The Interaction between ORF18 and ORF30 Is Required for Late Gene Expression in Kaposi's Sarcoma-Associated Herpesvirus

Castañeda, Angelica F.; Glaunsinger, Britt A.

doi:10.1128/jvi.01488-18

Cited by 22 publications

(52 citation statements)

References 31 publications

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“…Interaction of ORF66 and ORF34 and its function in virus production. ORF66 was reported to interact with other vPIC components, such as ORF31, ORF18, and ORF34, by us and others (17,19,21). We also found that ORF34 operated as the vPIC hub, by bridging ORF24 and vPIC components, including ORF66.…”

Section: P=103 X 10 -7supporting

confidence: 70%

Kaposi's Sarcoma-Associated Herpesvirus ORF66 Is Essential for Late Gene Expression and Virus Production via Interaction with ORF34

Watanabe

Nishimura

Izumi

et al. 2020

J Virol

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Kaposi’s sarcoma-associated herpesvirus (KSHV) is closely associated with B-cell and endothelial cell malignancies. After the initial infection, KSHV retains its viral genome in the nucleus of the host cell and establishes a lifelong latency. During lytic infection, KSHV-encoded lytic-related proteins are expressed in a sequential manner and are classified as immediate early, early, and late (L) gene transcripts. The transcriptional initiation of KSHV late genes is thought to require the complex formation of the viral preinitiation complex (vPIC), which may consist of at least 6 transcription factors (ORF18, -24, -30, -31, -34, and -66). However, the functional role of ORF66 in vPIC during KSHV replication remains largely unclear. Here, we generated ORF66-deficient KSHV using a bacterial artificial chromosome (BAC) system to evaluate its role during viral replication. While ORF66-deficient KSHV demonstrated mainly attenuated late gene expression and decreased virus production, viral DNA replication was unaffected. Chromatin immunoprecipitation analysis showed that ORF66 bound to the promoters of a late gene (K8.1) but did not bind to those of a latent gene (ORF72), an immediate early gene (ORF16), or an early gene (ORF46/47). Furthermore, we found that three highly conserved C-X-X-C sequences and a conserved leucine repeat in the C-terminal region of ORF66 were essential for the interaction with ORF34, the transcription of K8.1, and virus production. The interaction between ORF66 and ORF34 occurred in a zinc-dependent manner. Our data support a model in which ORF66 serves as a critical vPIC component to promote late viral gene expression and virus production. IMPORTANCE KSHV ORF66 is expressed during the early stages of lytic infection, and ORF66 and vPIC are thought to contribute significantly to late gene expression. However, the physiological importance of ORF66 in terms of vPIC formation remains poorly understood. Therefore, we generated an ORF66-deficient BAC clone and evaluated its viral replication. The results showed that ORF66 plays a critical role in virus production and the transcription of L genes. To our knowledge, this is the first report showing the function of ORF66 in virus replication using ORF66-deficient KSHV. We also clarified that ORF66 interacts with the transcription start site of the K8.1 gene, a late gene. Furthermore, we identified the ORF34-binding motifs in the ORF66 C terminus: three C-X-X-C sequences and a leucine-repeat sequence, which are highly conserved among beta- and gammaherpesviruses. Our study provides insights into the regulatory mechanisms of not only the late gene expression of KSHV but also those of other herpesviruses.

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Section: P=103 X 10 -7supporting

confidence: 70%

Kaposi's Sarcoma-Associated Herpesvirus ORF66 Is Essential for Late Gene Expression and Virus Production via Interaction with ORF34

Watanabe

Nishimura

Izumi

et al. 2020

J Virol

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“…We assessed the ability of the NTD chimeras to functionally complement ORF24 using an established transfection-based late gene transcription assay [13][14][15]. The six vTAs (ORFs 18, 30, 31, 34, 66 and either WT ORF24, its homologs, or chimeras) were co-transfected into HEK293T cells, along with a firefly luciferase reporter controlled by the K8.1 late gene promoter or, as a control, the early ORF57 promoter.…”

Section: Plos Pathogensmentioning

confidence: 99%

“…First, β/γ late gene transcription is regulated in part by a core promoter sequence 12-15 base pairs in length that has a TATT motif followed by a RVNYS motif in lieu of the canonical TATA box found in early viral promoters and in some cellular promoters [8][9][10][11]. Additionally, late gene expression requires at least six viral proteins, called viral transcriptional activators (vTAs), which form a complex at late gene promoters [12][13][14][15][16][17]. Little is known about the functional role these vTAs play in late gene transcription.…”

Section: Introductionmentioning

confidence: 99%

The gammaherpesviral TATA-box-binding protein directly interacts with the CTD of host RNA Pol II to direct late gene transcription

et al. 2020

Self Cite

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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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“…Despite the paucity of functional information on the individual vPIC components, protein-protein interaction mapping has provided insight into the overall complex organization [13,18]. Furthermore, point mutants that disrupt individual inter-subunit vPIC interactions abrogate late gene transcription, highlighting the fact that vPIC complex integrity is critical for function [13,20,21].…”

Section: Introductionmentioning

confidence: 99%

An integrative approach identifies direct targets of the late viral transcription complex and an expanded promoter recognition motif in Kaposi’s sarcoma-associated herpesvirus

Nandakumar

Glaunsinger

2019

PLoS Pathog

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The structural proteins of DNA viruses are generally encoded by late genes, whose expression relies on recruitment of the host transcriptional machinery only after the onset of viral genome replication. β and γ-herpesviruses encode a unique six-member viral pre-initiation complex (vPIC) for this purpose, although how the vPIC directs specific activation of late genes remains largely unknown. The specificity underlying late transcription is particularly notable given that late gene promoters are unusually small, with a modified TATA-box being the only recognizable element. Here, we explored the basis for this specificity using an integrative approach to evaluate vPIC-dependent gene expression combined with promoter occupancy during Kaposi’s sarcoma-associated herpesvirus (KSHV) infection. This approach distinguished the direct and indirect targets of the vPIC, ultimately revealing a novel promoter motif critical for KSHV vPIC binding. Additionally, we found that the KSHV vPIC component ORF24 is required for efficient viral DNA replication and identified a ORF24 binding element in the origin of replication that is necessary for late gene promoter activation. Together, these results identify an elusive element that contributes to vPIC specificity and suggest novel links between KSHV DNA replication and late transcription.

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The Interaction between ORF18 and ORF30 Is Required for Late Gene Expression in Kaposi's Sarcoma-Associated Herpesvirus

Cited by 22 publications

References 31 publications

Kaposi's Sarcoma-Associated Herpesvirus ORF66 Is Essential for Late Gene Expression and Virus Production via Interaction with ORF34

Kaposi's Sarcoma-Associated Herpesvirus ORF66 Is Essential for Late Gene Expression and Virus Production via Interaction with ORF34

The gammaherpesviral TATA-box-binding protein directly interacts with the CTD of host RNA Pol II to direct late gene transcription

An integrative approach identifies direct targets of the late viral transcription complex and an expanded promoter recognition motif in Kaposi’s sarcoma-associated herpesvirus

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