Abstract
Vesicular stomatitis virus (VSV) assembly requires condensation of the viral ribonucleoprotein (RNP) core with the matrix protein (M) during budding from the plasma membrane. The RNP core comprises the negative-sense genomic RNA completely coated by the nucleocapsid protein (N) and associated by a phosphoprotein (P) with the large polymerase protein (L). To study the assembly of single viral particles, we taggedMand P with fluorescent proteins. We selected from a library of viruses with insertions in theMgene a replication-competent virus containing a fluorescentMand combined that with our previously described virus containing fluorescent P. Virus particles containing those fusions maintained the same bullet shape appearance as wild-type VSV but had a modest increase in particle length, reflecting the increased genome size. Imaging of the released particles revealed a variation in the amount ofMand P assembled into the virions, consistent with a flexible packaging mechanism. We used the recombinants to further study the importance of the late domains in M, which serve to recruit the endosomal sorting complex required for transport (ESCRT) machinery during budding. Mutations in late domains resulted in the accumulation of virions that failed to pinch offfrom the plasma membrane. Imaging of single virions released from cells that were coinfected withMtagged with enhanced green fluorescent protein andMtagged with mCherry variants in which the late domains of one virus were inactivated by mutation showed a strong bias against the incorporation of the late-domain mutant into the released virions. In contrast, the intracellular expression and membrane association of the two variants were unaltered. These studies provide new tools for imaging particle assembly and enhance our resolution of existing models for assembly of VSV.
Original language | English |
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Pages (from-to) | 11750-11760 |
Number of pages | 11 |
Journal | Journal of virology |
Volume | 89 |
Issue number | 23 |
DOIs | |
State | Published - 2015 |