Structure‐based release analysis of the JC virus agnoprotein regions: A role for the hydrophilic surface of the major alpha helix domain in release

Abstract

Agnoprotein (Agno) is an important regulatory protein of JC virus (JCV), BK virus (BKV) and simian virus 40 (SV40) and these viruses are unable to replicate efficiently in the absence of this protein. Recent 3D-NMR structural data revealed that Agno contains two alpha-helices (a minor and a major) while the rest of the protein adopts an unstructured conformation (Coric et al., 2017, J Cell Biochem). Previously, release of the JCV Agno from the Agno-positive cells was reported. Here, we have further mapped the regions of Agno responsible for its release by a structure-based systematic mutagenesis approach. Results revealed that amino acid residues (Lys22, Lys23, Phe31, Glu34, and Asp38) located either on or adjacent to the hydrophilic surface of the major alpha-helix domain of Agno play critical roles in release. Additionally, Agno was shown to strongly interact with unidentified components of the cell surface when cells are treated with Agno, suggesting additional novel roles for Agno during the viral infection cycle.

Expression of novel proteins by polyomaviruses and recent advances in the structural and functional features of agnoprotein of JC virus, BK virus, and simian virus 40

“…This high level of conservation suggests that they also play similar roles in release of BKV and SV40 agnoproteins. Note that, in addition to JCV agnoprotein, the release of BKV and SV40 agnoprotein was also observed (unpublished data; Saribas, White, et al, ). The exact mechanism of agnoprotein release is currently unknown, however, the involvement of the ER to Golgi pathway was previously suspected using a specific inhibitor of this pathway, Brefeldin A (Otlu et al, ).…”

“…Agnoprotein was recently found to be released from the agnoprotein‐positive cells in relatively significant amounts (Otlu et al, ; Saribas, White, et al, ). The structure‐based release analysis studies clearly showed that the hydrophilic surface of the major α‐helix domain of the protein plays critical roles in this process (Saribas, White, et al, ).…”

“…The specific amino acid residues on each surface were analyzed for their contribution to agnoprotein release by site‐directed mutagenesis. Results demonstrated that two positively (Lys22 and Lys23) and two negatively (Glu34 and Asp38) charged residues located on the hydrophilic surface play critical roles in agnoprotein release (Saribas, White, et al, ). In addition, an aromatic residue, Phe31, located adjacent to the hydrophilic surface was also identified to be a significant contributor to this process (Saribas, White, et al, ; Figure ).…”

“…Results demonstrated that two positively (Lys22 and Lys23) and two negatively (Glu34 and Asp38) charged residues located on the hydrophilic surface play critical roles in agnoprotein release (Saribas, White, et al, ). In addition, an aromatic residue, Phe31, located adjacent to the hydrophilic surface was also identified to be a significant contributor to this process (Saribas, White, et al, ; Figure ). Interestingly, amino acid residues that are involved in agnoprotein release are all but Asp38 conserved among JCV, BKV, and SV40 agnoproteins.…”

Discovery and characterization of novel trans‐spliced products of human polyoma JC virus late transcripts from PML patients

“…The resulting plasmid was designated as Bluescript KS (+)-JCV Mad-1 (T524-Stop). The coding sequences of ORF1, ORF1M (Ile23Met), and ORF2 were subcloned into a pCGT7 vector (Saribas, White, & Safak, 2017) at XbaI/BamHI sites, where ORFs were tagged with a T7-tag sequence (MASMTGGQQMG) at their N-termini. The resulting expression plasmids were designated as pCGT7-JCV ORF1, pCGT7-JCV ORF1M (Ile23Met), and pCGT7-JCV ORF2.…”