Functional Genomics on Potato Virus A: Virus Genome-Wide Map of Sites Essential for Virus Propagation

Abstract

Transposition-based in vitro insertional mutagenesis strategies provide promising new approaches for functional characterization of any cloned gene or genome region. We have extended the methodology and scope of such analysis to a complete viral genome. To map genome regions both essential and nonessential for Potato virus A propagation, we generated a genomic 15-bp insertion mutant library utilizing the efficient in vitro DNA transposition reaction of phage Mu. We then determined the proficiency of 1125 mutants to propagate in tobacco protoplasts by using a genetic footprinting strategy that simultaneously mapped the genomic insertion sites. Over 300 sites critical for virus propagation were identified, and many of them were located in positions previously not assigned to any viral functions. Many genome regions tolerated insertions indicating less important sites for virus propagation and thus pinpointed potential locations for further genome manipulation. The methodology described is applicable to a detailed functional analysis of any viral nucleic acid cloned as DNA and can be used to address many different processes during viral infection cycles

Uridylylation of the Potyvirus VPg by Viral Replicase NIb Correlates with the Nucleotide Binding Capacity of VPg

“…The importance of this particular site in linking the genomic RNA of PVA to VPg is not proven, and therefore it is not ruled out that another tyrosine in PVA VPg may form the link. Adding support to this possibility is that the 5-amino acid insertion adjacent to Tyr-63 of VPg in the infectious cDNA of PVA had no influence on infectivity (33). However, other explanations seem more likely.…”

“…For example, the (Ϫ)-strand synthesis may require a different priming mechanism from that of the (ϩ)-strand synthesis or the in vitro system may not faithfully recapitulate in vivo events. All potyviral proteins are essential for virus propagation at a single cell level (33), and it therefore seems that the potyviral replication complex is formed via a complicated set of protein-protein interactions. The 6K2 protein directs the rep -FIG.…”

Phosphorylation of the Potyvirus Capsid Protein by Protein Kinase CK2 and Its Relevance for Virus Infection [W]

“…Another three sequences contained "atypical" CK2 phosphoacceptor sites. In a recent report, an in vitro DNA transposition-based strategy was used to generate a genome-wide insertion mutant library of PVA (Kekarainen et al, 2002). The virus could not tolerate a fiveamino acid insertion at Glu-245, disrupting the CK2 consensus sequence.…”

Plant biotechnology for deeper understanding, wider use and further development of agricultural and horticultural crops

“…The potyviral genome contains two positions (P1/ HC-Pro and NIb/CP) suitable for inserting foreign sequences without greatly affecting viral infectivity (Dolja et al 1992, Varrelman andMaiss 2000) and can be used also in PVA (Ivanov et al 2003, Kelloniemi et al 2006. A third, novel cloning site inside the P1 encoding region of PVA was detected by a transposone-based insertion mutagenesis approach (Kekarainen et al 2002).…”