Kaposi's Sarcoma-Associated Herpesvirus ORF18 and ORF30 Are Essential for Late Gene Expression during Lytic Replication

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with several human malignances. As saliva is likely the major vehicle for KSHV transmission, we studied in vitro KSHV infection of oral epithelial cells. Through infection of two types of oral epithelial cells, normal human oral keratinocytes (NHOKs) and papilloma-immortalized human oral keratinocyte (HOK16B) cells, we found that KSHV can undergo robust lytic replication in oral epithelial cells. By employing de novo lytic infection of HOK16B cells, we studied the functions of two previously uncharacterized genes, ORF18 and ORF30, during the KSHV lytic cycle. For this purpose, an ORF18-deficient virus and an ORF30-deficient virus were generated using a mutagenesis strategy based on bacterial artificial chromosome (BAC) technology. We found that neither ORF18 nor ORF30 is required for immediately early or early gene expression or viral DNA replication, but each is essential for late gene expression during both de novo lytic replication and reactivation. This critical role of ORF18 and ORF30 in late gene expression was also observed during KSHV reactivation. In addition, global analysis of viral transcripts by RNA sequencing indicated that ORF18 and ORF30 control the same set of viral genes. Therefore, we suggest that these two viral ORFs are involved in the same mechanism or pathway that coregulates the viral late genes as a group. IMPORTANCEWhile KSHV can infect multiple cell types in vitro, only a few can support a full lytic replication cycle with progeny virions produced. Consequently, KSHV lytic replication is mostly studied through reactivation, which requires chemicals to induce the lytic cycle or overexpression of the viral transcriptional activator, RTA. In this study, we present a robust de novo lytic infection system based on oral epithelial cells. Using this system, we demonstrate the role of two viral ORFs, ORF18 and ORF30, in regulating viral gene expression during KSHV lytic replication. As the major route of KSHV transmission is thought to be via saliva, this new KSHV lytic replication system will have important utility in the field.

Virus-Like Vesicles of Kaposi's Sarcoma-Associated Herpesvirus Activate Lytic Replication by Triggering Differentiation Signaling

“…Human papillomavirus 16-immortalized keratinocytes, (HOK16B cells) were maintained in keratinocyte growth medium (Lonza) (61). HOK16B.219 cells were established by infection with KSHV.219 and were further selected and maintained with 0.5 g/ml puromycin.…”

“…The number of PFU of MHV-68 virions was determined by infecting Vero cells (62). Infectious units (IU) of KSHV virions were quantified by counting GFP-positive clusters after infecting 293T cells (61). The concentration of KSHV VLVs was measured using a Bradford protein assay.…”

“…Three days postinfection, GFP-positive cell clusters containing two or more cells were counted under a fluorescence microscope to determine the titers of infectious KSHV viruses. Infectious units are expressed as the number of GFP-positive cell clusters in each well at the time of analysis (61).…”

“…For poly(A) RNA deep-sequencing analysis, HOK16B or HOK16B.219 cells were either mock treated or treated with VLVs for 1 or 2 days; then total RNA was purified with TRIzol, and RNA-seq libraries were prepared by a method described previously (61). Multiplex sequencing was performed on 50-bp single-end reads with an Illumina HiSeq 2000 machine at the UCLA Clinical Microarray Core.…”

CryoEM and mutagenesis reveal that the smallest capsid protein cements and stabilizes Kaposi's sarcoma-associated herpesvirus capsid

“…KSHV BAC16 genome was modified according to a previously described method (19). Briefly, for KSHV-SCPnull,…”