TY - JOUR
T1 - Display of complete life cycle of human papillomavirus type 16 in cultured placental trophoblasts
AU - Liu, Yong
AU - You, Hong
AU - Chiriva-Internati, Maurizio
AU - Korourian, Soheila
AU - Lowery, Curtis L.
AU - Carey, Martin J.
AU - Smith, Carl V.
AU - Hermonat, Paul L.
N1 - Funding Information:
This study was funded by a grant from the March of Dimes (G-FY99-188) to P.L.H. The authors thank Dr. Richard Schlegel for plasmid pAT-HPV-16 and Dr. Neil Christensen for the antibodies. The authors also thank Drs. Craig Meyers, Neil Christensen, and John Schiller for useful discussions.
PY - 2001/11/10
Y1 - 2001/11/10
N2 - Human papillomavirus (HPV) infection is threefold more prevalent in spontaneous abortion specimens compared to elective abortions, preferentially targeting the placental trophoblasts in these specimens. Here, by using infectious center and Southern blot analysis, we demonstrate that the transfected HPV-16 genome de novo replicates in 3A trophoblasts in culture. Peak DNA replication occurred 9-24 days posttransfection, showing classic DNA forms I, II, and III and an 8-kb monomer band upon DpnI/BamHI digestion. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of mRNA expression revealed that E6 and E2 were significantly expressed by day 9, coinciding with 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 confirmed by Western blot analysis. The production of HPV-16 virions was demonstrated by three techniques: The appearance of HPV-16 infectious units coinciding with L1 expression, the neutralization of these infectious units with known neutralizing anti-HPV-16 antibodies, and the appearance of spliced E1∧E4 and E6∧E7 transcripts (RT-PCR) in normal keratinocyte rafts infected with these trophoblast-produced HPV-16 infectious units. These data suggest that HPV-16 is carrying out its complete life cycle in trophoblasts. Previously, HPVs were known to productively replicate only in differentiating keratinocytes of skin. These findings expand HPV biology, support the hypothesis of a possible link between HPV and some spontaneous abortions, and present a new technology for studying HPV.
AB - Human papillomavirus (HPV) infection is threefold more prevalent in spontaneous abortion specimens compared to elective abortions, preferentially targeting the placental trophoblasts in these specimens. Here, by using infectious center and Southern blot analysis, we demonstrate that the transfected HPV-16 genome de novo replicates in 3A trophoblasts in culture. Peak DNA replication occurred 9-24 days posttransfection, showing classic DNA forms I, II, and III and an 8-kb monomer band upon DpnI/BamHI digestion. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of mRNA expression revealed that E6 and E2 were significantly expressed by day 9, coinciding with 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 confirmed by Western blot analysis. The production of HPV-16 virions was demonstrated by three techniques: The appearance of HPV-16 infectious units coinciding with L1 expression, the neutralization of these infectious units with known neutralizing anti-HPV-16 antibodies, and the appearance of spliced E1∧E4 and E6∧E7 transcripts (RT-PCR) in normal keratinocyte rafts infected with these trophoblast-produced HPV-16 infectious units. These data suggest that HPV-16 is carrying out its complete life cycle in trophoblasts. Previously, HPVs were known to productively replicate only in differentiating keratinocytes of skin. These findings expand HPV biology, support the hypothesis of a possible link between HPV and some spontaneous abortions, and present a new technology for studying HPV.
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U2 - 10.1006/viro.2001.1135
DO - 10.1006/viro.2001.1135
M3 - Article
C2 - 11887784
AN - SCOPUS:0035841619
SN - 0042-6822
VL - 290
SP - 99
EP - 105
JO - Virology
JF - Virology
IS - 1
ER -