The formation of viral particles requires the coordinated assembly of both nucleic acids and proteins. In the case of Rhabdoviruses, such as vesicular stomatitis virus (VSV), the particles display a characteristic bullet-shape. VSV virions consist of the matrix protein (M), glycoprotein (G), and viral ribonucleoprotein (RNP), which contains the nucleocapsid protein (N) coated RNA bound to the large polymerase protein (L) through the phosphoprotein (P). During assembly, these components are recruited to the plasma membrane where the viral RNP undergoes condensation by M and envelopment with G containing membranes. To address whether formation of the bullet-shape requires a consistent packaging of the viral proteins, the composition of single virions was measured with fluorescence microscopy. We generated autonomously replicating VSV bearing up to 3 fluorescent protein fusions in the disordered N-terminal region of M and N-terminus of P and G. Quantification of single particles reveals that VSV assembles with a range of M, P, and G molecules, suggesting a flexible packaging mechanism. The maintenance of the bullet-shape with significantly less M proposes that condensation does not require the particle to be saturated with M. Our fluorescent VSV clones permit the tracking of viral components in live cells. We observed that assembly of M into particles requires ~2 min and can be broken into 4 stages. First, M forms a small preassembly complex. Second, M rapidly assembles into particles where its incorporation initiates before P, although they are packaged concurrently. This is followed by a delay before final release of particles into the supernatant. Late domains in M were thought to only recruit the endosomal sorting complexes required for transport (ESCRT) pathway to mediate fission. However, using our M fusions we demonstrate that these motifs are required for efficient competition into released particles and a step in assembly prior to pinching off. These constructs have permitted the study of viral assembly at the single particle level and are useful tools for studying viral entry and egress. Specifically, VSV containing M-eGFP and the lassa virus glycoprotein instead of G was used to demonstrate the requirement of a host factor for lassa virus fusion. / Medical Sciences
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/17464089 |
| Date | 17 July 2015 |
| Creators | Soh, Timothy Kinshiong |
| Contributors | Whelan, Sean P., Cunningham, James M. |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | English |
| Detected Language | English |
| Type | Thesis or Dissertation, text |
| Format | application/pdf |
| Rights | open |
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