Characterization of mRNA interferases from Mycobacterium tuberculosis

mRNA interferases are sequence-specific endoribonucleases encoded by the toxin-antitoxin systems in the bacterial genomes. MazF from Escherichia coli has been shown to be an mRNA interferase that specifically cleaves at ACA sequences in single-stranded RNAs. It has been shown that MazF induction in E. coli effectively inhibits protein synthesis leading to cell growth arrest in the quasidormant state. Here we have demonstrated that Mycobacterium tuberculosis contains at least seven genes encoding MazF homologues (MazF-mt1 to -mt7), four of which (MazF-mt1, -mt3, -mt4, and -mt6) caused cell growth arrest when induced in E. coli. MazF-mt1 and MazF-mt6 were purified and characterized for their mRNA interferase specificities. We showed that MazF-mt1 preferentially cleaves the era mRNA between U and A in UAC triplet sequences, whereas MazF-mt6 preferentially cleaves U-rich regions in the era mRNA both in vivo and in vitro. These results indicate that M. tuberculosis contains sequence-specific mRNA interferases, which may play a role in the persistent dormancy of this devastating pathogen in human tissues.     

HIV protease cleaves poly(A)-binding protein

The PABP [poly(A)-binding protein] is able to interact with the 3' poly(A) tail of eukaryotic mRNA, promoting its translation. Cleavage of PABP by viral proteases encoded by several picornaviruses and caliciviruses plays a role in the abrogation of cellular protein synthesis. We report that infection of MT-2 cells with HIV-1 leads to efficient proteolysis of PABP. Analysis of PABP integrity was carried out in BHK-21 (baby-hamster kidney) and COS-7 cells upon individual expression of the protease from several members of the Retroviridae family, e.g. MoMLV (Moloney murine leukaemia virus), MMTV (mouse mammary tumour virus), HTLV-I (human T-cell leukaemia virus type I), SIV (simian immunodeficiency virus), HIV-1 and HIV-2. Moreover, protease activity against PABP was tested in a HeLa-cell-free system. Only MMTV, HIV-1 and HIV-2 proteases were able to cleave PABP in the absence of other viral proteins. Purified HIV-1 and HIV-2 proteases cleave PABP1 directly at positions 237 and 477, separating the two first RNA-recognition motifs from the C-terminal domain of PABP. An additional cleavage site located at position 410 was detected for HIV-2 protease. These findings indicate that some retroviruses may share with picornaviruses and caliciviruses the capacity to proteolyse PABP.     

Characterization of dual substrate binding sites in the homodimeric structure of Escherichia coli mRNA interferase MazF

MazF and MazE constitute a so-called addiction module that is critical for bacterial growth arrest and eventual cell death in response to stress. The MazF toxin was recently shown to possess mRNA interferase (MIase) activity, and acts as a protein synthesis inhibitor by cleaving cellular mRNA. As a cognate regulator, the short-lived antitoxin, MazE, inhibits MazF MIase activity and hence maintains the delicate homeostasis between these two components. In the present study, we have shown that the MazF homodimer contains two symmetric binding sites, each of which is capable of interacting with a MazE C-terminal peptide, MazEp(54-77). The slow exchange phenomenon between free and peptide-bound MazF on the NMR timescale indicates relatively high affinities for MazEp(54-77) at both sites (Kd,K'd < 10(-7) M). However, the observed sequential binding behavior suggests a negative cooperativity between the two sites (Kd < K'd). A 13 base single-stranded DNA, employed as an uncleavable RNA substrate analog, can also bind to both sites on the MazF homodimer with moderate affinity (Kd approximately 10(-5) -10(-6) M). Chemical shift perturbation data deduced from NMR experiments indicates that the two binding sites for the MazEp peptide coincided with those for the single-stranded DNA competitive inhibitor. These dual substrate-binding sites are located on the concave interface of the MazF homodimer, consisting of a highly basic region underneath the S1-S2 loop and two hydrophobic regions containing the H1 helix of one subunit and the S3-S4 loop of the opposing subunit. We show that the MazF homodimer is a bidentate endoribonuclease equipped with two identical binding sites for mRNA processing and that a single MazE molecule occupying one of the binding sites can affect the conformation of both sites, hence efficiently hindering the activity of MazF.     

