Size restriction is required for proper functioning of a bipartite begomovirus AC4 protein.

Many geminiviruses including members of the genus Begomovirus produce a protein known as C4 or AC4. Whereas C4/AC4 typically consists of over 80 amino acid residues, a few are much shorter. The significance of these shorter C4/AC4 proteins in viral infection and why the virus maintains their abbreviated length is not yet understood. The AC4 of the begomovirus tomato leaf curl Hsinchu virus (ToLCHsV) contains only 65 amino acids, but it extends to 96 amino acids when the natural termination codon is replaced with a normal codon. We discovered that both interrupting and extending AC4 were harmful to ToLCHsV. The extended AC4 (EAC4) also showed a lower ability to promote the infection of the heterologous virus potato virus X than the wildtype AC4. When the wildtype AC4 was fused with yellow fluorescent protein (AC4-YFP), it was predominantly found in chloroplasts, whereas EAC4-YFP was mainly localized to the cell periphery. These results suggest that ToLCHsV's AC4 protein is important for viral infection, and the virus may benefit from its abbreviated length because it may lead to chloroplast localization.