Determination of the a064r gene function produced by paramecium bursaria chlorella virus

The chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1), is a large dsDNA virus that infects the microalgae Chlorella variabilis NC64A.[1] It is a member of the giant virus group, a class of viruses that differ from the others in shape, genome size and number of encoded proteins.[2] It’s main feature is that unlike to other viruses, PBCV-1 encodes most, if not all, of the components required to glycosylate its major capsid protein (MCP), named Vp54 (gene a430l).[3] Noteworthy, Vp54 is decorated with four glycans, that are different from all other N-glycans known so far in the three domains of the life.[4]

Recent analysis about the PBCV-1 gene, has disclosed that it encodes, at least, six putative glycosyltransferases[1], presumably involved in glycosylation of the its major capsid protein Vp54. One of them, the A064R, catched our attention because 18 of 21 antigenic variants of PBCV-1 present mutations in this gene or it was deleted.[5] Comparing the wild-type glycan structures from PBCV-1 with those from a set of PBCV-1 mutants, that have truncated glycan structures, it was possible to address the role of the whole a064r gene. This gene encodes a protein of 638 amino acids organized in three domains [4], each with a particular function.

Combining structural analyses with enzymatic reactions, it is now established that the domain 1 encodes for a β-(1,4)-L-rhamnosyl transferase onto a xylose acceptor,[5] in contrast with previous data, which supposed that such domain encoded for a glucosyl-transferase.[6] 

Domain 2 has no resemblance with any known glycosyltransferase, and it encodes for a α-(1,2)-L-rhamnosyl transferase that recognizes rhamnose as acceptor, while domain 3 has a double methyltransferase activity and acts on a terminal rhamnose unit.

This communication will present the strategy used to evaluate the activity of this complex protein.