Thierry Michon

A virus-based biocatalyst

Virus particles are probably the most precisely defined nanometre-sized objects that can be formed by protein self-assembly. Although their natural function is the storage and transport of genetic material, they have more recently been applied as scaffolds for mineralization and as containers for the encapsulation of inorganic compounds. The reproductive power of viruses has been used to develop versatile analytical methods, such as phage display, for the selection and identification of (bio)active compounds. To date, the combined use of self-assembly and reproduction has not been used for the construction of catalytic systems. Here we describe a self-assembled system based on a plant virus that has its coat protein genetically modified to provide it with a lipase enzyme. Using single-object and bulk catalytic studies, we prove that the virus-anchored lipase molecules are catalytically active. This anchored biocatalyst, unlike man-made supported catalysts, has the capability to reproduce itself in vivo, generating many independent catalytically active copies.

The potyviral virus genome‐linked protein VPg forms a ternary complex with the eukaryotic initiation factors eIF4E and eIF4G and reduces eIF4E affinity for a mRNA cap analogue

The virus protein linked to the genome (VPg) of plant potyviruses is a 25‐kDa protein covalently attached to the genomic RNA 5′ end. It was previously reported that VPg binds specifically to eIF4E, the mRNAcap‐binding protein of the eukaryotic translation initiation complex. We performed a spectroscopic study of the interactions between lettuce eIF4E and VPg from lettuce mosaic virus (LMV). The cap analogue m7GDP and VPg bind to eIF4E at two distinct sites with similar affinity (Kd = 0.3 µm). A deeper examination of the interaction pathway showed that the binding of one ligand induces a decrease in the affinity for the other by a factor of 15. GST pull‐down experiments from plant extracts revealed that VPg can specifically trap eIF4G, the central component of the complex required for the initiation of protein translation. Our data suggest that eIF4G recruitment by VPg is indirectly mediated through VPg–eIF4E association. The strength of interaction between eIF4E and pep4G, the eIF4E‐binding domain on eIF4G, was increased significantly by VPg. Taken together these quantitative data show that VPg is an efficient modulator of eIF4E biochemical functions.

Mutational Analysis of Plant Cap-Binding Protein eIF4E Reveals Key Amino Acids Involved in Biochemical Functions and Potyvirus Infection

ABSTRACT The eukaryotic translation initiation factor 4E (eIF4E) (the cap-binding protein) is involved in natural resistance against several potyviruses in plants. In lettuce, the recessive resistance genes mo11 and mo12 against Lettuce mosaic virus (LMV) are alleles coding for forms of eIF4E unable, or less effective, to support virus accumulation. A recombinant LMV expressing the eIF4E of a susceptible lettuce variety from its genome was able to produce symptoms in mo11 or mo12 varieties. In order to identify the eIF4E amino acid residues necessary for viral infection, we constructed recombinant LMV expressing eIF4E with point mutations affecting various amino acids and compared the abilities of these eIF4E mutants to complement LMV infection in resistant plants. Three types of mutations were produced in order to affect different biochemical functions of eIF4E: cap binding, eIF4G binding, and putative interaction with other virus or host proteins. Several mutations severely reduced the ability of eIF4E to complement LMV accumulation in a resistant host and impeded essential eIF4E functions in yeast. However, the ability of eIF4E to bind a cap analogue or to fully interact with eIF4G appeared unlinked to LMV infection. In addition to providing a functional mutational map of a plant eIF4E, this suggests that the role of eIF4E in the LMV cycle might be distinct from its physiological function in cellular mRNA translation.

Virus scaffolds as enzyme nano-carriers

The cooperative organization of enzymes by cells is a key feature for the efficiency of living systems. In the field of nanotechnologies, effort currently aims at mimicking this natural organization. Nanoscale resolution and high-registration alignment are necessary to control enzyme distribution in nano-containers or on the surface of solid supports. Virus capsid self-assembly is driven by precise supramolecular combinations of protein monomers, which have made them attractive building blocks to engineer enzyme nano-carriers (ENCs). We discuss some examples of what in our opinion constitute the latest advances in the use of plant viruses, bacteriophages and virus-like particles (VLPs) as nano-scaffolds for enzyme selection, enzyme confinement and patterning, phage therapy, raw material processing, and single molecule enzyme kinetics studies.

