Abstract
To analyze the subcellular trafficking of herpesvirus capsids, the small capsid protein has been labeled with different fluorescent proteins. Here, we analyzed the infectivity of several HSV1(17+) strains in which the N-terminal region of the non-essential small capsid protein VP26 had been tagged at different positions. While some variants replicated with similar kinetics as their parental wild type strain, others were not infectious at all. Improper tagging resulted in the aggregation of VP26 in the nucleus, prevented efficient nuclear egress of viral capsids, and thus virion formation. Correlative fluorescence and electron microscopy showed that these aggregates had sequestered several other viral proteins, but often did not contain viral capsids. The propensity for aggregate formation was influenced by the type of the fluorescent protein domain, the position of the inserted tag, the cell type, and the progression of infection. Among the tags that we have tested, mRFPVP26 had the lowest tendency to induce nuclear aggregates, and showed the least reduction in replication when compared to wild type. Our data suggest that bona fide monomeric fluorescent protein tags have less impact on proper assembly of HSV1 capsids and nuclear capsid egress than tags that tend to dimerize. Small chemical compounds capable of inducing aggregate formation of VP26 may lead to new antiviral drugs against HSV infections.
Highlights
Single and dual-color fluorescently tagged strains are valuable tools to elucidate the intracellular trafficking of virions and subviral particles
Construction of herpes simplex virus type 1 (HSV1) Strains with Tagged VP26 To identify strains whose capsid formation and intracellular trafficking mimic that of their untagged parent, we compared several constructs with different fluorescent protein (FP) tags on VP26 that we had introduced into the HSV1(17+)blueLox bacterial artificial chromosome (BAC)
The extent of inhibition depended on the type of the tag with mRFP or CFP being almost non-invasive, on the location of the tag with XFPVP26Daa1–7 being better tolerated than XFPVP26Daa5–7, and on the cell line with Vero cells being less prone to aggregate formation than HeLa, Retinal pigment epithelial (RPE) or neuronal cells
Summary
Single and dual-color fluorescently tagged strains are valuable tools to elucidate the intracellular trafficking of virions and subviral particles. SCPs are essential for the replication of human and mouse cytomegalovirus, Epstein-Barr virus and Kaposis sarcoma-associated herpesvirus, but not for the alphaherpesviruses herpes simplex virus type 1 (HSV1), pseudorabiesvirus (PrV) or varizella zoster virus (VZV; [7,8,19,20,21,22,23]). HSV1 strains lacking the SCP yield lower titers than wild type in the murine eye and trigeminal ganglion after corneal infection as well as in BHK cells [8,20,24]. PrV lacking the SCP is less neuroinvasive and grows to lower titers in cell culture, while the SCP of VZV is essential for infection of the human skin xenograft murine model and of melanoma cells but not of embryonic lung fibroblasts [7,25]
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