EBV Conference 2008 Guangzhou - Baylor College of Medicine

65 (RegID: 1320)
Kudakwashe Mutyambizi
Institution: University of Cambridge/ Yale University School of Medicine
e-mail: km451@cam.ac.uk
AN IN VITRO MODEL FOR LATENCY IN THE GAMMAHERPESVIRUSES
K. Mutyambizi (1, 2), H. Coleman (1), V. Connor (1), S. Efstathiou (1).
Posterabstract:
Background: The human gammaherpesviruses EBV and KSHV realize their oncogenic potential during
latent infection. The species specificity of EBV and KSHV has hindered their study in animal models.
Murine gammaherpesvirus (MHV-68) efficiently infects the laboratory mouse providing a representative
gammaherpesvirus for the study of latency. Existing models for latency in MHV-68 have been confounded
by spontaneous reactivation. MHV68 ORF50, homologous to BRLF1 in EBV, is the major MHV68 viral
trans-activator mediating the switch between lytic and latent infection. To facilitate studies on viral
oncogenesis in gammaherpesvirus infection, an in vitro model of latency was established using replication
defective mutants (Δ50 MHV68) with the GFP reporter gene under ORF50 promoter control.
Methods: 3T3 fibroblasts, RAW macrophages and NS0 B cells were coordinately infected with Δ50
MHV-68 and screened for spontaneous reactivation using flow cytometry. Infected cultures were
artificially reactivated from latency by super-infection with WT MHV-68 to determine reactivation
efficiencies. Expression profiles of latent and lytic transcripts were established by quantitative real time
RT-PCR over a 3 day and 30 day time course to establish the transcriptional profile in early latency and
during maintenance of latency.
Results: Latency was readily established and maintained in 3T3 and RAW cells, with reactivation
efficiencies of 40-60%. The resistance to infection of NS0 cells by Δ50 MHV-68 restricted their further
study. Latently infected 3T3 and RAW cultures exhibited stable transcription of latency associated genes
including ORF73 indicating episomal maintenance. Lytic transcripts were negligibly transcribed with no
spontaneous reactivation detected.
Conclusion: We present a clean latency system devoid of contaminating lytic competent virus. It is a
tractable system enabling analysis during the establishment and maintenance phases of latency.
Furthermore, we distinguish the latent transcriptional profile from the lytic profile during early infection.
In this system, latent virus can be artificially reactivated, allowing diverse applications.
EBV Conference 2008 Guangzhou
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