ORF45-Mediated Prolonged c-Fos Accumulation Accelerates Viral Transcription during the Late Stage of Lytic Replication of Kaposi's Sarcoma-Associated Herpesvirus

Li, Xiaojuan; Du, Shiwei; Avey, Denis; Li, Yuqing; Zhu, Fanxiu; Kuang, Ersheng

doi:10.1128/jvi.00274-15

Cited by 27 publications

(36 citation statements)

References 63 publications

(113 reference statements)

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“…In addition to HCMV, other herpesviruses modulate AP-1 during infection. The Epstein Barr virus (EBV) protein, BGLF2, and the Kaposi’s sarcoma-associated herpesvirus (KSHV)-encoded ORF45 both activate the AP-1 signaling pathway to promote viral gene expression, replication, and survival (37, 38). EBV also encodes an AP-1 homolog, BZLF1, which, like AP-1, supports resting B cell proliferation and binds methylated EBV promoters critical for reactivation (39).…”

Section: Discussionmentioning

confidence: 99%

Activator protein-1 transactivation of the major immediate early locus is a determinant of cytomegalovirus reactivation from latency

Krishna

Wass

O’Connor

2020

Preprint

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Human cytomegalovirus (HCMV) is a ubiquitous pathogen that latently infects hematopoietic cells and has the ability to reactivate when triggered by immunological stress. This reactivation causes significant morbidity and mortality in immune-deficient patients, who are unable to control viral dissemination. While a competent immune system helps prevent clinically detectable viremia, a portrait of the factors that induce reactivation following the proper cues remains incomplete. Our understanding of the complex molecular mechanisms underlying latency and reactivation continue to evolve. We previously showed the HCMV-encoded G-protein coupled receptor US28 is expressed during latency and facilitates latent infection by attenuating the activator protein-1 (AP-1) transcription factor subunit, c-fos, expression and activity. We now show AP-1 is a critical component for HCMV reactivation. Pharmacological inhibition of c-fos significantly attenuates viral reactivation. In agreement, infection with a virus in which we disrupted the proximal AP-1 binding site in the major immediate early (MIE) enhancer results in inefficient reactivation compared to wild type. Concomitantly, AP-1 recruitment to the MIE enhancer is significantly decreased following reactivation of the mutant virus. Further, AP-1 is critical for de-repression of MIE-driven transcripts and downstream early and late genes, while immediate early genes from other loci remain unaffected. Our data also reveal MIE transcripts driven from the MIE promoter, the distal promoter, and the internal promoter, iP2, are dependent upon AP-1 recruitment, while iP1-driven transcripts are AP-1-independent. Collectively, our data demonstrate AP-1 binding to and activation of the MIE enhancer is a key molecular process controlling reactivation from latency.Significance StatementHuman cytomegalovirus (HCMV) is a common pathogen that infects the majority of the population for life. This infection poses little threat in immunologically healthy individuals, but can be fatal in people with compromised immune systems. Our understanding of the mechanisms underlying latency and reactivation remains incomplete. Here, we show the cellular transcription factor, AP-1, is a key to regulating HCMV reactivation. Our findings reveal AP-1 binding to the major immediate early enhancer/promoter is critical for switching this locus from one that is repressed during latency to one that is highly active following reactivation. Our work provides a novel mechanism HCMV exploits to reactivate, highlighting AP-1 as a potential target to prevent HCMV reactivation.

