Intramolecular Regulation of the Sequence-Specific mRNA Interferase Activity of MazF Fused to a MazE Fragment with a Linker Cleavable by Specific Proteases

Abstract

The genomes of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) consist of single-stranded RNA encoding polyproteins, which are processed to individual functional proteins by virus-encoded specific proteases. These proteases have been used as targets for drug development. Here, instead of targeting these proteases to inhibit viral infection, we utilized the protease activity to activate a toxic protein to prevent viral infection. We engineered the MazE-MazF antitoxin-toxin system of Escherichia coli to fuse a C-terminal 41-residue fragment of antitoxin MazE to the N-terminal end of toxin MazF with a linker having a specific protease cleavage site for either HIV PR (HIV-1 protease), NS3 protease (HCV protease), or factor Xa. These fusion proteins formed a stable dimer (instead of the MazF 2 -MazE 2 -MazF 2 heterohexamer in nature) to inactivate the ACA (sequence)-specific mRNA interferase activity of MazF. When the fusion proteins were incubated with the corresponding proteases, the MazE fragment was cleaved from the fusion proteins, releasing active MazF, which then acted as an ACA-specific mRNA interferase cleaving single-stranded MS2 phage RNA. The intramolecular regulation of MazF toxicity by proteases as demonstrated may provide a novel approach for preventive and therapeutic treatments of infection by HIV-1, HCV, and other single-stranded RNA viruses.

Regression of Solid Tumors by Induction of MazF, a Bacterial mRNA Endoribonuclease

“…For example, we showed that induction of the mazF gene from an HIV-1 promoter leads to degradation of viral mRNAs [Chono et al, 2011;Okamoto et al, 2013]. Results from our laboratory and another group [Park et al, 2012;Shapira et al, 2012] showed that MazF can be used to create novel tools against HIV-1 and hepatitis C viruses. In the future, it is possible to utilize MazF and other bacterial endoribonucleases as tissue-specific and selective anti-cancer agents by using tissue-specific promoters and tissue-specific delivery systems.…”

MLV based viral-like-particles for delivery of toxic proteins and nuclear transcription factors

“…5b&d). The packaging cells were also protected by truncated forms of MazE (MazE42 and MazE61 [11] but not as well as wild type MazE and only cells expressing low levels of Gag-MazF-2NES were able to survive after puromycin selection (Fig. 5b, lanes 3,4).…”

“…The MazE or truncated MazE sequences (MazE42-GFP and MazE61E–GFP) [11] were amplified from pCold-MazE (a gift from Dr. Masayori Inouye, Rutgers-RWJMS) by PCR and subcloned into pSin-EF1α-GFP-ΔKpnI at the SpeI restriction sites.…”

“…(b) Western blot of ecotropic pseudotyped VLPs from 293TCeB cells. Lane 1, MazF (E24A); lane 2, MazF + MazE-GFP; lane 3, MazF + MazE42-GFP; lane 4, MazF + MazE61E-GFP; lane 5, MazE-GFP [11]. (c) Schematic of the pL-MazE-GFP lentiviral vector.…”

Phylogenetic identification of bacterial MazF toxin protein motifs among probiotic strains and foodborne pathogens and potential implications of engineered probiotic intervention in food

“…MazEF is one of the most well-studied toxin–antitoxin (TA) systems and has been found on the chromosomes of many bacteria. The MazF protein has been recently categorized as an endoribonuclease [41,42] or as a type of RNA interference enzyme [43]. The link between this TA system and quorum sensing has also been explored recently through a small pentapeptide (NNWNN) called the Extracellular Death Factor (EDF) [44].…”