Fluorescence Microscopy Journal Club

Asymmetric packaging of polymerases within vesicular stomatitis virus

by Jeffery Hodges, Xiaolin Tang, Michael B. Landesman, John B. Ruedas, Anil Ghimire,
Manasa V. Gudheti, Jacques Perrault, Erik M. Jorgensen, Jordan M. Gerton, Saveez Saffarian

Biochemical and Biophysical Research Communications 2013, 440, 2, pp. 271-276

The vesicular stomatitis virus (VSV) a prototypical negative sense single-stranded RNA virus commonly used in the study of viral evolution and pathology. The study of viral behavior and structural organization in such viruses as VSV serves as a building block for the broader understanding of retroviral virus families, such as rabies and HIV. Furthermore, VSV has been targeted for its ability to be genetically modified for use in vaccines for Ebola and rabies, as well as its ability to be used in a variety of oncology treatments.

VSV is a bullet-shaped virus approximately 180 nm long by 80 nm wide, and contains multiple copies of L and P polymerase proteins that are used in transcribing and replicating the N-RNA of the virion. The polymerase engages with promoter sites located at the 3’ end of the RNA strand, determined previously by CryoEM, located at the bullet end of the virion. The authors use a combination of sub-diffraction techniques, namely super-resolution optical microscopy and atomic force microscopy (AFM) to determine the location and quantification of these polymerase proteins within the manifold of the virus to determine its spatial relationship to the transcriptional promoter site of the viral genome.

A series of super-resolution optical localization microscopy experiments on the VSV virus were conducted to measure the relative distance of the L and P proteins to the surface of the virus. This was accomplished by a novel combination of recombinant viruses encoding for the fluorescent protein eGFP for use in conventional diffraction-limited imaging to use as a center-of-mass marker for either the L or P protein, coupled with a super-resolution image of the virus membrane to determine the structural manifold of the VSV virus, leading to the conclusion that the placement of the L and P proteins being near the blunt end of the virus.

Verification of this phenomenon was corroborated via AFM in a fluorescent-free assay where the surface stiffness of the VSV virus was directly measured. The increase in the protein load at one end of the virus or the other will directly relate to a differing stiffness value for the virus surface, with the AFM being used as an active force sensor. These finding agreed well with the findings determined from imaging the virions with optical super-resolution techniques.

In conclusion, the authors determine that the L and P proteins, and hence the polymerase for the N-RNA, are packaged asymmetrically within the volume of the VSV virus. This has direct consequences for transcription events after viral entry into a host cell, and the knowledge of the location of the polymerase with respect to the position and layout of the N-RNA can help further the modeling of the replication of these viruses once they infect a host cell. This paper is a nice example of using super-resolution localization microscopy to determine not only spatial location but quantification as well of labeled protein, as well as incorporating another microscopy technique to further validate the optical results.