The 20S proteasome α5 subunit of Arabidopsis thaliana carries an RNase activity and interacts in planta with the lettuce mosaic potyvirus HcPro protein.

In plants, the ubiquitin/26S proteasome system (UPS) plays a central role in protein degradation and is involved in many steps of defence mechanisms, regardless of the types of pathogen targeted. In addition to its proteolytic activities, the UPS ribonuclease (RNase) activity, previously detected in 20S proteasome preparations from cauliflower and sunflower (Helianthus annuus), has been shown to specifically target plant viral RNAs in vitro. In this study, we show that recombinant Arabidopsis thaliana proteasomal α(5) subunit expressed in Escherichia coli harbours an RNase activity that degrades Tobacco mosaic virus (TMV, Tobamovirus)- and Lettuce mosaic virus (LMV, Potyvirus)-derived RNAs in vitro. The analysis of mutated forms of the α(5) subunit demonstrated that mutation of a glutamic acid at position 110 affects RNase activity. Furthermore, it was demonstrated, using a bimolecular fluorescence complement assay, that the multifunctional helper component proteinase (HcPro) of LMV, already known to interfere with the 20S proteasome RNase activity in vitro, can interact in vivo with the recombinant α(5) subunit. Further experiments demonstrated that, in LMV-infected lettuce cells, α(5) is partially relocalized to HcPro-containing infection-specific inclusions. Susceptibility analyses of Arabidopsis mutants, knocked out for each At-PAE gene encoding α(5) , showed that one (KO-pae1) of the two mutants exhibited a significantly increased susceptibility to LMV infection. Taken together, these results extend to A. thaliana α(5) the range of HcPro-interacting proteasomal subunits, and suggest that HcPro may modulate its associated RNase activity which may contribute to an antiviral response.

The C terminus of lettuce mosaic potyvirus cylindrical inclusion helicase interacts with the viral VPg and with lettuce translation eukaryotic initiation factor 4E

Recessive resistance to lettuce mosaic virus (LMV) is conferred in lettuce by the mo1 gene, encoding the eukaryotic translation initiation factor 4E (eIF4E). The C terminus of the viral cylindrical inclusion helicase (CI-Cter), together with the VPg, is involved directly in overcoming mo1 resistance. In this study, recombinant LMV VPg and CI-Cter proteins from wild-type or resistance-breaking isolates were expressed and purified from Escherichia coli. The allelic forms of eIF4E from susceptible or resistant lettuce cultivars were produced similarly and these proteins were used in ELISA-based assays to demonstrate the in vitro binding of the various forms of LMV CI-Cter to both lettuce eIF4E and LMV VPg proteins. All combinations tested displayed significant and specific interactions, and the interaction between the C-terminal part of the LMV CI and eIF4E was confirmed in vivo in bimolecular fluorescence complementation assays. Higher interaction signals for both CI-eIF4E and CI-VPg were observed for LMV-E, indicating that the eIF4E interaction network involving CI and VPg appears to be stronger in the case of this resistance-breaking isolate. This could suggest the need for a minimal interaction threshold for infection success in resistant lettuce, but more precise measurement of the interaction parameters linking eIF4E, VPg and CI is needed in order to reinforce such a hypothesis.

Stereoselective Incorporation of an Unsaturated Isoleucine Analogue into a Protein Expressed in E. coli

The unsaturated amino acid 2‐amino‐3‐methyl‐4‐pentenoic acid (E‐Ile) was prepared in the form of its (2S,3S),(2R,3R) and (2S,3R),(2R,3S) stereoisomeric pairs. The translational activities of SS‐E‐Ile and SR‐E‐Ile were assessed in an E. coli strain rendered auxotrophic for isoleucine. SS‐E‐Ile was incorporated into the test protein mouse dihydrofolate reductase (mDHFR) in place of isoleucine at a rate of up to 72 %; SR‐E‐Ile yielded no conclusive evidence for incorporation. ATP/PPi exchange assays indicated that SS‐E‐Ile was activated by the isoleucyl‐tRNA synthetase at a rate comparable to that characteristic of isoleucine; SR‐E‐Ile was activated approximately 100‐times more slowly than SS‐E‐Ile.

