Background:  Kaposi’s sarcoma-associated herpesvirus (KSHV; human herpesvirus-8, HHV-8)  follows an ordered lytic gene expression program after induction with tetra-decanoyl phorbol acetate. We produced an initial description of the KSHV expression program using viral microarrays, but precise kinetic assignments for some genes were unclear. Classically, late herpesvirus genes require viral DNA replication for maximal expression. We used cidofovir (CDR), a nucleotide-analogue KSHV DNA polymerase inhibitor, to dissect KSHV expression into 2 components:  genes expressed without viral DNA replication, and those requiring it.

Methods:  KSHV latently infected primary effusion lymphoma-derived BCBL-1 cells were induced into lytic replication by tetra-decanoyl phorbol acetate, with and without added CDR. KSHV replication and its suppression by CDR were verified by real-time PCR. Induction was assessed by RNA blots and real-time RT-PCR. RNA isolated from the cells was used as template for cDNA synthesis, labeled indirectly with Cy-3 and Cy-5 fluorochromes and hybridized to a specially constructed KSHV long oligonucleotide array containing detectors for essentially all known KSHV ORFs, with selected splice variants. KSHV expression was normalized against a cellular housekeeping gene set. Additional analyses, including quality control filtering, hierarchical clustering, and various statistical measures were performed.

Results:  RNA blot and real-time PCR analyses confirmed that tetra-decanoyl phorbol acetate induced lytic replication and cidofovir inhibited replication. Microarray analysis showed that expression of known immediate-early genes, (e.g., ORFs: 50, 57, and K8) serving lytic regulatory roles were relatively unaffected by CDR, while known late tegument and capsid structural genes, (e.g., ORFs 25, 26, 68 and assembly protein ORF 17) were CDR-sensitive. Latency associated transcript ORF 73 was unaffected by tetra-decanoyl phorbol acetate or CDR, suggesting it was constitutively expressed. Expression of several viral cell homologs, such as K2 (vIL-6), ORF 72 (vCyclin) and ORF 74 (vGPCR), were unaffected by CDR, while others, such as ORF 16 (vBCL-2), K11.1 (vIRF-2), K10.5 (LANA2, vIRF-3) were inhibited.

Conclusions:  CDR effectively enabled the discrimination between KSHV genes whose full expression required viral DNA replication and those that did not. The data provides additional insights into KSHV replication and pathogenesis strategies, particularly for KSHV genes with previously unclear kinetics, and helped clarify which viral cell homologs are expressed at particular times during replication.

Keywords: KSHV; HHV-8; Gene Expression