Understanding the dynamics of Zika virus transmission and formulating rational strategies for its control require precise diagnostic tools that are also appropriate for resource-poor environments. We have developed a rapid and sensitive loop-mediated isothermal amplification (LAMP) assay that distinguishes Zika viruses of Asian and African lineages. The assay does not detect chikungunya virus or flaviviruses such as dengue, yellow fever, or West Nile viruses. The assay conditions allowed direct detection of Zika virus RNA in cultured infected cells; in mosquitoes; in virus-spiked samples of human blood, plasma, saliva, urine, and semen; and in infected patient serum, plasma, and semen samples without the need for RNA isolation or reverse transcription. The assay offers rapid, specific, sensitive, and inexpensive detection of the Asian-lineage Zika virus strain that is currently circulating in the Western hemisphere, and can also detect the African-lineage Zika virus strain using separate, specific primers.
Walleye dermal sarcoma virus encodes a retroviral cyclin (rv-cyclin) with a cyclin box fold and transcription activation domain (AD). Co-immune precipitation (co-IP) identified an association of rv-cyclin with cyclin dependent kinase 8 (cdk8). Cdk8 is dependent upon cyclin C and regulates transcription with the Mediator complex, a co-activator of transcription. Mutation of cyclin residues, required for cdk binding, disrupt rv-cyclin-cdk8 co-IP. Mutation or removal of the AD has no effect on cdk8 interaction. Direct rv-cyclin-cdk8 binding is demonstrated by pulldown of active cdk8 and by GST-rv-cyclin binding to recombinant cdk8. Cdk3 is also activated by cyclin C and phosphorylates retinoblastoma protein to initiate entry into the cell division cycle. Co-IP and pulldowns demonstrate direct rv-cyclin binding to cdk3 as well. The rv-cyclin functions as a structural ortholog of cyclin C in spite of its limited amino acid sequence identity with C cyclins or with any known cyclins.
Walleye dermal sarcoma virus (WDSV) is a complex retrovirus associated with dermal sarcomas in walleye fish. Virus expression is tightly regulated and limited to accessory gene transcripts throughout tumour development. During tumour regression, this regulation is lost and the replication of virus is greatly enhanced. Cultured walleye fibroblasts infected in vitro do not produce significant quantities of infectious virus. Tissue culture cells established by explantation of tumour cells were found to harbour WDSV provirus and to express accessory and structural proteins. The sequence of the provirus showed little variation from a previous WDSV isolate. Retroviral particles were isolated from supernatants from these cells and were able to transfer infection to uninfected walleye fibroblasts. In addition to the virus present in supernatants, much of the virus was cell associated and liberated only by sonication. This virus was found at internal cellular membranes, including mitochondria, and was infectious.
Alterations in the functional levels of cyclin-dependent kinase-8 (CDK8) or its partner, cyclin C, have been clearly associated with cancers, including colon cancer, melanoma, and osteosarcoma. Walleye dermal sarcoma virus encodes a retroviral cyclin (RV-cyclin) that localizes to interchromatin granule clusters and binds CDK8. It also binds to the Aα subunit (PR65) of protein phosphatase 2A (PP2A). Binding to the Aα subunit excludes the regulatory B subunit, but not the catalytic C subunit, in a manner similar to that of T antigens of the small DNA tumor viruses. The expression of the RV-cyclin enhances the activity of immune affinity-purified CDK8 in vitro for RNA polymerase II carboxy-terminal domain (CTD) and histone H3 substrates. PP2A also enhances CDK8 kinase activity in vitro for the CTD but not for histone H3. The PP2A enhancement of CDK8 is independent of RV-cyclin expression and likely plays a role in the normal regulation of CDK8. The manipulation of endogenous PP2A activity by inhibition, amendment, or depletion confirmed its role in CDK8 activation by triggering CDK8 autophosphorylation. Although RV-cyclin and PP2A both enhance CDK8 activity, their actions are uncoupled and additive in kinase reactions. PP2A may be recruited to CDK8 in the Mediator complex by a specific PP2A B subunit or additionally by the RV-cyclin in infected cells, but the RV-cyclin appears to activate CDK8 directly and in a manner independent of its physical association with PP2A.
The retroviral cyclin protein (rv-cyclin) of walleye dermal sarcoma virus contains two known functional domains, a cyclin box motif and a carboxy terminal transcription activation domain (AD). The AD contacts TATA-binding protein-associated factor 9 (TAF9), and this action is necessary for both positive and negative regulation of transcription from host and viral promoters. Negative regulation occurs via interference with TAF9 binding by transcriptional activators. Transcription factors that share a functional TAF9-binding motif include NF-κB. Rv-cyclin down regulates NF-κB-dependent transcription, whether induced by TNFαor by direct phosphorylation of IκB by expressed MEKK1. In rv-cyclin-expressing cells NF-κB p65 is phosphorylated and translocates to the nucleus, where it forms heterodimers with p50 and binds NF-κB response elements. Furthermore, interference with NF-kB is dependent upon an intact TAF9-binding motif in rv-cyclin. The outcome of this NF-κB down regulation is likely to be important in the control of virus replication and tumorigenesis.
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