Quantitative RT-PCR assay for HPV infection in cultured cells.
Early events in the life cycle of the human papillomaviruses (HPV) have been difficult to investigate due to both the scarcity of authentic HPV virions and limitations in assays to detect and quantify nonpermissive infections in monolayer cell culture. We have developed a quantitative reverse transcription-PCR (QRT-PCR) assay for the E1( wedge )E4 transcript of HPV-11. This assay is both sensitive, and capable of differentiating between infections caused by a wide range of virus input. The QRT-PCR assay measured accurately the relative amount of viral transcripts present in samples during validation experiments using RNAs from three cell lines. Infections in all three cell lines, using titrations of HPV-11 virions ranging from 20 to 600 particles per cell, produced linear expression profiles suggesting that these multiplicities of infection are below the saturation level for viral uptake and transcription. Comparison of the QRT-PCR assay with the commonly used nested RT-PCR assay revealed that although the nested RT-PCR assay was more sensitive, it did not differentiate between infections caused by >1000-fold difference in viral inputs. Potential applications of the QRT-PCR assay are demonstrated in experiments measuring the ability of a capsid-specific monoclonal antibody and a nonspecific microbicide to block HPV-11 infection.
Culp
Timothy D
TD
Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.
Christensen
Neil D
ND
D000818:Animals; D019556:COS Cells; D005260:Female; D006801:Humans; D015514:Oncogene Proteins, Fusion / Q000235:genetics / Q000378:metabolism*; D027383:Papillomaviridae / Q000235:genetics / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000821:virology*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D014764:Viral Proteins
D015514:Oncogene Proteins, Fusion; D012367:RNA, Viral; D014764:Viral Proteins; C072777:oncogene protein E1--E4, Human papillomavirus type 16