scholarly journals Association of Kaposi's Sarcoma-Associated Herpesvirus ORF31 with ORF34 and ORF24 Is Critical for Late Gene Expression

2015 ◽  
Vol 89 (11) ◽  
pp. 6148-6154 ◽  
Author(s):  
Kevin Brulois ◽  
Lai-Yee Wong ◽  
Hye-Ra Lee ◽  
Priyanka Sivadas ◽  
Armin Ensser ◽  
...  
Keyword(s):  
Gene Expression ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Late Gene ◽  
Late Gene Expression ◽  
Reading Frame ◽  
Amino Terminal ◽  
Late Genes ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

Transcription of herpesvirus late genes depends on several virus-encoded proteins whose function is not completely understood. Here, we identify a viral trimeric complex of Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 31 (ORF31), ORF24, and ORF34 that is required for late gene expression but not viral DNA replication. We found that (i) ORF34 bridges the interaction between ORF31 and ORF24, (ii) the amino-terminal cysteine-rich and carboxyl-terminal basic domains of ORF31 mediate the ORF31-ORF34 interaction required for late gene expression, and (iii) a complex consisting of ORF24, ORF31, and ORF34 specifically binds to the K8.1 late promoter. Together, our results support the model that a subset of lytic viral proteins assembles into a transcriptional activator complex to induce expression of late genes.

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

2018 ◽  
Vol 93 (1) ◽  
Author(s):  
Angelica F. Castañeda ◽  
Britt A. Glaunsinger
Keyword(s):  
Gene Expression ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Late Gene ◽  
Central Component ◽  
Gene Promoters ◽  
Late Gene Expression ◽  
Late Genes ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTIn 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.IMPORTANCEKaposi’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.

scholarly journals Interaction between ORF24 and ORF34 in the Kaposi's Sarcoma-Associated Herpesvirus Late Gene Transcription Factor Complex Is Essential for Viral Late Gene Expression

2015 ◽  
Vol 90 (1) ◽  
pp. 599-604 ◽  
Author(s):  
Zoe H. Davis ◽  
Charles R. Hesser ◽  
Jimin Park ◽  
Britt A. Glaunsinger
Keyword(s):  
Gene Expression ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Late Gene ◽  
Late Gene Expression ◽  
Reading Frame ◽  
Interaction Interface ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Transcription Factor Complex

Transcription of herpesviral late genes is stimulated after the onset of viral DNA replication but otherwise restricted. Late gene expression in gammaherpesviruses requires the coordination of six early viral proteins, termed viral transactivation factors (vTFs). Here, we mapped the organization of this protein complex for Kaposi's sarcoma-associated herpesvirus. Disruption of this complex via point mutation of the interaction interface between the open reading frame 24 (ORF24) and ORF34 vTFs ablated both late gene expression and viral replication.

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

2019 ◽  
Author(s):  
Divya Nandakumar ◽  
Britt Glaunsinger
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Gene Transcription ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Late Gene ◽  
Late Genes ◽  
Late Transcription ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

AbstractThe 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. Together, these results identify an elusive element that contributes to vPIC specificity and suggest novel links between KSHV DNA replication and late transcription.Author summaryGene expression in DNA viruses often occurs in temporal waves, with expression of essential structural proteins occurring late in infection, after viral genome replication has begun. Strategies underlying expression of these viral late genes are often sophisticated; for example, the β- and γ-herpesviruses encode a six-component viral complex that directs late gene transcription, largely by unknown mechanisms. Here, we evaluated how this complex specifically recognizes late promoters during infection with the oncogenic human γ-herpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV). We found that one of the components of the late transcription complex was required for robust viral DNA replication, suggesting new links between KSHV replication and transcription. Combined measurements of late gene expression and promoter occupancy then revealed which KHSV genes are directly controlled by the late gene transcription complex, leading to identification of a key new regulatory element in KSHV late promoters. Together, these data help explain how the late gene transcription complex is able to bind seemingly minimal promoters with high specificity, ensuring robust expression of viral factors necessary for assembly of progeny virions.

