The minor capsid protein L2 of papillomaviruses (PVs) likely plays a role in the selective encapsidation of PV DNA in viral capsids and in the infectivity of PV virions. The L2 protein also can cause the relocalization of the PV early protein, E2TA, to nuclear subdomains known as promyelocytic leukemia oncogenic domains (PODs) in which it is localized. E2TA is a transcriptional transactivator that also plays a critical role in viral DNA replication. In this study, we investigated whether L2, in causing the relocalization of E2TA, alters the activities of E2TA. We provide evidence that L2 inhibits the transcriptional transactivation function of E2, but it does not specifically inhibit the capacity of E2 to support viral DNA replication. We also investigated whether the colocalization of E2 and L2 to PODs and the ability of L2 to inhibit the transcriptional transactivation activity of E2TA might be mediated through a direct interaction between these two proteins. Using an in vitro protein-protein association assay, we found that L2 binds to E2TA. Two regions in E2TA were found to mediate this interaction. One of those domains is present in an alternative E2 gene product, E2TR, which is an antagonist to E2TA. Here we show that the L2 protein also relocalizes the E2 transcriptional repressor, E2TR, to the nuclear subdomains. These data suggest that the ability of L2 to relocalize E2 proteins to PODs is mediated through a direct interaction with L2.

The study of viral infectivity and detection of viral capsid antigens of the major cervical cancer-associated human papillomavirus (HPV) type, HPV-16, requires knowledge of which epitopes are exposed in clinical specimens of infected tissue or on intact capsids. To define the antigenic epitopes ofHPV-16, antisera to 66 overlapping synthetic peptides corresponding to the HPV-16 capsid proteins L1 and L2 and to seven peptide analogues were tested in immunoperoxidase stainings of consecutive sections from formalin-fixed, paraffin-embedded HPV infected tissue. Antisera against eleven different peptides from L1 and against seven different peptides from L2 recognized the HPV capsid antigen. Most epitopes were only found on the capsid antigen of certain genital HPV types, but four antigenic epitopes in L1 were detectable also in cutaneous wart specimens. All antigenic epitopes in L2 were restricted to genital HPV types and four L2 epitopes were only detectable in HPV-16 or HPV-33 positive specimens. The surface exposure of the antigenie epitopes was investigated by comparing the reactivity of the antipeptide antisera with intact or disrupted virions or capsids of HPV-11, HPV-16 and bovine papillomavirus (BPV). Twenty antipeptide sera from L1 and seven antipeptide sera from L2 were reactive with intact HPV-16 capsids at titres up to 1 : 146000. Sixteen of these antisera were also reactive with disrupted HPV-16 capsids. Cross-reactivity with disrupted HPV-11 and BPV was detected for eleven and six antisera, respectively, whereas intact HPV-11 or BPV virions showed only weak cross-reactivity. In conclusion, the HPV-16 L1 and L2 capsid proteins contained multiple antigenic epitopes, most of which were shared with one or several additional HPV types.

Exposure to HPV16 or related viruses appears to be a major risk factor in the majority of anal cancers.

All types of papillomaviruses (PV) share common, so-called group-specific epitopes. To identify the major group-specific epitopes, we immunized 26 guinea pigs or rabbits with purified bovine PV type 1 (BPV), canine PV, or avian PV from the common chaffinch. The resulting hyperimmune sera, as well as a commercially available rabbit antiserum to BPV and seven monoclonal antibodies to BPV, were tested in an enzyme-linked immunosorbent assay with a set of 66 overlapping 20-amino-acid peptides representing the complete sequence of the major capsid proteins (Li and L2) of human PV type 16 (HPV 16). Sera from the same animals before immunization were used as controls. The minimal reactive epitopes within each peptide were further characterized by testing of truncated peptides. The cross-reactive epitopes were clustered in two regions of Li, an internal region (at positions 171 to 235), which contained three epitopes, and the more reactive region at the carboxy terminus (at positions 411 to 475), which contained six epitopes. The most reactive of the HPV 16 broadly cross-reactive epitopes was a carboxy-terminal epitope which had the sequence DTYRF and which reacted with nine of the antisera to BPV, canine PV, or avian PV, with the commercially available rabbit antiserum to BPV, and also with a mouse monoclonal antibody to BPV. Antipeptide antisera to all of the HPV 16 Li peptides and to the most antigenically reactive of their truncated analogs were made in guinea pigs. Antipeptide antisera reactive with BPV were obtained for three of the cross-reactive epitopes, and one of these antisera allowed highly sensitive detection of group-specific PV antigen by immunoperoxidase staining.

Several neutralizing sites of the human papillomavirus (HPV) capsid are known to be critically dependent on the conformation of the capsid. However, efficient production of HPV16 capsids in mammalian cells has been difficult, possibly because the HPV genome contains negative regulatory elements. To circumvent these problems, we cloned the HPV16 L1 and L2 genes from a healthy HPV16-infected woman into a Semliki Forest virus based expression vector (P. Liljeström and H. Garoff, Biotechnology 9, 1356-1361, 1991). Recombinant HPV16 L1- or L2-producing Semliki Forest virus was generated and used for infection of mammalian cells. The HPV16 L1 and L2 proteins were efficiently expressed and the majority of the L1 protein self assembled into virus-like particles (VLPs). Coexpression of L1 and L2 resulted in incorporation of L2 into the VLPs. The particles had a density of approximately 1.3 g/ml as determined by density gradient centrifugation. Transmission electron microscopy revealed that the particles had a morphology similar to native virions. The HPV16 VLPs produced by the Semliki Forest virus expression system may be useful as a conformationally correctly assembled target for studies of HPV attachment, assembly, serology, or vaccination.