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. We found that VACV decapping enzymes are required for selective translation of viral post-replicative mRNAs (transcribed after viral DNA replication) independent of PKR-and RNase L-mediated translation repression. Further molecular characterization demonstrated that VACV decapping enzymes are necessary for efficient translation of mRNA with a 5'-poly(A) leader, which are present in all viral post-replicative mRNAs. Inactivation of D10 alone in VACV significantly impairs poly(A)-leader-mediated translation. Remarkably, D10 stimulates mRNA translation in the absence of VACV infection with a preference for RNA containing a 5'-poly(A) leader. We further revealed that VACV decapping enzymes are needed for 5'-poly(A) leader-mediated cap-independent translation enhancement during infection. Our findings identified a mechanism by which VACV mRNAs are selectively translated through subverting viral decapping enzymes to stimulate 5'-poly(A) leader-mediated translation.

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. Computational analysis of gene expression in CaCx transcriptomic datasets identified a frequent increased expression of TLS genes. However, the essential TLS polymerases did not follow this pattern. These data were confirmed with in vitro and ex vivo systems. Further interrogation of TLS, using POLη as a representative TLS polymerase, demonstrated that α-HPV16 E6 blocks TLS polymerase induction by degrading p53. This doomed the pathway, leading to increased replication fork collapse and sensitivity to treatments that cause replication stress (e.g., UV and Cisplatin). This sensitivity could be overcome by the addition of exogenous POLη.

Intraflagellar transport (IFT) is essential for the assembly and maintenance of flagella and cilia. Recent biochemical studies have shown that IFT complex B (IFT-B) is comprised of two subcomplexes, IFT-B1 and IFT-B2. The IFT-B2 subunit IFT57 lies at the interface between IFT-B1 and IFT-B2. Here, using a Chlamydomonas reinhardtii mutant for IFT57, we tested whether IFT57 is required for IFT-B complex assembly by bridging IFT-B1 and IFT-B2 together. In the ift57-1 mutant, levels of IFT57 and other IFT-B proteins were greatly reduced at the whole-cell level. However, strikingly, in the protease-free flagellar compartment, while the level of IFT57 was reduced, the levels of other IFT particle proteins were not concomitantly reduced but were present at the wild-type level. The IFT movement of the IFT57-deficient IFT particles was also unchanged. Moreover, IFT57 depletion disrupted the flagellar waveform, leading to cell swimming defects. Analysis of the mutant flagellar protein composition showed that certain axonemal proteins were altered. Taken together, these findings suggest that IFT57 does not play an essential structural role in the IFT particle complex but rather functions to prevent it from degradation. Additionally, IFT57 is involved in transporting specific motility-related proteins.

Decapping enzymes comprise many members from various organisms, ranging from plants, animals, and viruses. The mechanisms regulating their functions vary and are still largely unknown.

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. The RNA-based luciferase reporter assay described here circumvents issues of plasmid replication in poxvirus-infected cells and cryptic transcription from the plasmid. This protocol can be used to determine translation regulation by cis-elements in an mRNA including 5′-UTR and 3′-UTR in systems other than poxvirus-infected cells. Moreover, different modes of translation initiation like cap-dependent, cap-independent, reinitiation, and internal initiation can be investigated using this method.