S-like-phase cyclin-dependent kinases stabilize the Epstein-Barr virus BDLF4 protein to temporally control late gene transcription

Yoshitaka Sato, Takahiro Watanabe, Chihiro Suzuki, Yuichi Abe, H. M. Abdullah Al Masud, Tomoki Inagaki, Masahiro Yoshida, Takeshi Suzuki, Fumi Goshima, Jun Adachi, Takeshi Tomonaga, Takayuki Murata, Hiroshi Kimura

Research output: Contribution to journalArticle

Abstract

Temporally controlled gene expression is necessary for the propagation of herpesviruses. To achieve this, herpesviruses encode several transcriptional regulators. In Epstein-Barr virus, BcRF1 associates with five viral proteins (BDLF4, BGLF3, BFRF2, BVLF1, and BDLF3.5) to form the viral late (L) gene regulatory complex, which is called the viral preinitiation complex (vPIC), on TATT-containing promoters. However, regulation of the vPIC has been largely unexplored. In this study, we performed two screens using a kinase inhibitor library and identified a series of cyclin-dependent kinase (CDK) inhibitors that downregulated the expression of L genes without any impact on viral DNA replication through destabilization of the BDLF4 protein. Knockdown of CDK2 by short hairpin RNA (shRNA) and proteasome inhibitor treatment showed that phosphorylation of the BDLF4 protein prevented ubiquitin-mediated degradation. Moreover, we demonstrated that cyclin A- and E-associated CDK2 complexes phosphorylated BDLF4 in vitro, and we identified several serine/threonine phosphorylation sites in BDLF4. Phosphoinactive and phosphomimic mutants revealed that phosphorylation at threonine 91 plays a role in stabilizing BDLF4. Therefore, our findings indicate that S-like-phase CDKs mediate the regulation of L gene expression through stabilization of the BDLF4 protein, which makes the temporal L gene expression system more robust. IMPORTANCE Late (L) genes represent more than one-third of the herpesvirus genome, suggesting that many of these genes are indispensable for the life cycle of the virus. With the exception of BCRF1, BDLF2, and BDLF3, Epstein-Barr virus L genes are transcribed by viral regulators, which are known as the viral preinitiation complex (vPIC) and the host RNA polymerase II complex. Because the vPIC is conserved in beta- and gammaherpesviruses, studying the control of viral L gene expression by the vPIC contributes to the development of drugs that specifically inhibit these processes in beta- and gammaherpesvirus infections/diseases. In this study, we demonstrated that CDK inhibitors induced destabilization of the vPIC component BDLF4, leading to a reduction in L gene expression and subsequent progeny production. Our findings suggest that CDK inhibitors may be a therapeutic option against beta- and gammaherpesviruses in combination with existing inhibitors of herpesvirus lytic replication, such as ganciclovir.

Original languageEnglish
Article numberY
JournalJournal of Virology
Volume93
Issue number8
DOIs
Publication statusPublished - 01-01-2019

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Genetic Transcription
Human herpesvirus 4
cyclin-dependent kinase
Cyclin-Dependent Kinases
Human Herpesvirus 4
S Phase
Herpesviridae
transcription (genetics)
Gene Expression
gene expression
Viral Genes
Phosphorylation
Threonine
Proteins
genes
proteins
threonine
phosphorylation
Cyclin A
Cyclin E

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

Cite this

Sato, Yoshitaka ; Watanabe, Takahiro ; Suzuki, Chihiro ; Abe, Yuichi ; Abdullah Al Masud, H. M. ; Inagaki, Tomoki ; Yoshida, Masahiro ; Suzuki, Takeshi ; Goshima, Fumi ; Adachi, Jun ; Tomonaga, Takeshi ; Murata, Takayuki ; Kimura, Hiroshi. / S-like-phase cyclin-dependent kinases stabilize the Epstein-Barr virus BDLF4 protein to temporally control late gene transcription. In: Journal of Virology. 2019 ; Vol. 93, No. 8.
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Sato, Y, Watanabe, T, Suzuki, C, Abe, Y, Abdullah Al Masud, HM, Inagaki, T, Yoshida, M, Suzuki, T, Goshima, F, Adachi, J, Tomonaga, T, Murata, T & Kimura, H 2019, 'S-like-phase cyclin-dependent kinases stabilize the Epstein-Barr virus BDLF4 protein to temporally control late gene transcription', Journal of Virology, vol. 93, no. 8, Y. https://doi.org/10.1128/JVI.01707-18