ACA‐specific RNA sequence recognition is acquired via the loop 2 region of MazF mRNA interferase

MazF is an mRNA interferase that cleaves mRNAs at a specific RNA sequence. MazF from E. coli (MazF‐ec) cleaves RNA at A and CA. To date, a large number of MazF homologs that cleave RNA at specific three‐ to seven‐base sequences have been identified from bacteria to archaea. MazF‐ec forms a dimer, in which the interface between the two subunits is known to be the RNA substrate‐binding site. Here, we investigated the role of the two loops in MazF‐ec, which are closely associated with the interface of the MazF‐ec dimer. We examined whether exchanging the loop regions of MazF‐ec with those from other MazF homologs, such as MazF from Myxococcus xanthus (MazF‐mx) and MazF from Mycobacterium tuberculosis (MazF‐mt3), affects RNA cleavage specificity. We found that exchanging loop 2 of MazF‐ec with loop 2 regions from either MazF‐mx or MazF‐mt3 created a new cleavage sequence at (A/U)(A/U)AA and C in addition to the original cleavage site, A and CA, whereas exchanging loop 1 did not alter cleavage specificity. Intriguingly, exchange of loop 2 with 8 or 12 consecutive Gly residues also resulted in a new RNA cleavage site at (A/U)(A/U)AA and C. The present study suggests a method for expanding the RNA cleavage repertoire of mRNA interferases, which is crucial for potential use in the regulation of specific gene expression and for biotechnological applications. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

GCN2 Has Inhibitory Effect on Human Immunodeficiency Virus-1 Protein Synthesis and Is Cleaved upon Viral Infection

The reversible phosphorylation of the alpha-subunit of eukaryotic translation initiation factor 2 (eIF2alpha) is a well-characterized mechanism of translational control in response to a wide variety of cellular stresses, including viral infection. Beside PKR, the eIF2alpha kinase GCN2 participates in the cellular response against viral infection by RNA viruses with central nervous system tropism. PKR has also been involved in the antiviral response against HIV-1, although this antiviral effect is very limited due to the distinct mechanisms evolved by the virus to counteract PKR action. Here we report that infection of human cells with HIV-1 conveys the proteolytic cleavage of GCN2 and that purified HIV-1 and HIV-2 proteases produce direct proteolysis of GCN2 in vitro, abrogating the activation of GCN2 by HIV-1 RNA. Transfection of distinct cell lines with a plasmid encoding an HIV-1 cDNA clone competent for a single round of replication resulted in the activation of GCN2 and the subsequent eIF2alpha phosphorylation. Moreover, transfection of GCN2 knockout cells or cells with low levels of phosphorylated eIF2alpha with the same HIV-1 cDNA clone resulted in a marked increase of HIV-1 protein synthesis. Also, the over-expression of GCN2 in cells led to a diminished viral protein synthesis. These findings suggest that viral RNA produced during HIV-1 infection activates GCN2 leading to inhibition of viral RNA translation, and that HIV-1 protease cleaves GCN2 to overcome its antiviral effect.     

Insights into the mRNA cleavage mechanism by MazF, an mRNA interferase

MazF is an Escherichia coli toxin that is highly conserved among the prokaryotes and plays an important role in growth regulation. When MazF is induced, protein synthesis is effectively inhibited. However, the mechanism of MazF action has been controversial. Here we unequivocally demonstrate that MazF is an endoribonuclease that specifically cleaves mRNAs at ACA sequences. We then demonstrate its enzymatic specificity using short RNA substrates. MazF cleaves RNA at the 5'-end of ACA sequences, yielding a 2',3'-cyclic phosphate at one side and a free 5'-OH group at the other. Using DNA-RNA chimeric substrates containing XACA, the 2'-OH group of residue X was found absolutely essential for MazF cleavage, whereas all the other residues may be deoxyriboses. Therefore, MazF exhibits exquisite site specificity and has utility as an RNA-restriction enzyme for RNA structural studies or as an mRNA interferase to regulate cell growth in prokaryotic and eukaryotic cells.     

