In the beta- and gammaherpesviruses, a specialized complex of viral transcriptional activators (vTAs) coordinate to direct expression of virus-encoded late genes, which are critical for viral assembly and whose transcription initiates only after the onset of viral DNA replication. The vTAs in Kaposi’s sarcoma-associated herpesvirus (KSHV) are ORF18, ORF24, ORF30, ORF31, ORF34, and ORF66. While the general organization of the vTA complex has been mapped, the individual roles of these proteins and how they coordinate to activate late gene promoters remain largely unknown. Here, we performed a comprehensive mutational analysis of the conserved residues in ORF18, which is a highly interconnected vTA component. Surprisingly, the mutants were largely selective for disrupting the interaction with ORF30 but not the other three ORF18 binding partners. Furthermore, disrupting the ORF18-ORF30 interaction weakened the vTA complex as a whole, and an ORF18 point mutant that failed to bind ORF30 was unable to complement an ORF18 null virus. Thus, contacts between individual vTAs are critical as even small disruptions in this complex result in profound defects in KSHV late gene expression. IMPORTANCE Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi’s sarcoma and other B-cell cancers and remains a leading cause of death in immunocompromised individuals. A key step in the production of infectious virions is the transcription of viral late genes, which generates capsid and structural proteins and requires the coordination of six viral proteins that form a complex. The role of these proteins during transcription complex formation and the importance of protein-protein interactions are not well understood. Here, we focused on a central component of the complex, ORF18, and revealed that disruption of its interaction with even a single component of the complex (ORF30) prevents late gene expression and completion of the viral lifecycle. These findings underscore how individual interactions between the late gene transcription components are critical for both the stability and function of the complex.
Late gene transcription in the beta- and gammaherpesviruses depends on a set of virally encoded transcriptional activators (vTAs) that hijack the host transcriptional machinery and direct it to a subset of viral genes that are required for completion of the viral replication cycle and capsid assembly. In Kaposi’s sarcoma-associated herpesvirus (KSHV), these vTAs are encoded by ORF18, ORF24, ORF30, ORF31, ORF34, and ORF66. Assembly of the vTAs into a complex is critical for late gene transcription, and thus, deciphering the architecture of the complex is central to understanding its transcriptional regulatory activity. Here, we generated an ORF66-null virus and confirmed that it fails to produce late genes and infectious virions. We show that ORF66 is incorporated into the vTA complex primarily through its interaction with ORF34, which is dependent upon a set of four conserved cysteine-rich motifs in the C-terminal domain of ORF66. While both ORF24 and ORF66 occupy the canonical K8.1 late gene promoter, their promoter occupancy requires the presence of the other vTAs, suggesting that sequence-specific, stable binding requires assembly of the entire complex on the promoter. Additionally, we found that ORF24 expression is impaired in the absence of a stable vTA complex. This work extends our knowledge about the architecture of the KSHV viral preinitiation complex and suggests that it functions as a complex to recognize late gene promoters. IMPORTANCE Kaposi’s sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is an oncogenic gammaherpesvirus that is the causative agent of multiple human cancers. The release of infectious virions requires the production of capsid proteins and other late genes, whose production is transcriptionally controlled by a complex of six virally encoded proteins that hijack the host transcription machinery. It is poorly understood how this complex assembles or what function five of its six components play in transcription. Here, we demonstrate that ORF66 is an essential component of this complex in KSHV and that its inclusion in the complex depends upon its C-terminal domain, which contains highly conserved cysteine-rich motifs reminiscent of zinc finger motifs. Additionally, we examined the assembly of the viral preinitiation complex at late gene promoters and found that while sequence-specific binding of late gene promoters requires ORF24, it additionally requires a fully assembled viral preinitiation complex.
