could enhance L1 mRNA production indirectly by inhibiting the early 3′-splice site SA3358, which competed with the late 3′-splice site SA5639. SRp30c bound directly to sequences downstream of SA3358, suggesting that SRp30c inhibited the enhancer at SA3358 and caused a redirection of splicing to the late 3′-splice site SA5639. This inhibitory effect of SRp30c was independent of its RS domain. These results suggest that SRp30c can activate HPV-16 L1 mRNA expression via a bimodal mechanism: directly by stimulating splicing to late splice sites and indirectly by inhibiting competing early splice sites.

Human papillomavirus (HPV) life cycle requires extensive manipulation of cell signalling to provide conditions adequate for viral replication within the stratified epithelia. In this regard, we show that the E2 regulatory protein of α, β and μ-HPV genotypes enhances tumour necrosis factor (TNF)-induced activation of nuclear factor kappa B (NF-κB). This activation is mediated by the N-terminal domain of E2, but does not rely on its transcriptional properties. It is independent of the NF-κB regulator Tax1BP1, which nevertheless interacts with all the E2 proteins. E2 specifically activates NF-κB pathways induced by TNF, while interleukin-1-induced pathways are not affected. E2 stimulates the activating K63-linked ubiquitination of TRAF5, and interacts with both TRAF5 and TRAF6. Our data suggest that E2 potentiates TNF-induced NF-κB signalling mediated by TRAF5 activation through direct binding. Since NF-κB controls epithelial differentiation, this activity may be involved in the commitment of infected keratinocytes to proliferation arrest and differentiation, both required for the implementation of the productive viral cycle.

We previously isolated human papillomavirus 83 (HPV83m) from a cervical smear. Sequence analysis of E6 and E7 proteins highlighted five mutations located in the second putative zinc-finger region of E6 (E6m), an important domain for protein–protein or protein–DNA interactions. Here, we show that E6m of HPV83m can trigger human primary cell proliferation and anchorage-independent growth properties, similarly to E6 of HPV16, a high-risk HPV (HR-HPV). Interestingly, we demonstrate that, in contrast to E6 of HPV16, E6m corrupts neither p53 stability nor telomerase activity, but acts as a specific modulator of the transcriptional machinery. By studying E6m reversion mutants, we confirmed the importance of the second zinc-finger domain in triggering the observed upregulation of cell growth and of the transcriptional machinery. Reversion of these mutations in E6m (to yield strain E6r) fully abolished the oncogenic potential of E6m, transforming the phenotype of E6 from a high-risk to a low-risk phenotype. Importantly, our data define the importance of a cluster of mutations in the second zinc finger of E6m in increasing the oncogenic potential of HPV83.

Bovine papillomavirus types 1 and 2 (BPV-1 and BPV-2) are known to induce common equine skin tumours, termed sarcoids. Recently, it was demonstrated that vaccination with BPV-1 virus-like particles (VLPs) is safe and highly immunogenic in horses. To establish a BPV-1 challenge model for evaluation of the protective potential of BPV-1 VLPs, four foals were injected intradermally with infectious BPV-1 virions and with viral genome-based and control inocula, and monitored daily for tumour development. Blood was taken before inoculation and at weekly intervals. BPV-1-specific serum antibodies were detected by a pseudo-virion neutralization assay. Total nucleic acids extracted from tumours, intact skin and PBMCs were tested for the presence of BPV-1 DNA and mRNA using PCR and RT-PCR, respectively. Intralesional E5 oncoprotein expression was determined by immunofluorescence. Pseudo-sarcoids developed exclusively at sites inoculated with virions. Tumours became palpable 11–32 days after virion challenge, reached a size of ≤20 mm in diameter and then resolved in ≤6 months. No neutralizing anti-BPV-1 serum antibodies were detectable pre- or post-challenge. BPV-1 DNA was present in lesions but not in intact skin. In PBMCs, viral DNA was already detectable before lesions were first palpable, in concentrations correlating directly with tumour growth kinetics. PBMCs from two of two foals also harboured E5 mRNA. Immunofluorescence revealed the presence of the E5 protein in tumour fibroblasts, but not in the apparently normal epidermis overlying the lesions. Together with previous findings obtained in horses and cows, these data suggest that papillomavirus infection may include a viraemic phase.

In the present study, the expression, generation and subcellular localization of Torque teno sus virus (TTSuV) proteins were characterized into two genetically distinct TTSuV species (TTSuV1 and TTSuV2). Following transfection of three TTSuV1 and TTSuV2 full-length ORF (ORF1, ORF2 and ORF3) expression constructs into porcine kidney cells, alternative splice variants encoding new TTSuV protein isoforms were identified for the first time. Proteins encoded from ORF1 and ORF3 were localized in the nucleoli of porcine kidney cells and that of ORF2 in the cytoplasm and nucleus excluding the nucleoli. The subcellular localization of the different protein isoforms was not only similar between distinct TTSuV species but also to the ones described in human Torque teno virus (TTV). Results of the present in vitro study were not based on full-length viral clones but suggested that alternative splicing strategy to generate TTSuV protein isoforms probably occurs in vivo. Obtained data provide new information on molecular biology of TTSuV and anelloviruses, which until now has been solely based on results obtained from human TTV.