Characterization of ChpBK, an mRNA interferase from Escherichia coli

Escherichia coli contains a number of antitoxin-toxin modules on its chromosome, which are responsible for cell growth arrest and possible cell death. ChpBK is a toxin encoded by the ChpBIK antitoxin-toxin module. This module consists of a pair of genes, chpBI and chpBK encoding antitoxin ChpBI and toxin ChpBK, respectively. ChpBK consists of 116 amino acid residues, and its sequence shows 35% identity and 52% similarity to MazF, another E. coli toxin. MazF has been shown to be a sequence-specific (ACA) endoribonuclease that cleaves cellular mRNAs and effectively blocks protein synthesis and is thus termed as an mRNA interferase. Here we demonstrate that ChpBK is another mRNA interferase in E. coli whose induction effectively blocks cell growth in a manner similar to that of MazF. The protein synthesis as judged by incorporation of [35S]methionine was, however, reduced by only 60% upon ChpBK induction. We demonstrate that ChpBK is a new sequence-specific endoribonuclease that cleaves mRNAs both in vivo and in vitro at the 5'-or3'-side of the A residue in ACY sequences (Y is U, A, or G). The ChpBK cleavage of a synthetic RNA substrate generated a 2',3'-cyclic phosphate group at the 3'-end of the 5'-end product and a 5'-OH group at the 5'-end of the 3'-end product in a manner identical to that of MazF.     

Translation of Sindbis virus 26S mRNA does not require intact eukariotic initiation factor 4G

The infection of baby hamster kidney (BHK) cells by Sindbis virus gives rise to a drastic inhibition of cellular translation, while under these conditions the synthesis of viral structural proteins directed by the subgenomic 26S mRNA takes place efficiently. Here, the requirement for intact initiation factor eIF4G for the translation of this subgenomic mRNA has been examined. To this end, SV replicons that contain the protease of human immunodeficiency virus type 1 (HIV-1) or the poliovirus 2A(pro) replacing the sequences of SV glycoproteins have been constructed. BHK cells electroporated with the different RNAs synthesize protein C and the corresponding protease at late times. Notably, the proteolysis of eIF4G by both proteases has little effect on the translation of the 26S mRNA. In addition, recombinant viable SVs were engineered that encode HIV-1 PR or poliovirus 2A protease under the control of a duplicated late promoter. Viral protein synthesis at late times of infection by the recombinant viruses is slightly affected in BHK cells that contain proteolysed eIF4G. The translatability of SV genomic 49S mRNA was assayed in BHK cells infected with a recombinant virus that synthesizes luciferase and transfected with a replicon that expresses poliovirus 2Apro. Under conditions where eIF4G has been hydrolysed significantly the translation of genomic SV RNA was deeply inhibited. These findings indicate a different requirement for intact eIF4G in the translation of genomic and subgenomic SV mRNAs. Finally, the translation of the reporter gene that encodes green fluorescent protein, placed under the control of a second duplicate late promoter, is also resistant to the cleavage of eIF4G. In conclusion, despite the presence of a cap structure in the 5' end of the subgenomic SV mRNA, intact eIF4G is not necessary for its translation.     

Inhibition of HIV-1 gene expression by a fragment of hnRNP U

Cellular proteins are now appreciated as critically involved in all steps of the human immunodeficiency virus type 1 (HIV-1) life cycle, and disrupting host functions essential for virus replication may provide novel antiviral approaches. Selection from a human complementary DNA (cDNA) library for clones able to induce resistance to infection by recombinant HIV-1 genomes resulted in the identification of a gene fragment that potently restricts HIV-1 activity. The active cDNA encodes an N-terminal fragment of the heterogeneous nuclear ribonuclear protein U (hnRNP U). The gene fragment specifically targets the 3' long terminal repeat (3'LTR) in the viral mRNA and blocks the cytoplasmic accumulation of HIV-1 mRNAs. The results suggest that HIV-1 requires machinery for the nuclear export of viral mRNAs that can be specifically blocked by an interfering gene.     