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

2018 ◽  
Author(s):  
Angelica F Castañeda ◽  
Britt A Glaunsinger
Keyword(s):  
Gene Expression ◽  
Kaposi's Sarcoma ◽  
Mutational Analysis ◽  
Kaposi’S Sarcoma ◽  
Late Gene ◽  
Gene Promoters ◽  
Late Gene Expression ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
The Individual ◽  
Kaposi’S Sarcoma Associated Herpesvirus

AbstractIn 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, 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.

scholarly journals The Kaposi’s Sarcoma-Associated Herpesvirus ORF34 Protein Interacts and Stabilizes HIF-2α via Binding to the HIF-2α bHLH and PAS Domains

2019 ◽  
Vol 93 (17) ◽  
Author(s):  
Muzammel Haque ◽  
K. G. Kousoulas
Keyword(s):  
Gene Expression ◽  
Life Cycle ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Late Gene Expression ◽  
Lytic Gene ◽  
Lytic Gene Expression ◽  
Proteasome Pathway ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTHypoxia and hypoxia inducible factors (HIFs) play important roles in the Kaposi’s sarcoma-associated herpesvirus (KSHV) life cycle. KSHV is the causative agent of Kaposi’s sarcoma (KS) and other AIDS-related malignancies. Kaposi’s sarcoma is a highly vascular tumor, which preferentially develops in the lower extremities of the body where blood vessels are often poorly oxygenated. The main cellular responses to hypoxia are mediated mainly by two isoforms of HIF, HIF-1α and HIF-2α. HIF-1α and HIF-2α have common as well as distinct functions, although they are similar in structure and function. Previously, we showed that the KSHV ORF34 protein binds HIF-1α and facilitates its degradation through the ubiquitin-proteasome pathway causing negative regulation of HIF-1α-dependent genes (Haque and Kousoulas, J Virol 87:2164-2173, 2013, https://www.doi.org/10.1128/JVI.02460-12). Herein, we show that theORF34gene is involved in the regulation of KSHV lytic gene expression, since deletion ofORF34resulted in reduced immediate early and early lytic gene expression and blocked late gene expression. Coimmunoprecipitation experiments revealed that the ORF34 protein physically interacted with HIF-2α in transfected as well as in KSHV-infected cells. Utilization of ORF34 truncations revealed that three distinct domains bind HIF-2α and that both bHLH and PAS domains of HIF-2α interacted with ORF34. Unlike HIF-1α, dose-dependent coexpression of ORF34 stabilized the HIF-2α protein, ensuring HIF-2α-dependent transcriptional activity. The ORF34 protein enhanced HIF-2α ubiquitination at the bHLH and PAS domains. The results show that the KSHV ORF34 protein is involved in the KSHV life cycle by regulating the expression of HIF-1α and HIF-2α proteins.IMPORTANCEHypoxia inducible factor 1α (HIF-1α) and HIF-2α are transcription factors which play important roles in the Kaposi’s sarcoma-associated herpesvirus (KSHV) latent and lytic gene replication. Herein, we show that theORF34gene is involved in the regulation of KSHV lytic gene expression, since deletion ofORF34resulted in reduced immediate early and early lytic gene expression and blocked late gene expression. In addition, we demonstrate that the KSHV ORF34 protein binds and stabilizes HIF-2α, in contrast to its role in binding HIF-1α and causing its degradation via the proteasome pathway. Thus, the KSHV ORF34 protein plays a regulatory role in the KSHV life cycle by regulating HIF-1α and HIF-2α expression.

scholarly journals Conserved CxnC Motifs in Kaposi’s Sarcoma-Associated Herpesvirus ORF66 Are Required for Viral Late Gene Expression and Are Essential for Its Interaction with ORF34

2019 ◽  
Vol 94 (2) ◽  
Author(s):  
Allison L. Didychuk ◽  
Angelica F. Castañeda ◽  
Lola O. Kushnir ◽  
Carolyn J. Huang ◽  
Britt A. Glaunsinger
Keyword(s):  
Gene Transcription ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Late Gene ◽  
Gene Promoters ◽  
Preinitiation Complex ◽  
Terminal Domain ◽  
Late Genes ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT 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.

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

2019 ◽  
Author(s):  
Allison L. Didychuk ◽  
Angelica F. Castañeda ◽  
Lola O. Kushnir ◽  
Carolyn J. Huang ◽  
Britt A. Glaunsinger
Keyword(s):  
Gene Transcription ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Late Gene ◽  
Gene Promoters ◽  
Terminal Domain ◽  
Late Genes ◽  
Virally Encoded ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTLate 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, 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.IMPORTANCEKaposi’s 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.

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