Electrochemical Atomic Force Microscopy Imaging of Redox-Immunomarked Proteins on Native Potyviruses: From Subparticle to Single-Protein Resolution

We show herein that electrochemical atomic force microscopy (AFM-SECM), operated in molecule touching (Mt) mode and combined with redox immunomarking, enables the in situ mapping of the distribution of proteins on individual virus particles and makes localization of individual viral proteins possible. Acquisition of a topography image allows isolated virus particles to be identified and structurally characterized, while simultaneous acquisition of a current image allows the sought after protein, marked by redox antibodies, to be selectively located. We concomitantly show that Mt/AFM-SECM, due to its single-particle resolution, can also uniquely reveal the way redox functionalization endowed to viral particles is distributed both statistically among the viruses and spatially over individual virus particles. This possibility makes Mt/AFM-SECM a unique tool for viral nanotechnology.

Cotranslational coat protein-mediated inhibition of potyviral RNA translation.

ABSTRACT Potato virus A (PVA) is a single-stranded positive-sense RNA virus and a member of the family Potyviridae. The PVA coat protein (CP) has an intrinsic capacity to self-assemble into filamentous virus-like particles, but the mechanism responsible for the initiation of viral RNA encapsidation in vivo remains unclear. Apart from virion assembly, PVA CP is also involved in the inhibition of viral RNA translation. In this study, we show that CP inhibits PVA RNA translation in a dose-dependent manner, through a mechanism involving the CP-encoding region. Analysis of this region, however, failed to identify any RNA secondary structure(s) preferentially recognized by CP, suggesting that the inhibition depends on CP-CP rather than CP-RNA interactions. In agreement with this possibility, insertion of an in-frame stop codon upstream of the CP sequence led to a marked decrease in the inhibition of viral RNA translation. Based on these results, we propose a model in which the cotranslational interactions between excess CP accumulating in trans and CP translated from viral RNA in cis are required to initiate the translational repression. This model suggests a mechanism for how viral RNA can be sequestered from translation and specifically selected for encapsidation at the late stages of viral infection. IMPORTANCE The main functions of the CP during potyvirus infection are to protect viral RNA from degradation and to transport it locally, systemically, and from host to host. Although virion assembly is a key step in the potyviral infectious cycle, little is known about how it is initiated and how viral RNA is selected for encapsidation. The results presented here suggest that CP-CP rather than CP-RNA interactions are predominantly involved in the sequestration of viral RNA away from translation. We propose that the cotranslational nature of these interactions may represent a mechanism for the selection of viral RNA for encapsidation. A better understanding of the mechanism of virion assembly may lead to development of crops resistant to potyviruses at the level of viral RNA encapsidation, thereby reducing the detrimental effects of potyvirus infections on food production.

Further studies on the human pancreatic binary complexes involving procarboxypeptidase A.

In contrast to procarboxypeptidase B which has always been reported to be secreted by the pancreas as a monomer, procarboxypeptidase A occurs as a monomer and/or associated to one or two functionally different proteins, depending on the species. Recent studies showed that, in the human pancreatic secretion, procarboxypeptidase A is mainly secreted as a 44 kDa protein involved in at least three different binary complexes. As previously reported, two of these complexes associated procarboxypeptidase A to either a glycosylated truncated protease E or zymogen E. In this paper, we identified proelastase 2 as the partner of procarboxypeptidase A in the third complex, thus reporting for the first time the occurrence of a proelastase 2/procarboxypeptidase A binary complex in vertebrates. Moreover, from N‐terminal sequence analyses, the 44 kDa procarboxypeptidase A involved in these complexes was identified as being of the A1 type. Only one type of procarboxypeptidase B, the B1 type, has been detected in the analyzed pancreatic juices, thus emphasizing the previously observed genetic differences between individuals.