Inhibition of HIV type 1 reverse transcriptase assay by nucleases produced by contaminating mycoplasmas.
Mycoplasmal contamination of HIV-1-infected cells has been found to induce reduction of reverse transcriptase (RT) activity; however, the exact mechanism of this phenomenon was not clearly elucidated. Our results indicate that the apparent reduction in RT activity is due to a calcium-dependent nuclease(s) that is (are) produced by contaminating mycoplasmas. The interference with the RT assay was found to be due to the degradation of products of the RT activity. Addition of EGTA at a 1 mM concentration was sufficient to remove the inhibitory effect. The particular HIV-1-producing cell line that was under study was found to be contaminated with Mycoplasma fermentans and Mycoplasma pirum and the latter was isolated in pure culture. Nuclease activity was also observed with pure cultures of mycoplasmas from different species. The activity was found to be of the endonuclease type because it was active with both supercoiled and linear DNAs.
Quillent
C
C
Département du SIDA et des Rétrovirus, Institut Pasteur, U.R.A. CNRS 1157, Paris, France.
Grau
O
O
Clavel
F
F
Montagnier
L
L
Blanchard
A
A
D002460:Cell Line; D046508:Culture Techniques; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D003851:Deoxyribonucleases / Q000096:biosynthesis / Q000494:pharmacology*; D004866:Equipment Contamination; D024841:Fluoroquinolones; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D009174:Mycoplasma / Q000187:drug effects / Q000201:enzymology / Q000302:isolation & purification*; D016858:Mycoplasma fermentans / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D015363:Quinolones / Q000494:pharmacology; D012194:RNA-Directed DNA Polymerase / Q000032:analysis; D018894:Reverse Transcriptase Inhibitors; D013379:Substrate Specificity; D013601:T-Lymphocytes
D017931:DNA Primers; D004269:DNA, Bacterial; D024841:Fluoroquinolones; D015363:Quinolones; D018894:Reverse Transcriptase Inhibitors; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D003851:Deoxyribonucleases; C061363:sparfloxacin