Display of complete life cycle of human papillomavirus type 16 in culturedplacental trophoblasts

Human papillomavirus (HPV) infection is threefold more prevalent in spontan eous abortion specimens compared to elective abortions, preferentially targ eting the placental trophoblasts in these specimens. Here, by using infecti ous center and Southern blot analysis, we demonstrate that the transfected HPV-16 genome de novo replicates in 3A trophoblasts in culture. Peak DNA re plication occurred 9-24 days posttransfection, showing classic DNA forms I, II, and III and an 8-kb monomer band upon DpnI/BamHI digestion. Reverse tr anscription-polymerase chain reaction (RT-PCR) analysis of mRNA expression revealed that E6 and E2 were significantly expressed by day 9, coinciding w ith HPV-16 DNA replication. However, significant L1 expression was delayed until day 18. L1 protein expression on day 18, but not day 9, was also conf irmed by Western blot analysis. The production of HPV-16 virions was demons trated by three techniques: the appearance of HPV-16 infectious units coinc iding with L1 expression, the neutralization of these infectious units with known neutralizing anti-HPV-16 antibodies, and the appearance of spliced E 1<^>E4 and E6<^>E7 transcripts (RT-PCR) in normal keratinocyte rafts infect ed with these trophoblast-produced HPV-16 infectious units. These data sugg est that HPV-16 is carrying out its complete life cycle in trophoblasts. Pr eviously, HPVs were known to productively replicate only in differentiating keratinocytes of skin. These findings expand HPV biology, support the hypo thesis of a possible link between HPV and some spontaneous abortions, and p resent a new technology for studying HPV. (C) 2001 Academic Press.