Biology subjects

Castaneda, A. F.

Publications and source records attributed to Castaneda, A. F..

3 recordsLinked to original sources

bioRxiv2020

The γ-herpesviral TATA box binding protein directly interacts with RNA Polymerase II to direct late gene transcription

{beta}- and {gamma}-herpesviruses include the oncogenic human viruses Kaposis 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 {beta}- and {gamma}-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. SIGNIFICANCE STATEMENTThe {beta}- and {gamma}-herpesviruses mediate their late gene transcription through a set of viral transcriptional activators (vTAs). One of these vTAs, ORF24 in Kaposis sarcoma-associated herpesvirus (KSHV), is a mimic of host TATA-box-binding protein (TBP). We demonstrate that the N-terminal domain of ORF24 and its homologs from other {beta}- and {gamma}-herpesviruses directly bind the unstructured C-terminal domain (CTD) of RNA Pol II. This functionally distinguishes the viral TBP mimic from cellular TBP, which does not bind Pol II. Thus, herpesviruses encode a transcription factor that has the dual ability to directly interact with promoter DNA and the polymerase, a property which is unique in eukaryotic transcription and is conceptually akin to prokaryotic transcription factors.

molecular biology↗

bioRxiv2019

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

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 Kaposis sarcoma-associated herpesvirus (KSHV), these vTAs are encoded by ORF18, ORF24, ORF30, ORF31, ORF34, 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 mediated by 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 find that ORF24 expression is impaired in the absence of a stable vTA complex. This work extends our knowledge about the architecture of the KSHV vPIC and suggests that it functions as a complex to recognize late gene promoters.\n\nIMPORTANCEKaposis sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is an oncogenic gammaherpesvirus that is the causative agent of multiple human cancers. Release of infectious virions requires production of capsid proteins and other late genes, whose production are 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 is mediated through its C-terminal domain, which contains highly conserved cysteine-rich motifs reminiscent of zinc finger motifs. Additionally, we examine assembly of the viral pre-initiation complex at late gene promoters and find that while sequence-specific binding of late gene promoters requires ORF24, it additionally requires a fully assembled viral pre-initation complex.

molecular biology↗

bioRxiv2018

The interaction between ORF18 and ORF30 is required for late gene transcription in Kaposi’s sarcoma-associated herpesvirus

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 Kaposis 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, remains 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.

molecular biology↗