Conserved Cx_nC motifs in Kaposi’s sarcoma-associated herpesvirus ORF66 are required for viral late gene expression and mediate its interaction with ORF34

Abstract

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

The viral packaging motor potentiates late gene expression in Kaposi’s sarcoma-associated herpesvirus

“…As anticipated, knockout of the viral primase ORF56 caused a total loss of DNA replication, as did knockout of ORF75. Loss of the vTA ORF30 caused a ~6-fold defect in viral DNA replication, in accordance with the replication defects we have previously observed when knocking out other vTAs (2,8). CRISPR knockout of ORF29 caused a DNA replication defect comparable to that caused by the loss of ORF30, while knockouts for all cellular genes were largely similar to the controls.…”

“…TurboID-ORF34 was subcloned into the NotI and XhoI sites of N-term 3xHA pcDNA4. Plasmids pcDNA4/TO-ORF18-2xStrep (Addgene 120372), pcDNA4/TO-ORF24-2xStrep (Addgene 129742), pcDNA4/TO-ORF30-2xStrep (Addgene 129743), pcDNA4/TO-ORF31-2xStrep (Addgene plasmid 129744), pcDNA4/TO-ORF66-2xStrep (Addgene 130953) and pcDNA4/TO-2xStrep-ORF34 (Addgene plasmid 120376) have been previously described (7,8). Plasmid K8.1 Pr pGL4.16 + Ori (Addgene 131038) and ORF57 Pr pGL4.16 (Addgene 120378) have been previously described (7).…”

“…In contrast, for the -and herpesviruses like cytomegalovirus (CMV) and Kaposi's sarcoma-associated herpesvirus (KSHV), late genes have strikingly minimal promoters, consisting only of a TATT sequence followed by a short degenerate motif in lieu of a canonical TATA box (2,3). Late gene transcription additionally requires a dedicated set of six essential viral transcriptional activator (vTA) proteins that form a complex at late gene promoters (4)(5)(6)(7)(8)(9)(10). In KSHV, the vTAs are encoded by ORF18, ORF24, ORF30, ORF31, ORF34, and ORF66.…”

“…The best understood of these factors is ORF24, a unique transcriptional coordinator that acts as a viral mimic of cellular TATA-binding protein (TBP) and directly binds both late gene promoters and RNA polymerase II (5,(11)(12)(13). The other vTAs contact one another extensively within the complex, forming interactions that are critical for late gene transcription (6)(7)(8)(14)(15)(16). Several additional factors in -and -herpesviruses have been implicated in late gene expression.…”

“…Late gene production is essential for production of progeny virions (5,8,14,21,22), yet the principles that define the broader regulation of late gene expression remain elusive. We sought to address this gap by identifying viral and cellular factors functionally associated with the vTA complex during late gene transcription.…”