Human papillomavirus type 18 chimeras containing the L2/L1 capsid genes from evolutionarily diverse papillomavirus types generate infectious virus

Abstract

Papillomaviruses (PVs) comprise a large family of viruses infecting nearly all vertebrate species, with more than 100 human PVs identified. Our previous studies showed that a mutant chimera HPV18/16 genome, consisting of the upper regulatory region and early ORFs of HPV18 and the late ORFs of HPV16, was capable of producing infectious virus in organotypic raft cultures. We were interested in determining whether the ability of this chimeric genome to produce infectious virus was the result of HPV18 and HPV16 being similarly oncogenic, anogenital types and whether more disparate PV types could also interact functionally. To test this we created a series of HPV18 chimeric genomes where the ORFs for the HPV18 capsid genes were replaced with the capsid genes of HPV45, HPV39, HPV33, HPV31, HPV11, HPV6b, HPV1a, CRPV, and BPV1. All chimeras were able to produce infectious chimeric viral particles, although with lower infectivity than wild-type HPV18. Steps in the viral life cycle and characteristics of the viral particles were examined to identify potential causes for the decrease in infectivity.

FIGURE 1

(A) Phylogenetic tree of PVs based on L1 sequence homology. PVs used in this study are bolded and boxed. The figure was adopted and modified (Bravo and Alonso, 2004). (B) Creation of chimeric PV recombinant plasmids. The areas representing the early genes (grey) and the late genes (black) of HPV18 are shown. The approximate positions of the ORFs are labeled. HPV18 was cloned into pBSSK(+) (dotted area) at the unique EcoRI site. The HPV18 late gene ORFs were removed and replaced with the corresponding sequences from the panel of PVs.

FIGURE 2

Southern blot hybridizations of DNA from chimeric PV infected cell lines grown in undifferentiated monolayer cultures. Each chimeric PV cell line was analyzed for the episomal maintenance, copy number, and integrity of the chimeric PV genome. The blot was first probed with an HPV18-specific probe (top panels), stripped and reprobed with the chimeric PV-specific probe (bottom panels). Lanes 1, undigested DNA form I supercoiled (FI) and form II nicked (FII) DNA. Lanes 2, DNA digested with EcoRI to linearize the viral genome (FIII). Lanes 3, DNA digested with BglII to separate the HPV18 sequences (early ORFs) from the chimeric PV sequences (late ORFs). HPV18 genomic 100- and 10-copy number standards (left side) and the chimeric PV genomic 100- and 10-copy number standards (right side) are shown. Note extensive nicking of the supercoiled DNA during DNA isolation from tissues has converted the majority of the supercoiled viral genomes to the nicked form.

FIGURE 3

Immunohistochemical analyses of chimeric PV-infected tissues. Chimeric infected, fully stratified and differentiated raft culture tissue sections were stained with (A) hematoxylin and eosin or (B) immunostained with a keratin 10-specific MAb (AM201-5M, BioGenex).

FIGURE 3

Immunohistochemical analyses of chimeric PV-infected tissues. Chimeric infected, fully stratified and differentiated raft culture tissue sections were stained with (A) hematoxylin and eosin or (B) immunostained with a keratin 10-specific MAb (AM201-5M, BioGenex).

FIGURE 4

Southern blot analyses of chimeric PV genome amplification. Each chimeric PV cell line was analyzed for genome amplification during differentiation of the host tissue. The blot was probed with an HPV18-specific probe. Odd numbered lanes: DNA isolated from undifferentiated monolayer cells and digested with EcoRI to linearize the viral genome. Even numbered lanes: DNA isolated from differentiated raft culture tissues and digested with EcoRI to linearize the viral genome. Lanes 1–2, HPV18/45; Lanes 3–4, HPV18/39; Lanes 5–6, HPV18/33; Lanes 7–8, HPV18/31; Lanes 9–10, HPV18/11; Lanes 11–12, HPV18/6b; Lanes 13–14, HPV18/1a; Lanes 15–16, HPV18/CRPV; Lanes 17–18, HPV18/BPV1.

Figure 5

In situ hybridization analysis for the presence of chimeric PV DNA amplification in differentiating raft tissues harboring chimeric PV genomes. (A) wild-type HPV11 xenograft tissue, (B) wild-type HPV18, (C) HPV18/45, (D) HPV18/11, (E) HPV18/CRPV, (F) HPV18/BPV1. Arrows indicate cells stained positive for genome amplification. HPV18/45, HPV18/11, and HPV18/CRPV showed no positive staining, whereas HPV18/BPV1 showed only one positively stained cell.

FIGURE 6

Relative virus titers. Quantification of the amounts of encapsidated chimeric PV genomes by qPCR was performed as described in the Materials and Methods. Each endonuclease-resistant viral genome preparation was analyzed in triplicate. In addition, three endonuclease-resistant viral genome preparations obtained from three independent cell lines were analyzed for each mutant virus. Data are presented as a fold change in titer compared with wild-type HPV18.

FIGURE 7

Neutralization analyses of chimeric PV. Neutralization of chimeric HPV18/31 (lanes 1 and 2), HPV18/11 (lane 3), HPV18/45 (lane 4), HPV18/CRPV (lane 5), HPV18/BPV1 (lane 6), HPV18/33 (lane 7) and HPV18/6b (lane 8) with MAbs H31.A6, H11.B2, H45.L10, CRPV.4B, B1.A1, H33.B6 and H6.N8, respectively. Shown is a 2% agarose gel of nested RT-PCR-amplified HPV 18 E1^E4 and β-actin. The arrowhead indicates β-actin and the arrow indicates HPV18 E1^E4 amplified products. The chimeric PV dilution was 1:20 and each MAb was diluted 1:20.

FIGURE 8

Relative binding of chimeric PV-specific MAbs to chimeric PV particles by ELISA assay. Reading for each chimeric PV was defined as the difference between wells probed with a chimera-specific and an irrelevant MAb. Values are means + standard error bars of the means (S.E.M.)