Abstract
The L-protein of vesicular stomatitis virus (VSV) is a single-chain multi-domain RNA-dependent RNA polymerase. Previously reported attempts of intramolecular insertions of fluorescent proteins into the L-protein resulted in temperature-sensitive and highly attenuated polymerase activity. Here, we describe a novel insertion site that was selected based on in silico prediction. Of five preselected locations, insertion of the fluorescent protein mCherry in the VSV polymerase between amino acids 1620 and 1621 preserved polymerase function even after extended passaging and showed only mild attenuation compared to wildtype VSV polymerase. High magnification fluorescence imaging revealed a corpuscular cytosolic pattern for the L-protein. To confirm that the insertion site tolerates inclusion of proteins others than mCherry, we cloned mWasabi into the same position in L, generating a VSV-LmWasabi, which was also functional. We also generated a functional dual-color-dual-insertion VSV construct with intramolecularly labeled P and L-proteins. Together, our data present an approach to tag VSV polymerase intramolecularly without perturbing enzymatic activity. This L fusion protein might enable future tracing studies to monitor intracellular location of the VSV transcription and replication machinery in real-time life-imaging studies.
Highlights
Vesicular stomatitis virus (VSV) is a prototypical member of the family Rhabdoviridae and is widely studied as a model for viruses with nonsegmented negative-sense RNA genomes [1]
Using in silico prediction and recent information on the 3D structure of the L-protein, we focused our search for potential insertion sites on the connector domain (CD; 1358–1557) and the methyltransferase domain (MT; 1598–1892) (Figure 1A,B)
We selected regions located at the surface and in flexible loops, which should minimize the possibility of steric clashes. This led to the identification of the following candidate insertion sites: CD1506,Viruses
Summary
Vesicular stomatitis virus (VSV) is a prototypical member of the family Rhabdoviridae and is widely studied as a model for viruses with nonsegmented negative-sense RNA genomes [1]. The VSV L-protein is a single-chain multi-domain RNA-dependent RNA polymerase, which catalyzes mRNA 50 -capping, cap methylation, and mRNA 30 polyadenylation [4]. It is responsible for genome replication as well. A structural organization of five distinct domains and two linker regions was described: an RNA-dependent RNA polymerase spanning amino acid positions 35–865; a capping domain at positions 866–1334; a linker 1 at positions 1335–1357; a connector domain at positions 1358–1557;
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