Kaposi’s sarcoma-associated herpesvirus ORF34 is essential for late gene expression and virus production

Abstract

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman’s disease. KSHV establishes a life-long infection in its host and alternates between a latent and lytic infection state. During lytic infection, lytic-related genes are expressed in a temporal manner and categorized as immediate early, early, and late gene transcripts. ORF34 is an early-late gene that interacts with several viral transcription-associated factors, however its physiological importance remains poorly understood. Here, we investigated the role of ORF34 during KSHV infection by generating ORF34-deficient KSHV, using a bacterial artificial chromosome system. Our results reveal that ORF34-deficient KSHV exhibited significantly attenuated late gene expression and viral production but did not affect viral DNA replication. ORF34 interacted with transcription factors ORF18, ORF24, ORF31, and ORF66, and a novel ORF34-interaction partner, ORF23. The C-terminal region of ORF34 was important for interaction with ORF24 and viral production. Our data support a model, in which ORF34 serves as a hub for recruiting a viral transcription complex to ORF24 to promote late viral gene expression.

Transcriptional and Post-Transcriptional Regulation of Viral Gene Expression in the Gamma-Herpesvirus Kaposi’s Sarcoma-Associated Herpesvirus

“…Homologous viral PICs were identified in beta- and gamma-herpesviruses, and consist of proteins that are required for replication and have no homologs in alpha-herpesviruses [68]. In KSHV, the viral PIC is composed of ORF18, ORF24, ORF30, ORF31, ORF34 and ORF66 [69–73] (Figure 1). One study suggested that ORF23 may also be part of the complex [69], but its role is in question because ORF23 activity is dispensable for replication in the murine gamma-herpesvirus MHV68 [74].…”

“…In KSHV, the viral PIC is composed of ORF18, ORF24, ORF30, ORF31, ORF34 and ORF66 [69–73] (Figure 1). One study suggested that ORF23 may also be part of the complex [69], but its role is in question because ORF23 activity is dispensable for replication in the murine gamma-herpesvirus MHV68 [74]. Although viral DNA replication is required for late gene transcription during infection, co-expression of the six proteins is sufficient to drive transcription of a reporter with a late gene promoter [70].…”

“…cells in which the alternative PIC was present [75]. The six proteins are also all necessary for late gene expression, as omission of any of them from transfection experiments [70] or deletion of most of them from the genome using the BAC16 system [16] prevents late gene expression (ORF18, ORF30: [71]; ORF24: [72]; ORF31: [73]; ORF34: [69]; ORF66 not yet tested). Interestingly, ORF24 appears to provide two key functions of the complex: DNA binding and RNAPII recruitment.…”

“…Instead, ORF24 substitutes for TBP in the recruitment of other cellular general transcription factors, while also directly recruiting RNAPII [72]. ORF24 only binds one other member of the viral PIC, ORF34, via amino acids 323–339 of ORF24 and the C-terminal domain of ORF34 [69, 70]. In turn, ORF34 may serve as a scaffold, as it interacts with all of the other proteins through a central domain [69, 70].…”

“…ORF24 only binds one other member of the viral PIC, ORF34, via amino acids 323–339 of ORF24 and the C-terminal domain of ORF34 [69, 70]. In turn, ORF34 may serve as a scaffold, as it interacts with all of the other proteins through a central domain [69, 70]. The exact function of the other four components of the viral PIC remains unclear.…”