Removal of hepatitis C virus-infected cells by a zymogenized bacterial toxin

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and has become a global health threat. No HCV vaccine is currently available and treatment with antiviral therapy is associated with adverse side effects. Moreover, there is no preventive therapy for recurrent hepatitis C post liver transplantation. The NS3 serine protease is necessary for HCV replication and represents a prime target for developing anti HCV therapies. Recently we described a therapeutic approach for eradication of HCV infected cells that is based on protein delivery of two NS3 protease-activatable recombinant toxins we named “zymoxins”. These toxins were inactivated by fusion to rationally designed inhibitory peptides via NS3-cleavable linkers. Once delivered to cells where NS3 protease is present, the inhibitory peptide is removed resulting in re-activation of cytotoxic activity. The zymoxins we described suffered from two limitations: they required high levels of protease for activation and had basal activities in the un-activated form that resulted in a narrow potential therapeutic window. Here, we present a solution that overcame the major limitations of the “first generation zymoxins” by converting MazF ribonuclease, the toxic component of the E. coli chromosomal MazEF toxin-antitoxin system, into an NS3-activated zymoxin that is introduced to cells by means of gene delivery. We constructed an expression cassette that encodes for a single polypeptide that incorporates both the toxin and a fragment of its potent natural antidote, MazE, linked via an NS3-cleavable linker. While covalently paired to its inhibitor, the ribonuclease is well tolerated when expressed in naïve, healthy cells. In contrast, activating proteolysis that is induced by even low levels of NS3, results in an eradication of NS3 expressing model cells and HCV infected cells. Zymoxins may thus become a valuable tool in eradicating cells infected by intracellular pathogens that express intracellular proteases.     

Bacillus subtilis MazF-bs (EndoA) is a UACAU-specific mRNA interferase

MazF is an mRNA interferase which cleaves mRNAs at a specific sequence. Here, we show that in contrast to MazF-ec from Escherichia coli, which specifically cleaves ACA sequences, MazF-bs from Bacillus subtilis is an mRNA interferase that specifically cleaves a five-base sequence, UACAU. MazF homologues widely prevailing in Gram-positive bacteria were found to be highly homologous to MazF-bs, suggesting that they may also have similar cleavage specificity. This cleavage site is over-represented in the B. subtilis genes associated with biosynthesis of secondary metabolites, suggesting that MazF-bs may be involved in the regulation of the production of secondary metabolites.     

Structural and functional characterization of nonstructural protein 2 for its role in hepatitis C virus assembly

The hepatitis C virus (HCV) is a flavivirus replicating in the cytoplasm of infected cells. The HCV genome is a single-stranded RNA encoding a polyprotein that is cleaved by cellular and viral proteases into 10 different products. While the structural proteins core protein, envelope protein 1 (E1) and E2 build up the virus particle, most nonstructural (NS) proteins are required for RNA replication. One of the least studied proteins is NS2, which is composed of a C-terminal cytosolic protease domain and a highly hydrophobic N-terminal domain. It is assumed that the latter is composed of three trans-membrane segments (TMS) that tightly attach NS2 to intracellular membranes. Taking advantage of a system to study HCV assembly in a hepatoma cell line, in this study we performed a detailed characterization of NS2 with respect to its role for virus particle assembly. In agreement with an earlier report ( Jones, C. T., Murray, C. L., Eastman, D. K., Tassello, J., and Rice, C. M. (2007) J. Virol. 81, 8374-8383 ), we demonstrate that the protease domain, but not its enzymatic activity, is required for infectious virus production. We also show that serine residue 168 in NS2, implicated in the phosphorylation and stability of this protein, is dispensable for virion formation. In addition, we determined the NMR structure of the first TMS of NS2 and show that the N-terminal segment (amino acids 3-11) forms a putative flexible helical element connected to a stable alpha-helix (amino acids 12-21) that includes an absolutely conserved helix side in genotype 1b. By using this structure as well as the amino acid conservation as a guide for a functional study, we determined the contribution of individual amino acid residues in TMS1 for HCV assembly. We identified several residues that are critical for virion formation, most notably a central glycine residue at position 10 of TMS1. Finally, we demonstrate that mutations in NS2 blocking HCV assembly can be rescued by trans-complementation.     