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Section: Discussionmentioning

confidence: 99%

Activator protein-1 transactivation of the major immediate early locus is a determinant of cytomegalovirus reactivation from latency

Krishna

Wass

O’Connor

2020

Preprint

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“…In previous studies, we revealed that ORF45 induces sustained ERK-RSK activation and plays an essential role in KSHV lytic replication, and an ORF45-null or F66A mutation in ORF45 abolishes its activation and significantly decreases lytic replication (12)(13)(14)(15). Similarly, the inhibition of RSK activation by inhibitors or short hairpin RNAs (shRNAs) blocks both latent and lytic cycles (12,16), indicating that RSK is an effective target for disrupting KSHV lytic replication and persistent infection.…”

Section: Discussionmentioning

confidence: 99%

“…4C, middle). Interestingly, although the expression of the latent gene LANA was not affected, the expression of the latent genes vCyclin and vFLIP was inhibited to the same extent as the lytic genes, probably because their expression was increased during lytic replication similar to the lytic genes (20,21) and ORF45-induced c-Fos activation upregulated their transcription (14); thus, the TAT-10F10 peptide inhibits ORF45-mediated activation of the ERK-RSK-c-Fos pathway and, consequently, viral transcription. In addition, vFLIP is translated through a viral internal ribosome entry site (IRES) (22) that is upregulated by eIF4B phosphorylation (13,23), and inhibition of eIF4B phosphorylation by TAT-10F10 decreases vFLIP translation.…”

Section: Development Of a Nontoxic Cell-permeable Orf45 Tat-10f10 Pepmentioning

confidence: 96%

“…Multiple cellular pathways are activated during KSHV lytic replication, and they play critical roles in viral infection, replication, and pathogenesis (11). KSHV lytic replication requires the sustained ERK-RSK activation that is induced by tegument protein ORF45 during the late stage (12) to promote late transcription and translation through the phosphorylation of c-Fos and eIF4B, respectively (13,14). ORF45-null or ORF45 F66A mutagenesis results in the depletion of sustained ERK-RSK activation and decreases lytic replication, and RSK inhibitors similarly suppress both KSHV latent and lytic replication (12,15,16).…”

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confidence: 99%

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Development of an ORF45-Derived Peptide To Inhibit the Sustained RSK Activation and Lytic Replication of Kaposi's Sarcoma-Associated Herpesvirus

Xiao

Yao

et al. 2019

J Virol

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The lytic replication of Kaposi’s sarcoma-associated herpesvirus (KSHV) requires sustained extracellular signal-regulated kinase (ERK)-p90 ribosomal S6 kinase (RSK) activation, which is induced by an immediate early (IE) gene-encoded tegument protein called ORF45, to promote the late transcription and translation of viral lytic genes. An ORF45-null or single-point F66A mutation in ORF45 abolishes ORF45-RSK interaction and sustained ERK-RSK activation during lytic reactivation and subsequently results in a significant decrease in late lytic gene expression and virion production, indicating that ORF45-mediated RSK activation plays a critical role in KSHV lytic replication. Here, we demonstrate that a short ORF45-derived peptide in the RSK-binding region is sufficient for disrupting ORF45-RSK interaction, consequently suppressing lytic gene expression and virion production. We designed a nontoxic cell-permeable peptide derived from ORF45, TAT-10F10, which is composed of the ORF45 56 to 76 amino acid (aa) region and the HIV Tat protein transduction domain, and this peptide markedly inhibits KSHV lytic replication in iSLK.219 and BCBL1 cells. Importantly, this peptide enhances the inhibitory effect of rapamycin on KSHV-infected cells and decreases spontaneous and hypoxia-induced lytic replication in KSHV-positive lymphoma cells. These findings suggest that a small peptide that disrupts ORF45-RSK interaction might be a promising agent for controlling KSHV lytic infection and pathogenesis. IMPORTANCE ORF45-induced RSK activation plays an essential role in KSHV lytic replication, and ORF45-null or ORF45 F66A mutagenesis that abolishes sustained RSK activation and RSK inhibitors significantly decreases lytic replication, indicating that the ORF45-RSK association is a unique target for KSHV-related diseases. However, the side effects, low affinity, and poor efficacy of RSK modulators limit their clinical application. In this study, we developed a nontoxic cell-permeable ORF45-derived peptide from the RSK-binding region to disrupt ORF45-RSK associations and block ORF45-induced RSK activation without interfering with S6K1 activation. This peptide effectively suppresses spontaneous, hypoxia-induced, or chemically induced KSHV lytic replication and enhances the inhibitory effect of rapamycin on lytic replication and sensitivity to rapamycin in lytic KSHV-infected cells. Our results reveal that the ORF45-RSK signaling axis and KSHV lytic replication can be effectively targeted by a short peptide and provide a specific approach for treating KSHV lytic and persistent infection.

