Mimivirus collagen is modified by bifunctional lysyl hydroxylase and glycosyltransferase enzyme.

Collagens, the most abundant proteins in animals, are modified by hydroxylation of proline and lysine residues and by glycosylation of hydroxylysine. Dedicated prolyl hydroxylase, lysyl hydroxylase, and collagen glycosyltransferase enzymes localized in the endoplasmic reticulum mediate these modifications prior to the formation of the collagen triple helix. Whereas collagen-like proteins have been described in some fungi, bacteria, and viruses, the post-translational machinery modifying collagens has never been described outside of animals. We demonstrate that the L230 open reading frame of the giant virus Acanthamoeba polyphaga mimivirus encodes an enzyme that has distinct lysyl hydroxylase and collagen glycosyltransferase domains. We show that mimivirus L230 is capable of hydroxylating lysine and glycosylating the resulting hydroxylysine residues in a native mimivirus collagen acceptor substrate. Whereas in animals from sponges to humans the transfer of galactose to hydroxylysine in collagen is conserved, the mimivirus L230 enzyme transfers glucose to hydroxylysine, thereby defining a novel type of collagen glycosylation in nature. The presence of hydroxylysine in mimivirus proteins was confirmed by amino acid analysis of mimivirus recovered from A. polyphaga cultures. This work shows for the first time that collagen post-translational modifications are not confined to the domains of life. The utilization of glucose instead of the galactose found throughout animals as well as a bifunctional enzyme rather than two separate enzymes may represent a parallel evolutionary track in collagen biology. These results suggest that giant viruses may have contributed to the evolution of collagen biology.

Virulence of a mouse-adapted Semliki Forest virus strain is associated with reduced susceptibility to interferon.

Type I interferons (IFNs) are essential components of the innate immune system. This study characterized the distinct IFN sensitivities of two closely related Semliki Forest virus (SFV) strains in cell culture. The virulent L10 strain was derived from the original virus isolate by propagation in mice. In contrast, the avirulent SFV strain, designated V42, was derived from an earlier passage of the original virus isolated from mosquitoes. The virulent L10 strain produced a cytopathic effect (CPE) in IFN-treated cells and the production of infectious virus was only two orders of magnitude lower compared with untreated cells. In contrast, the avirulent V42 exerted no CPE in IFN-treated cells and production of infectious virus was four orders of magnitude lower compared with untreated cells. The reduced CPE in IFN-treated cells infected with the avirulent V42 strain was due to inhibition of productive infection and not to reduced cell death. The virulent L10 strain synthesized less genomic RNA but more non-structural proteins than the avirulent V42 strain, suggesting more efficient translation of the L10 genomic RNA. Using a cell line unable to produce IFN, it was shown that the reduced susceptibility of the L10 strain to the action of IFN was not due to reduced IFN induction. Hence, the reduced susceptibility of the virulent L10 strain to the action of IFN allows it to overcome the established IFN-induced antiviral state of the cell, thereby increasing its virulence.

Rapid selection of specific MAP kinase-binders from designed ankyrin repeat protein libraries.

We describe here the rapid selection of specific MAP-kinase binders from a combinatorial library of designed ankyrin repeat proteins (DARPins). A combined in vitro/in vivo selection approach, based on ribosome display and the protein fragment complementation assay (PCA), yielded a large number of different binders that are fully functional in the cellular cytoplasm. Ribosome-display selection pools of four successive selection rounds were examined to monitor the enrichment of JNK2-specific DARPins. Surprisingly, only one round of ribosome display with subsequent PCA selection of this pool was necessary to isolate a first specific binder with micromolar affinity. After only two rounds of ribosome-display selection followed by PCA, virtually all DARPins showed JNK2-specific binding, with affinities in the low nanomolar range. The enrichment factor of ribosome display thus approaches 10(5) per round. In a second set of experiments, similar results were obtained with the kinases JNK1 and p38 as targets. Again, almost all investigated DARPins obtained after two rounds of ribosome display showed specific binding to the targets used, JNK1 or p38. In all three selection experiments the identified DARPins possess very high specificity for the target kinase. Taken together, the combination of ribosome display and PCA selections allowed the identification of large pools of binders at unparalleled speed. Furthermore, DARPins are applicable in intracellular selections and immunoprecipitations from the extract of eukaryotic cells.