Biochemical characterization of a reverse transcriptase activity associated with retroviral-like particles isolated from human placental villous tissue.
The presence of budding type-C retroviral-like particles in normal placental trophoblast, particularly at the basal surface of the placental syncytiotrophoblast, is well documented. Retroviral-like particles were isolated from human placental villous tissues using isopycnic sucrose gradient centrifugation. Reverse transcriptase activity (RTase) associated with isolated retroviral-like particles was characterised using a combination of synthetic template-primers. These studies showed that RTase activity was more specific with poly(rC).oligo(dG)12-18 than poly(dC).oligo(dG)12-18. Furthermore, activity was detected with poly(rCm).oligo(dG)12-18, a template-primer which has previously been shown to be specific for retroviral RTase. Maximum activity appeared at a sucrose density between 1.15-1.17 g/ml, characteristic of enveloped retroviral particles. Electron microscopy examination of the gradient purified particles revealed morphology and size similar to other retroviruses. Endogenous retroviral particles were isolated from 26 out of 32 (81%) first-trimester placental villous tissue extracts. These particles are likely to be product of endogenous proviral sequences present in the germline of humans. Although these studies showed presence of intact retroviral particles in placental tissues, it was not possible to propagate the isolated particles in vitro. All attempts to propagate placental retroviral particles by co-cultivation with human cells (U937 and JAr choriocarcinoma cells) and long term placental villous tissue explant cultures were unsuccessful. Subsequently, there was no evidence of retroviral-like particles or RTase activity in these cell cultures, including after stimulation with 5'-azacytidine or dexamethasone, chemical agents known to stimulate particle production in virus-infected lines.
Mwenda
J M
JM
Institute of Primate Research, National Museums of Kenya, Karen, Nairobi.
D002499:Centrifugation, Density Gradient; D002824:Chorionic Villi / Q000382:microbiology*; D005260:Female; D006801:Humans; D009843:Oligoribonucleotides / Q000378:metabolism; D011247:Pregnancy; D012194:RNA-Directed DNA Polymerase / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012190:Retroviridae / Q000201:enzymology* / Q000254:growth & development / Q000302:isolation & purification / Q000648:ultrastructure; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D013698:Templates, Genetic; D014776:Virus Cultivation
D009843:Oligoribonucleotides; D012194:RNA-Directed DNA Polymerase