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

Abstract

We reported previously that the capsid protein (CP) of Potato virus A (PVA) is phosphorylated both in virus-infected plants and in vitro. In this study, an enzyme that phosphorylates PVA CP was identified as the protein kinase CK2. The ␣ -catalytic subunit of CK2 (CK2 ␣ ) was purified from tobacco and characterized using in-gel kinase assays and liquid chromatographytandem mass spectrometry. The tobacco CK2 ␣ gene was cloned and expressed in bacterial cells. Specific antibodies were raised against the recombinant enzyme and used to demonstrate the colocalization of PVA CP and CK2 ␣ in infected tobacco protoplasts. A major site of CK2 phosphorylation in PVA CP was identified by a combination of mass spectrometric analysis, radioactive phosphopeptide sequencing, and mutagenesis as Thr-242 within a CK2 consensus sequence. Amino acid substitutions that affect the CK2 consensus sequence in CP were introduced into a full-length infectious cDNA clone of PVA tagged with green fluorescent protein. Analysis of the mutant viruses showed that they were defective in cell-to-cell and longdistance movement. Using in vitro assays, we demonstrated that CK2 phosphorylation inhibited the binding of PVA CP to RNA, suggesting a molecular mechanism of CK2 action. These results suggest that the phosphorylation of PVA CP by CK2 plays an important regulatory role in virus infection.

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

“…A full-length, infectious cDNA is now available from two PVA strains with a slightly different host range (Puurand et al 1996, Paalme et al 2004). 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).…”

Purification of viral genome-linked protein VPg from potato virus A-infected plants reveals several post-translationally modified forms of the protein

“…A six-Hishaemagglutinin (HisHA) tag was attached to the 39 end of the VPg encoding gene in a vector containing the full-length infectious cDNA of PVA B11 (Puurand et al, 1996) tagged with green fluorescent protein (GFP) and cloned under the cauliflower mosaic virus 35S promoter (35S-PVA-GFP; Ivanov et al, 2003) using standard procedures. The previously described PVA infectious cDNA fragment (nt 5163-6370) in pBlueScript (Rajamäki & Valkonen, 1999) was engineered by site-directed PCR mutagenesis to introduce the coding sequence for the HisHA tag (HHHHHHYPYDVPDYA).…”

Design, synthesis and biological evaluation of 2-aminopyrimidinones and their 6-aza-analogs as a new class of CK2 inhibitors

“…CK2 overexpression and hyper-activation are observed in a large number of tumors 2 , including breast cancer 3 , lung 4 , prostate 5 , colon 6 , kidney 7 and hematological malignancies 8 . Recently, it was reported that the decrease in CK2 regulation plays a critical role in inflammatory 9 and infectious diseases 10 . Altogether, these facts raised the importance of finding pharmacologically useful inhibitors of this kinase.…”