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:10.1101/401976

Cited by 4 publications

(14 citation statements)

References 32 publications

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“…To assess the contacts that ORF66 makes within the vTA complex, we tested its pairwise interactions with other components by co-IP. As has been previously observed, ORF66 interacts with ORF18 ( Figure 2C ) (11), ORF31 ( Figure 2D ), and most robustly with ORF34 ( Figure 2E ) (10). We were unable to detect direct protein-protein interactions between ORF66 and ORF24 or ORF30 (data not shown).…”

Section: Resultssupporting

confidence: 85%

“…Based on our observation that ORF66 is essential for late gene transcription and our previous observation that ORF66 interacts with ORF18 (11), we sought to further characterize the interactions of ORF66 within the vTA complex ( Figure 2A ). We began by assessing its association with the complex as a whole upon immunoprecipitation of different vTA components in transiently transfected HEK293T cells.…”

Section: Resultsmentioning

confidence: 99%

“…To determine if the Cx n C motifs were necessary for late gene transcription, we used a previously described reporter assay wherein firefly luciferase gene is under the control of an early gene promoter (from ORF57) or a late gene promoter (K8.1) (11). In addition to the firefly luciferase reporter, the six vTAs (including wild-type or mutant ORF66) were transiently transfected into HEK293T cells along with a renilla luciferase reporter to control for transfection efficiency.…”

Section: Resultsmentioning

confidence: 99%

“…We also generated KSHV with N-terminally HA tagged ORF24 (HA24; (1)) and lacking either ORF66 (HA24/66S) or ORF30 (H24/30S) to determine whether promoter binding by ORF24 is influenced by other vTAs. Unlike ORF66, the ORF30 vTA is a small protein with no predicted nucleic acid binding properties and its only connection with the complex occurs through ORF18 (see Figure 2A ) (11). Thus, ORF30 deletion should enable evaluation of the general importance of the vTA complex in ORF24 late gene promoter binding.…”

Section: Resultsmentioning

confidence: 99%

“…Stop mutants of five of the six KSHV vTAs have been generated and tested, revealing that they share a common phenotype in which late gene transcription fails to occur, ultimately preventing release of infectious virions (6, 8-10). Disrupting protein-protein contacts within the complex also prevents late gene transcription, as preventing the incorporation of even the smallest vPIC component, ORF30 (77 amino acids) by disrupting its interaction with its binding partner ORF18 completely prevents late gene transcription in both KSHV and HCMV (11, 12). Although deletion of KSHV ORF66 has not been tested, an EBV mutant lacking the ORF66 homolog (BFRF2) fails to produce infectious virions due to a defect in late gene transcription (4).…”

Section: Introductionmentioning

confidence: 99%

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

Didychuk¹,

Castañeda²,

Kushnir

et al. 2019

Preprint

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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

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Section: Resultssupporting

confidence: 85%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Didychuk¹,

Castañeda²,

Kushnir

et al. 2019

Preprint

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Kaposi’s sarcoma-associated herpesvirus ORF66 is essential for late gene expression and virus production via interaction with ORF34

Watanabe

Nishimura

Izumi

et al. 2019

Preprint

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25Kaposi's sarcoma-associated herpesvirus (KSHV) is closely 26 associated with B-cell and endothelial cell malignancies. After the initial 27 infection, KSHV retains its viral genome in the nucleus of the host cell and 28 establishes a lifelong latency. During lytic infection, KSHV encoded lytic-related 29 proteins are expressed in a sequential manner and are classified as immediate 30 early, early, and late gene transcripts. The transcriptional initiation of KSHV late 31 genes is thought to require the complex formation of the virus specific pre-32 initiation complex (vPIC), which may consist of at least 6 transcription factors 33 (ORF18, 24, 30, 31, 34, and 66). However, the functional role of ORF66 in vPIC 34 during KSHV replication remains largely unclear. Here, we generated ORF66-35 deficient KSHV using a BAC system to evaluate its role during viral replication. 36While ORF66-deficient KSHV demonstrated mainly attenuated late gene 37 expression and decreased viral production, viral DNA replication was 38 unaffected. CHIP analysis showed that ORF66 bound to the promoters of late 39 gene (K8.1), but did not to those of latent gene (ORF72), immediate early gene 40 (ORF16) and early gene (ORF46/47). Furthermore, we found that three highly 41 conserved C-X-X-C sequences and a conserved leucine-repeat in the C-42 terminal region of ORF66 were essential for interaction with ORF34 and viral 43 production. The interaction between ORF66 and ORF34 occurred in a zinc-44 dependent manner. Our data support a model, in which ORF66 serves as a 45 critical vPIC component to promote late viral gene expression and viral 46 production. 47 IMPORTANCE 49 KSHV ORF66, a late gene product, and vPIC are thought to contribute 50 significantly to late gene expression during the lytic replication. However, the 51 physiological importance of ORF66 in terms of viral replication and vPIC 52 formation remains poorly understood. Therfore, we generated a ORF66-53 deficient BAC clone and evaluated its viral replication. Results showed that 54 ORF66 played a critical role in virus production and the transcription of L genes. 55To our knowledge, this is the first report showing ORF66 function in virus 56 replication using ORF66-deficient KSHV. We also clarified that ORF66 57 interacted with the transcription start site of K8.1 gene, a late gene. 58 Furthermore, we identified the ORF34-binding motifs in the ORF66 C-terminus: 59 three C-X-X-C sequences and a leucine-repeat sequence, which are highly 60 conserved among -and -herpesviruses. Our study provides insights into the 61 regulatory mechanisms of not only the late gene expression of KSHV but also 62 those of other herpesviruses. 63 64 65 replication. Finally, the transcriptional products of L genes, contribute to viral 90 particle formation by its encoding viral structure proteins (15). 91The viral pre-initiation complex (vPIC) has recently been proposed to 92 regulate L gene expression(16). vPIC is composed of several viral proteins 93 conserved among -and -herpesviruses (17), and has f...

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

Castañeda

Glaunsinger

2019

J Virol

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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.

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

Cited by 4 publications

References 32 publications

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

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

Kaposi’s sarcoma-associated herpesvirus ORF66 is essential for late gene expression and virus production via interaction with ORF34

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

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

Cited by 4 publications

References 32 publications

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

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

Kaposi’s sarcoma-associated herpesvirus ORF66 is essential for late gene expression and virus production via interaction with ORF34

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

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

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