Abstract:
Geminiviruses are a family of plant-infecting viruses characterized by twin
icosahedral capsids and circular single-stranded (ss) DNA genomes. Members
of this family cause devastating diseases in crops worldwide. Geminiviruses
replicate in the cell nucleus of the infected plant mainly through a rolling-circle
replication (RCR) mechanism. As none of the viral proteins possesses DNA
polymerase activity, these viruses heavily rely on the host replication machinery
for successful replication. As viral replication is a fundamental step in the
infection cycle, the host factors essential for this process can serve as potential
targets to develop strategies to control infection. We have previously shown
that DNA polymerase α and δ are required for viral replication. However, the
composition of the viral replisome remains mostly elusive. The viral replication
associated (Rep) protein is highly conserved, and the only virus-encoded
protein essential for this process. Here, we used Rep from the geminivirus
tomato yellow leaf curl virus (TYLCV) as a bait to capture host factors involved
in viral replication in Nicotiana benthamiana via TurboID-based proximity
labelling (PL) followed by mass spectrometry (MS) analysis. Our data
demonstrate that geminiviruses exploit the molecular machinery mediating
eukaryotic leading-strand, but not lagging-strand, DNA synthesis in the
bidirectional replication fork. An exception is the DNA polymerases, which are
swapped. Furthermore, our findings suggest that the composition of the viral
replisome is conserved within the phylum Circular Rep-Encoding Single
Stranded (CRESS) DNA viruses. In addition, our work shows that eukaryotic
DNA helicase loaders, but not the helicase itself, contribute to the viral genome
replication, underscoring a mechanistic divergence in replication initiation
between geminiviruses and their plant hosts. Structural prediction indicates that
geminiviral Rep assembles into a homo-hexameric complex, analogous to the
hetero-hexameric eukaryotic helicase. Taken together, our results demonstrate
that geminiviruses selectively repurpose the plant DNA replication machinery
to carry out RCR. Collectively, our study provides new insight into viral
replication strategies and lays the foundation for the identification of new
antiviral targets.
geminiviruses