S-like-phase cyclin-dependent kinases stabilize the Epstein-Barr virus BDLF4 protein to temporally control late gene transcription. / Sato, Yoshitaka; Watanabe, Takahiro; Suzuki, Chihiro; Abe, Yuichi; Abdullah Al Masud, H. M.; Inagaki, Tomoki; Yoshida, Masahiro; Suzuki, Takeshi; Goshima, Fumi; Adachi, Jun; Tomonaga, Takeshi; Murata, Takayuki; Kimura, Hiroshi.

In: Journal of Virology, Vol. 93, No. 8, Y, 01.01.2019.

Research output: Contribution to journalArticle

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AU - Sato, Yoshitaka

AU - Watanabe, Takahiro

AU - Suzuki, Chihiro

AU - Abe, Yuichi

AU - Abdullah Al Masud, H. M.

AU - Inagaki, Tomoki

AU - Yoshida, Masahiro

AU - Suzuki, Takeshi

AU - Goshima, Fumi

AU - Adachi, Jun

AU - Tomonaga, Takeshi

AU - Murata, Takayuki

AU - Kimura, Hiroshi

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N2 - Temporally controlled gene expression is necessary for the propagation of herpesviruses. To achieve this, herpesviruses encode several transcriptional regulators. In Epstein-Barr virus, BcRF1 associates with five viral proteins (BDLF4, BGLF3, BFRF2, BVLF1, and BDLF3.5) to form the viral late (L) gene regulatory complex, which is called the viral preinitiation complex (vPIC), on TATT-containing promoters. However, regulation of the vPIC has been largely unexplored. In this study, we performed two screens using a kinase inhibitor library and identified a series of cyclin-dependent kinase (CDK) inhibitors that downregulated the expression of L genes without any impact on viral DNA replication through destabilization of the BDLF4 protein. Knockdown of CDK2 by short hairpin RNA (shRNA) and proteasome inhibitor treatment showed that phosphorylation of the BDLF4 protein prevented ubiquitin-mediated degradation. Moreover, we demonstrated that cyclin A- and E-associated CDK2 complexes phosphorylated BDLF4 in vitro, and we identified several serine/threonine phosphorylation sites in BDLF4. Phosphoinactive and phosphomimic mutants revealed that phosphorylation at threonine 91 plays a role in stabilizing BDLF4. Therefore, our findings indicate that S-like-phase CDKs mediate the regulation of L gene expression through stabilization of the BDLF4 protein, which makes the temporal L gene expression system more robust. IMPORTANCE Late (L) genes represent more than one-third of the herpesvirus genome, suggesting that many of these genes are indispensable for the life cycle of the virus. With the exception of BCRF1, BDLF2, and BDLF3, Epstein-Barr virus L genes are transcribed by viral regulators, which are known as the viral preinitiation complex (vPIC) and the host RNA polymerase II complex. Because the vPIC is conserved in beta- and gammaherpesviruses, studying the control of viral L gene expression by the vPIC contributes to the development of drugs that specifically inhibit these processes in beta- and gammaherpesvirus infections/diseases. In this study, we demonstrated that CDK inhibitors induced destabilization of the vPIC component BDLF4, leading to a reduction in L gene expression and subsequent progeny production. Our findings suggest that CDK inhibitors may be a therapeutic option against beta- and gammaherpesviruses in combination with existing inhibitors of herpesvirus lytic replication, such as ganciclovir.

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