Session 43-Themed Discussion
TD: XMRV: New Findings and Controversies
Wednesday, 1-2 pm; Room 302-304
Paper # 215
Development of a GFP-indicator Cell Line for the Detection of XMRV
KyeongEun Lee, F Ruscetti, P Lloyd, A Rein, G Fanning-Heidecker, and V KewalRamani
NCI-Frederick, MD, US
Background: HIV titer can be estimated using indicator cell lines within days of infection. HIV indicator cells rely on production of tat to transactivate expression of a reporter gene under the control of HIV LTR sequences. Simple retroviruses typically do not encode transcriptional transactivators. For simple retroviruses that lack transformational or cytopathic activity, their titers are measured by endpoint dilution and assaying for virus proliferation after weeks of cell culture. Replication-dependent vectors have been used to monitor mobilization by retrotransposable elements or the replication of retroviruses. Here we describe an indicator cell line for the detection of infectious xenotropic murine leukemia virus-related virus (XMRV) that relies on the propagation of a vector, which leads to expression of a GFP reporter.
Methods: We constructed a murine leukemia virus vector encoding puromycin resistance and a CMV enhancer/promoter driven GFP reporter gene whose transcription was antisense to the vector mRNA. The GFP reporter sequence (iGFP) was interrupted by an intron placed in the sense direction relative to the vector. The prostate cell line, LNCaP, was stably transfected with the above construct and drug-resistant clones were isolated. GFP was detected by fluorescence microscopy or FACS analysis.
Results: Several LNCaP-iGFP cell clones displaying sensitivity to XMRV infection after endpoint dilution were isolated and designated Detectors of Exogenous Retroviral Sequence Elements (DERSE) cells. GFP signal could be detected within 3 days of infection, with the number of GFP+ cells increasing over time. GFP signal after virus inoculation was dose-dependent and could be impaired by heat inactivation or the addition of zidovudine to cultures at the time of infection.
Conclusions: In principle, DERSE cells should also detect other gammaretroviruses capable of infecting human cell lines. Because this indicator cell system utilizes GFP as a reporter, infection can be monitored in live cultures by fluorescence microscopy. DERSE cells provide a facile assay to assess antiviral or antibody mediated neutralization of XMRV, and should be useful in assessing the presence of infectious XMRV in patient samples.