10In the beta-and gammaherpesviruses, a specialized complex of viral transcriptional activators 11 (vTAs) coordinate to direct expression of virus-encoded late genes, which are critical for viral 12 assembly and whose transcription initiates only after the onset of viral DNA replication. The 13 vTAs in Kaposi's sarcoma-associated herpesvirus (KSHV) are ORF18, ORF24, ORF30, ORF31, 14 ORF34, and ORF66. While the general organization of the vTA complex has been mapped, the 15 individual roles of these proteins, and how they coordinate to activate late gene promoters, 16 remains largely unknown. Here, we performed a comprehensive mutational analysis of the 17 conserved residues in ORF18, a highly interconnected vTA component. Surprisingly, the 18 mutants were largely selective for disrupting the interaction with ORF30 but not the other 19 three ORF18 binding partners. Furthermore, disrupting the ORF18-ORF30 interaction weakened 20 the vTA complex as a whole, and an ORF18 point mutant that failed to bind ORF30 was unable interactions between the late gene transcription components are critical for both the stability 35 and function of the complex. 36 37 genes (20, 21). 62Studies in both beta-and gammaherpesviruses indicate that the vTAs form a complex, 63 the general organization of which has been mapped in MCMV and KSHV (7, 9, 10, 19) (Figure 64 1A). Notably, several recent reports demonstrate that specific interactions between the vTAs
11Late gene transcription in the beta-and gammaherpesviruses depends on a set of virally-12 encoded transcriptional activators (vTAs) that hijack the host transcriptional machinery and direct 13 it to a subset of viral genes that are required for completion of the viral replication cycle and capsid 14 assembly. In Kaposi's sarcoma-associated herpesvirus (KSHV), these vTAs are encoded by 15 ORF18, ORF24, ORF30, ORF31, ORF34, ORF66. Assembly of the vTAs into a complex is critical 16 for late gene transcription, and thus deciphering the architecture of the complex is central to 17 understanding its transcriptional regulatory activity. Here, we generated an ORF66-null virus and 18 confirmed that it fails to produce late genes and infectious virions. We show that ORF66 is 19 incorporated into the vTA complex primarily through its interaction with ORF34, which is mediated 20 by a set of four conserved cysteine-rich motifs in the C-terminal domain of ORF66. While both 21 ORF24 and ORF66 occupy the canonical K8.1 late gene promoter, their promoter occupancy 22requires the presence of the other vTAs, suggesting that sequence-specific, stable binding 23 requires assembly of the entire complex on the promoter. Additionally, we find that ORF24 24 expression is impaired in the absence of a stable vTA complex. This work extends our knowledge 25 about the architecture of the KSHV vPIC and suggests that it functions as a complex to recognize 26 late gene promoters. 27 28 IMPORTANCE 29Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is an oncogenic 30 gammaherpesvirus that is the causative agent of multiple human cancers. Release of infectious 31 virions requires production of capsid proteins and other late genes, whose production are 32 transcriptionally controlled by a complex of six virally-encoded proteins that hijack the host 33 transcription machinery. It is poorly understood how this complex assembles or what function five 34 of its six components play in transcription. Here, we demonstrate that ORF66 is an essential 35 component of this complex in KSHV and that its inclusion in the complex is mediated through its 36 C-terminal domain, which contains highly conserved cysteine-rich motifs reminiscent of zinc finger 37 motifs. Additionally, we examine assembly of the viral pre-initiation complex at late gene 38 promoters and find that while sequence-specific binding of late gene promoters requires ORF24, 39 it additionally requires a fully assembled viral pre-initation complex. 40 41
β-and γ-herpesviruses include the oncogenic human viruses Kaposi's sarcoma-associated virus (KSHV) and Epstein-Barr virus (EBV), and human cytomegalovirus (HCMV), which is a significant cause of congenital disease. Near the end of their replication cycle, these viruses transcribe their late genes in a manner distinct from host transcription. Late gene transcription requires six virally encoded proteins, one of which is a functional mimic of host TATA-box-binding protein (TBP) that is also involved in recruitment of RNA polymerase II (Pol II) via unknown mechanisms. Here, we applied biochemical protein interaction studies together with electron microscopy-based imaging of a reconstituted human preinitiation complex to define the mechanism underlying Pol II recruitment. These data revealed that the herpesviral TBP, encoded by ORF24 in KSHV, makes a direct protein-protein contact with the C-terminal domain of host RNA polymerase II (Pol II), which is a unique feature that functionally distinguishes viral from cellular TBP. The interaction is mediated by the N-terminal domain (NTD) of ORF24 through a conserved motif that is shared in its β-and γ-herpesvirus homologs. Thus, these herpesviruses employ an unprecedented strategy in eukaryotic transcription, wherein promoter recognition and polymerase recruitment are facilitated by a single transcriptional activator with functionally distinct domains.
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