Identification of an L-rhamnose synthetic pathway in two nucleocytoplasmic large DNA viruses

Abstract

Nucleocytoplasmic large DNA viruses (NCLDVs) are characterized by large genomes that often encode proteins not commonly found in viruses. Two species in this group are Acanthocystis turfacea chlorella virus 1 (ATCV-1) (family Phycodnaviridae, genus Chlorovirus) and Acanthamoeba polyphaga mimivirus (family Mimiviridae), commonly known as mimivirus. ATCV-1 and other chlorovirus members encode enzymes involved in the synthesis and glycosylation of their structural proteins. In this study, we identified and characterized three enzymes responsible for the synthesis of the sugar L-rhamnose: two UDP-D-glucose 4,6-dehydratases (UGDs) encoded by ATCV-1 and mimivirus and a bifunctional UDP-4-keto-6-deoxy-D-glucose epimerase/reductase (UGER) from mimivirus. Phylogenetic analysis indicated that ATCV-1 probably acquired its UGD gene via a recent horizontal gene transfer (HGT) from a green algal host, while an earlier HGT event involving the complete pathway (UGD and UGER) probably occurred between a protozoan ancestor and mimivirus. While ATCV-1 lacks an epimerase/reductase gene, its Chlorella host may encode this enzyme. Both UGDs and UGER are expressed as late genes, which is consistent with their role in posttranslational modification of capsid proteins. The data in this study provide additional support for the hypothesis that chloroviruses, and maybe mimivirus, encode most, if not all, of the glycosylation machinery involved in the synthesis of specific glycan structures essential for virus replication and infection.

FIG. 1.

l-Rhamnose biosynthetic pathways in bacterial and plant cells. In bacteria, the three enzymatic activities are on separate polypeptides. In plants, UGD and UGER are fused into a single polypeptide (RHM isoforms) (34).

FIG. 2.

Maximum-likelihood tree of UDP-d-glucose 4,6-dehydratase (UGD) for 14 taxa, including the viruses ATCV-1 and mimivirus. Only posterior probabilities of ≥50% are indicated to the left of each node. The distance bar represents 0.1 amino acid substitution per site. Accession numbers follow each of the taxon names. Accession numbers with five or six digits can be obtained at the JGI Genome portal at http://genome.jgi-psf.org for the respective species. ▪, plant species; •, green algal species; ▴, nonplant/algal species; +, bacterial species (used as the outgroup, TDP-glucose 4,6-dehydratase). The two viruses are in italics.

FIG. 3.

Maximum-likelihood tree of UDP-4-keto-6-deoxy-d-glucose epimerase/reductase (UGER) for 12 taxa, including mimivirus. Only posterior probabilities of ≥50% are indicated to the left of each node. The distance bar represents 0.2 amino acid substitution per site. Accession numbers follow each of the taxon names. Accession numbers with five or six digits can be obtained at the JGI Genome portal at http://genome.jgi-psf.org for the respective species. ▪, plant species; •, green algal species; ▴, nonplant/algal species; +, bacterial species (used as the outgroup, dTDP-4-deoxyrhamnose reductase). Mimivirus is in italics.

FIG. 4.

Size exclusion chromatography of UGDs and UGER. (A) ATCV-1 UGD; (B) mimivirus UGD; (C) mimivirus UGD preincubated with 10 μM NADH; (D) mimivirus UGER. Proteins were kept in 50 mM Tris-HCl-150 mM NaCl, pH 7.5. Mimivirus UGD and UGER proteins also contained 1 mM Na EDTA and 1 mM DTT (indicated as buffer). Detection was performed at 220 nm. Molecular masses (kDa) were calculated after column calibration using known standards, as indicated in the Materials and Methods section.

FIG. 5.

Products of the enzymatic activity of UGD and UGER determined by anion-exchange chromatography. (A) UGD activity by ATCV-1; (B) production of the intermediate UDP-4-keto-6-deoxy-d-glucose by ATCV-1 UGD; (C) same as panel B but incubated with mimivirus UGER and NADPH.

FIG. 6.

Electrospray spectrum of UGD and UGER products. (A) Analysis of the UGD product, corresponding to chromatogram shown in Fig. 5A. (B) Analysis of the UGER product, corresponding to Fig. 5C.

FIG. 7.

GC-MS analysis of acetylated alditols. Extracted ion currents (XIC) of the ion at 157 m/z are shown for the standards rhamnose (Rha) and fucose (Fuc) and for the UGER product (sample).

FIG. 8.

(A) Expression profiles of the ATCV-1 Z544R (UGD) gene determined by real-time PCR at different times p.i. ATCV-1 DNA replication begins at 60 to 90 min p.i. (B) Expression profiles of mimivirus R141 (UGD) and L780 (UGER) genes.