A Poxvirus Decapping Enzyme Colocalizes with Mitochondria To Regulate RNA Metabolism and Translation and Promote Viral Replication.

Decapping enzymes remove the 5' cap of eukaryotic mRNA, leading to accelerated RNA decay. They are critical in regulating RNA homeostasis and play essential roles in many cellular and life processes. They are encoded in many organisms and viruses, including vaccinia virus, which was used as the vaccine to eradicate smallpox.

High Risk α-HPV E6 Impairs Translesion Synthesis by Blocking POLη Induction.

High risk genus α human papillomaviruses (α-HPVs) express two versatile oncogenes (α-HPV E6 and E7) that cause cervical cancer (CaCx) by degrading tumor suppressor proteins (p53 and RB). α-HPV E7 also promotes replication stress and alters DNA damage responses (DDR). The translesion synthesis pathway (TLS) mitigates DNA damage by preventing replication stress from causing replication fork collapse.

Poxvirus-encoded decapping enzymes promote selective translation of viral mRNAs.

Cellular decapping enzymes negatively regulate gene expression by removing the methylguanosine cap at the 5' end of eukaryotic mRNA, rendering mRNA susceptible to degradation and repressing mRNA translation. Vaccinia virus (VACV), the prototype poxvirus, encodes two decapping enzymes, D9 and D10, that induce the degradation of both cellular and viral mRNAs. Using a genome-wide survey of translation efficiency, we analyzed vaccinia virus mRNAs in cells infected with wild type VACV and mutant VACVs with inactivated decapping enzymes.

In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells.

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5'-poly(A) leader in the 5'-UTR. To dissect the role of 5'-poly(A) leader in mRNA translation during poxvirus infection we developed an in vitro transcribed RNA-based luciferase reporter assay. This reporter assay comprises of four core steps: (1) PCR to amplify the DNA template for in vitro transcription; (2) in vitro transcription to generate mRNA using T7 RNA polymerase; (3) Transfection to introduce in vitro transcribed mRNA into cells; (4) Detection of luciferase activity as the indicator of translation.