Structural basis of HIV-1 and HIV-2 protease inhibition by a monoclonal antibody

BACKGROUND: Since the demonstration that the protease of the human immunodeficiency virus (HIV Pr) is essential in the viral life cycle, this enzyme has become one of the primary targets for antiviral drug design. The murine monoclonal antibody 1696 (mAb1696), produced by immunization with the HIV-1 protease, inhibits the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates with inhibition constants in the low nanomolar range. The antibody cross-reacts with peptides that include the N terminus of the enzyme, a region that is highly conserved in sequence among different viral strains and that, furthermore, is crucial for homodimerization to the active enzymatic form. RESULTS: We report here the crystal structure at 2.7 A resolution of a recombinant single-chain Fv fragment of mAb1696 as a complex with a cross-reactive peptide of the HIV-1 protease. The antibody-antigen interactions observed in this complex provide a structural basis for understanding the origin of the broad reactivity of mAb-1696 for the HIV-1 and HIV-2 proteases and their respective N-terminal peptides. CONCLUSION: A possible mechanism of HIV-protease inhibition by mAb1696 is proposed that could help the design of inhibitors aimed at binding inactive monomeric species.     

A p6Pol-protease fusion protein is present in mature particles of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) protease (PR) and p6(Pol) are translated as part of the Gag-Pol polyprotein after a ribosomal frameshift. PR is essential to virus replication and is responsible for cleaving Gag and Gag-Pol precursors, but the role of p6(Pol) in HIV-1 infection is poorly understood. Here, we report that (i) PR is present in mature HIV-1 virions primarily as a p6(Pol)-PR fusion protein; (ii) HIV-1 PR cleaves viral precursor proteins expressed in bacterial cells at the Phe-Leu bond (positions 1639 to 1642) located at the junction of the NC and p6(Pol) proteins, releasing the p6(Pol)-PR fusion protein; and (iii) purified p6(Pol)-PR fusion protein undergoes autocleavage in vitro at at least three sites.     

Synthetic HIV-2 protease cleaves the GAG precursor of HIV-1 with the same specificity as HIV-1 protease

A 99-amino acid protein having the deduced sequence of the protease from human immunodeficiency virus type 2 (HIV-2) was synthesized by the solid phase method and tested for specificity. The folded peptide catalyzes specific processing of a recombinant 43-kDa GAG precursor protein (F-16) of HIV-1. Although the protease of HIV-2 shares only 48% amino acid identity with that of HIV-1, the HIV-2 enzyme exhibits the same specificity toward the HIV-1 GAG precursor. Fragments of 34, 32, 24, 10, and 9 kDa were generated from F-16 GAG incubated with the protease. N-terminal amino acid sequence analysis of proteolytic fragments indicate that cleavage sites recognized by HIV-2 protease are identical to those of HIV-1 protease. The verified cleavage sites in F-16 GAG appear to be processed independently, as indicated by the formation of the intermediate fragments P32 and P34 in nearly equal ratios. The site nearest the amino terminus is quite conserved between the two viral GAG proteins (...VSQNY-PIVQN...in HIV-1,...KGGNY-PVQHV...in HIV-2). In contrast, the putative second site (...IPFAA-AQQKG...) of HIV-2 GAG shares minimal sequence identity with site 2 of HIV-1 GAG (...SATIM-MQRGN...). These sequence variations in the substrates suggest higher order structural features that may influence recognition by the proteases. Pepstatin A inhibits HIV-2 protease, whereas 1,10-phenanthroline and phenylmethylsulfonylfluoride do not; these results are in agreement with the finding that proteases of HIV and other retroviruses are aspartyl proteases.     

Crystal structure of a cross-reaction complex between an anti-HIV-1 protease antibody and an HIV-2 protease peptide

The monoclonal antibody 1696, elicited by HIV-1 protease, inhibits the activity of both HIV-1 and HIV-2 proteases with inhibition constants in the low nanomolar range. The antibody cross-reacts with peptides derived from the N-terminal region of both proteases. The crystal structure of the recombinant single-chain Fv fragment of 1696 complexed with an N-terminal peptide from the HIV-2 protease has been determined at 1.88A resolution. Interactions of the peptide with scFv1696 are compared with the previously reported structure of scFv1696 in complex with the corresponding peptide from HIV-1 protease. The origin of cross-reactivity of mAb1696 with HIV proteases is discussed.