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

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

Immacolata Speciale 1 , Garry Duncan 2 , Michela Tonetti 3 , Maria Elena Laugieri 3 , Todd Lowary 4 , Sicheng Lin 4 , Antonio Molinaro 1 , Cristina De Castro 5 , James Van Etten 2

1 Department of Chemical Sciences, University of Naples Federico II, Napoli, Italy, 2 Department of Plant Pathology and Nebraska Center for Virology, Lincoln, United States of America, 3 Department of Experimental Medicine and Center of Excellence for Biomedical Research, Genova, Italy, 4 Department of Chemistry, University of Alberta, Edmonton, United States of America, 5 Department of Agricultural Sciences, University of Napoli Federico II, Portici, Italy

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.

Van Etten, JL, Agarkova, I, Dunigan, DD, Tonetti, M, De Castro, C, Duncan, GA. Viruses, 2017, 9:88

De Castro, C, Duncan, GA, Garozzo, D, Molinaro, A, Sturiale, L, Tonetti, M, Van Etten, JL. Glycobiophysics. Advances in Experimental Medicine and Biology, 2018, vol. 1104:237-257. Springer, Singapore

Van Etten, JL, Gurnon, J, Yanai-Balser, G, Dunigan, D, Graves, MV. Biochem Biophys Acta, 2010, 1800 (2):152-159

De Castro, C, Molinaro, A, Piacente, F, Gurnon, JR, Sturiale, L, Palmigiano, A, Lanzetta, R, Parrilli, M, Garozzo, D, Tonetti, MG, Van Etten, JL. PNAS, 2013 110: 13956-13960.

Speciale, I, Duncan, GA, Unione, L, Agarkova, I, Garozzo, D, Barbero, JJ, Lin, S, Lowary, TL, Molinaro, A, Noel, E, Laugieri, ME, Tonetti, MG, Van Etten, JL, De Castro, C. JBC, 2019, doi/10.1074/jbc.RA118.007182

Zhang, Y, Xiang, Y, Van Etten, JL, Rossmann, MG. NIH Public Access, 2007, 15(9): 1031-1039

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