Abstract
Since Kaposi's sarcoma associated herpesvirus (KSHV) establishes a persistent infection in human B cells, B cells are a critical compartment for viral pathogenesis. RTA, the replication and transcription activator of KSHV, can either directly bind to DNA or use cellular DNA binding factors including CBF1/CSL as DNA adaptors. In addition, the viral factors LANA1 and vIRF4 are known to bind to CBF1/CSL and modulate RTA activity. To analyze the contribution of CBF1/CSL to reactivation in human B cells, we have successfully infected DG75 and DG75 CBF1/CSL knock-out cell lines with recombinant KSHV.219 and selected for viral maintenance by selective medium. Both lines maintained the virus irrespective of their CBF1/CSL status. Viral reactivation could be initiated in both B cell lines but viral genome replication was attenuated in CBF1/CSL deficient lines, which also failed to produce detectable levels of infectious virus. Induction of immediate early, early and late viral genes was impaired in CBF1/CSL deficient cells at multiple stages of the reactivation process but could be restored to wild-type levels by reintroduction of CBF1/CSL. To identify additional viral RTA target genes, which are directly controlled by CBF1/CSL, we analyzed promoters of a selected subset of viral genes. We show that the induction of the late viral genes ORF29a and ORF65 by RTA is strongly enhanced by CBF1/CSL. Orthologs of ORF29a in other herpesviruses are part of the terminase complex required for viral packaging. ORF65 encodes the small capsid protein essential for capsid shell assembly. Our study demonstrates for the first time that in human B cells viral replication can be initiated in the absence of CBF1/CSL but the reactivation process is severely attenuated at all stages and does not lead to virion production. Thus, CBF1/CSL acts as a global hub which is used by the virus to coordinate the lytic cascade.
Highlights
Kaposi’s sarcoma associated herpesvirus (KSHV) establishes a persistent infection in the human host
CBF1 deficient and proficient human B cell lines can be infected with recombinant KSHV, express viral latent antigens and maintain the viral genome in the presence of selective cell culture medium In order to analyze the contribution of CBF1 to viral reactivation in KSHV infected B cells we used a CBF1 negative human somatic B cell line
The recombinant virus rKSHV.219 is a derivative of the KSHV strain obtained from JSC-1 cells [20]. rKSHV.219 encodes for red fluorescent protein (RFP) under the control of a CBF1 independent fragment of the KSHV early lytic PAN promoter, constitutively expresses green fluorescent protein (GFP), and carries a puromycin resistance cassette as a selectable marker
Summary
Kaposi’s sarcoma associated herpesvirus (KSHV) establishes a persistent infection in the human host. Infected human B cells in the circulation of the infected host are likely to constitute a major latent reservoir, from where KSHV can reactivate and spread. Infection and virus production of CBF1 proficient and deficient murine fibroblasts was studied in isogenic systems. These studies suggested lytic reactivation is blocked while establishment of latency is not impaired in cells lacking CBF1 [8,9]. Activation of viral targets by activated Notch defined further sets of CBF1 dependent promoters. These studies convincingly showed, that CBF1 contributes to the activation of ORF6, ORF8, ORF19, Author Summary
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
