A phage single-stranded DNA (ssDNA) binding protein complements ssDNA accumulation of a geminivirus and interferes with viral movement

Geminiviruses are plant viruses with circular single-stranded DNA (ssDNA) g enomes encapsidated in double icosahedral particles. Tomato leaf curl gemin ivirus (ToLCV) requires coat protein (CP) for the accumulation of ssDNA in protoplasts and in plants but not for systemic infection and symptom develo pment in plants. In the absence of CP, infected protoplasts accumulate redu ced levels of ssDNA and increased amounts of double-stranded DNA (dsDNA), c ompared, to accumulation in the presence of wild-type virus. To determine w hether the gene 5 protein (g5p), a ssDNA binding protein from Escherichia c oli phage M13, could restore the accumulation of ssDNA, ToLCV that lacked t he CP gene was modified to express g5p or g5p fused to the N-terminal 66 am ino acids of CP (CP66:6G:g5). The modified viruses led to the accumulation of wild-type levels of ssDNA and high levels of dsDNA The accumulation of s sDNA was apparently due to stable binding of g5p to viral ssDNA. The high l evels of dsDNA accumulation during infections with the modified viruses sug gested a direct role for CP in viral DNA replication. ToLCV that produced t he CP66:6G:g5 protein did not spread efficiently in Nicotiana benthamiana p lants, and inoculated plants developed only very mild symptoms. In infected protoplasts, the CP66:6G:g5 protein was immunolocalized to nuclei. We prop ose that the fusion protein interferes with the function of the BV1 movemen t protein and thereby prevents spread of the infection.