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“…KSHV ORF45 inhibits type I interferon by blocking phosphorylation and nuclear accumulation of IRF-7 (14,(33)(34)(35). It can efficiently activate RSK, eIF4B, and ERK (36)(37)(38)(39)(40)(41). The interaction between KSHV ORF45 and SIAH-1 promotes ubiquitination and proteasomal degradation of the protein (42).…”

Section: Discussionmentioning

confidence: 99%

Epstein-Barr Virus BKRF4 Gene Product Is Required for Efficient Progeny Production

Masud

Watanabe

Yoshida

et al. 2017

J Virol

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Epstein-Barr virus (EBV), a member of human gammaherpesvirus, infects mainly B cells. EBV has two alternative life cycles, latent and lytic, and is reactivated occasionally from the latent stage to the lytic cycle. To combat EBV-associated disorders, understanding the molecular mechanisms of the EBV lytic replication cycle is also important. Here, we focused on an EBV lytic gene, BKRF4. Using our anti-BKRF4 antibody, we revealed that the BKRF4 gene product is expressed during the lytic cycle with late kinetics. To characterize the role of BKRF4, we constructed BKRF4-knockout mutants using the bacterial artificial chromosome (BAC) and CRISPR/Cas9 systems. Although disruption of the BKRF4 gene had almost no effect on viral protein expression and DNA synthesis, it significantly decreased progeny virion levels in HEK293 and Akata cells. Furthermore, we show that BKRF4 is involved not only in production of progeny virions but also in increasing the infectivity of the virus particles. Immunoprecipitation assays revealed that BKRF4 interacted with a virion protein, BGLF2. We showed that the C-terminal region of BKRF4 was critical for this interaction and for efficient progeny production. Immunofluorescence analysis revealed that BKRF4 partially colocalized with BGLF2 in the nucleus and perinuclear region. Finally, we showed that BKRF4 is a phosphorylated, possible tegument protein and that the EBV protein kinase BGLF4 may be important for this phosphorylation. Taken together, our data suggest that BKRF4 is involved in the production of infectious virions. Although the latent genes of EBV have been studied extensively, the lytic genes are less well characterized. This study focused on one such lytic gene, BKRF4, which is conserved only among gammaherpesviruses (ORF45 of Kaposi's sarcoma-associated herpesvirus or murine herpesvirus 68). After preparing the BKRF4 knockout virus using B95-8 EBV-BAC, we demonstrated that the BKRF4 gene was involved in infectious progeny particle production. Importantly, we successfully generated a BKRF4 knockout virus of Akata using CRISPR/Cas9 technology, confirming the phenotype in this separate strain. We further showed that BKRF4 interacted with another virion protein, BGLF2, and demonstrated the importance of this interaction in infectious virion production. These results shed light on the elusive process of EBV progeny maturation in the lytic cycle. Notably, this study describes a successful example of the generation and characterization of an EBV construct with a disrupted lytic gene using CRISPR/Cas9 technology.

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ORF45-Mediated Prolonged c-Fos Accumulation Accelerates Viral Transcription during the Late Stage of Lytic Replication of Kaposi's Sarcoma-Associated Herpesvirus

Cited by 27 publications

References 63 publications

Activator protein-1 transactivation of the major immediate early locus is a determinant of cytomegalovirus reactivation from latency

Activator protein-1 transactivation of the major immediate early locus is a determinant of cytomegalovirus reactivation from latency

Development of an ORF45-Derived Peptide To Inhibit the Sustained RSK Activation and Lytic Replication of Kaposi's Sarcoma-Associated Herpesvirus

Epstein-Barr Virus BKRF4 Gene Product Is Required for Efficient Progeny Production

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