Retroviral Cyclin Controls Cyclin-Dependent Kinase 8-Mediated Transcription Elongation and Reinitiation

Abstract

Walleye dermal sarcoma virus (WDSV) infection is associated with the seasonal development and regression of walleye dermal sarcoma. Previous work showed that the retroviral cyclin (RV-cyclin), encoded by WDSV, has separable cyclin box and transcription activation domains. It binds to cyclin-dependent kinase 8 (CDK8) and enhances its kinase activity. CDK8 is evolutionarily conserved and is frequently overexpressed in human cancers. It is normally activated by cyclin C and is required for transcription elongation of the serum response genes (immediate early genes [IEGs]) FOS, EGR1, and cJUN. The IEGs drive cell proliferation, and their expression is brief and highly regulated. Here we show that constitutive expression of RV-cyclin in the HCT116 colon cancer cell line significantly increases the level of IEG expression in response to serum stimulation. Quantitative reverse transcription-PCR (RT-PCR) and nuclear run-on assays provide evidence that RV-cyclin does not alter the initiation of IEG transcription but does enhance the overall rate of transcription elongation and maintains transcription reinitiation. RVcyclin does not increase activating phosphorylation events in the mitogen-activated protein kinase pathway and does not inhibit decay of IEG mRNAs. At the EGR1 gene locus, RV-cyclin increases and maintains RNA polymerase II (Pol II) occupancy after serum stimulation, in conjunction with increased and extended EGR1 gene expression. The RV-cyclin increases CDK8 occupancy at the EGR1 gene locus before and after serum stimulation. Both of RV-cyclin's functional domains, i.e., the cyclin box and the activation domain, are necessary for the overall enhancement of IEG expression. RV-cyclin presents a novel and ancient mechanism of retrovirus-induced oncogenesis. IMPORTANCEThe data reported here are important to both virology and cancer biology. The novel mechanism pinpoints CDK8 in the development of walleye dermal sarcoma and sheds light on CDK8's role in many human cancers. CDK8 controls expression from highly regulated genes, including the interferon-stimulated genes. Its function is likely the target of many viral interferon-resistance mechanisms. CDK8 also controls cellular responses to metabolic stimuli, stress, and hypoxia, in addition to the serum response. The retroviral cyclin (RV-cyclin) represents a highly selected probe of CDK8 function. RV-cyclin does not control CDK8 specificity but instead enhances CDK8's effects on regulated genes, an important distinction for its use to delineate natural CDK8 targets. The outcomes of this research are applicable to investigations of normal and abnormal CDK8 functions. The mechanisms defined here will contribute directly to the dermal sarcoma model in fish and clarify an important path for oncogenesis and innate resistance to viruses. T he complex retrovirus walleye dermal sarcoma virus (WDSV) is associated with the seasonal development and regression of dermal sarcoma in walleye fish (Sander vitreus) (1-7). The seasonal nature of the tumor is coupled with a...

Cyclin-Dependent Kinases 8 and 19 Regulate Host Cell Metabolism during Dengue Virus Serotype 2 Infection

“…More investigation into the global picture of CDK8/19-regulated gene expression during DENV2 infection is warranted to understand their complete role. We previously showed that CDK8 activity is enhanced by a retroviral cyclin [31,32,35]. Interaction between the retroviral cyclin and CDK8 enhances CDK8 function in the serum response to the advantage of the virus, which requires cell proliferation for transmission.…”

“…Knockdown of either CDK8 or CDK19 has previously been shown to reduce the proliferation rate of cultured cells [31,54,55], and we observed reduced proliferation of CDK8 and CDK19 These data demonstrate that CDK8 mRNA and protein levels increase after DENV2 infection and suggest active CDK8 phosphorylation of histone H3 at serine 10 in association with increased transcriptional activity. CDK8 chromatin occupancy is also increased during DENV2 infection, an outcome associated with enhanced CDK8-dependent gene expression [27,28,31,32].…”

“…CDK8 regulates transcriptional elongation by phosphorylation of the C-terminal domain of the largest subunit of RNA Pol II, a variety of transcription factors, and serine 10 on histone H3 (H3S10) [26][27][28][29][30]. CDK8 regulates gene expression preferentially during induced states such as hypoxia and serum starvation [27,28,31,32] and has recently been implicated in the transcription of glycolytic and autophagic genes [33,34].…”

“…We have previously shown that CDK8 is directly and very specifically targeted by the oncogenic retrovirus, walleye dermal sarcoma virus [31,32,35,36], and reasoned that CDK8 is likely targeted, directly or indirectly, by many viruses with metabolic and autophagic demands, as exemplified by DENV2 infection. We found that expression of CDK8 is upregulated during DENV2 infection of Huh7 human hepatocellular carcinoma cells, and that this precedes increased expression of two key genes, hexokinase 2 (HK2) and microtubule-associated protein 1 light chain 3 (LC3).…”

RNA Polymerase II Promoter-Proximal Pausing and Release to Elongation Are Key Steps Regulating Herpes Simplex Virus 1 Transcription

“…Forward primer Reverse primer GAPDH (95) 5=-GCCATCAATGACCCCCAT-3= 5=-CGCTCCTGGAAGATGGTG-3= ␣0 5 =-CCCACTATCAGGTACACCAGCTT-3= 5=-CTGCGCTGCGACACCTT-3= UL54 5=-GCATCCTTCGTGTTTGTCATTCTG-3= 5=-GCATCTTCTCTCCGACCCCG-3= UL29 5=-GGGCGCAACTTTCGCAATCAATTC-3= 5=-GGCCGACAGAAACCCGTTGTTAAA-3= UL51 5=-GCCAGTCGTTCTAGGTTCAC-3= 5=-GTTAACGCGCTACTTCCCG-3= UL31 5=-GGTCTATGAGCCGGTAGTGC-3= 5=-CTCTTGGCCGGGATTCTTGG-3=…”