Asymmetric packaging of polymerases within vesicular stomatitis virus

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

doi:10.1016/j.bbrc.2013.09.064

Cited by 17 publications

(18 citation statements)

References 32 publications

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“…It is therefore not surprising that the abundance of P per particle can vary. An earlier study using a virus with a fluorescent L or P suggested that a variable amount of both of these proteins is packaged into single particles (40). The fluorescent M fusions result in viruses that are attenuated with regard to their growth, which may reflect the fact that the presence of the fluorophore itself influences the packaging of M.…”

Section: Discussionmentioning

confidence: 99%

Tracking the Fate of Genetically Distinct Vesicular Stomatitis Virus Matrix Proteins Highlights the Role for Late Domains in Assembly

Soh

Whelan

2015

J Virol

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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 tagged M and P with fluorescent proteins. We selected from a library of viruses with insertions in the M gene a replication-competent virus containing a fluorescent M and 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 of M and 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 off from the plasma membrane. Imaging of single virions released from cells that were coinfected with M tagged with enhanced green fluorescent protein and M tagged 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. IMPORTANCEAssembly of vesicular stomatitis virus (VSV) particles requires the separate trafficking of the viral replication machinery, a matrix protein (M) and a glycoprotein, to the plasma membrane. The matrix protein contains a motif termed a "late domain" that engages the host endosomal sorting complex required for transport (ESCRT) machinery to facilitate the release of viral particles. Inactivation of the late domains through mutation results in the accumulation of virions arrested at the point of release. In the study described here, we developed new tools to study VSV assembly by fusing fluorescent proteins to M and to a constituent of the replication machinery, the phosphoprotein (P). We used those tools to show that the late domains of M are required for efficient incorporation into viral particles and that the particles contain a variable quantity of M and P. V esicular stomatitis virus (VSV) forms particles with an ordered bullet shape. Those particles contain a lipid envelope modified by the insertion of the viral attachment and fusion glycoprotein (G). Inside the particle is a ribonucleoprotein (RNP) core that is delivered to the cytoplasm to initiate the process ...

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Section: Discussionmentioning

confidence: 99%

Tracking the Fate of Genetically Distinct Vesicular Stomatitis Virus Matrix Proteins Highlights the Role for Late Domains in Assembly

Soh

Whelan

2015

J Virol

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“…Both GSD and SIM indicate that the M matrix protein is more closely associated with the ribonucleoprotein complex (RNP) containing the RNA genome than with the virus envelope. A similar assessment of the sub-virion localization of the polymerase complex subunits, L and P, in the vesicular stomatitis virus (VSV) [83] indicated the proteins were located toward one end of the asymmetrically shaped virus and occupied ~50 nm of a 150 nm cavity inside the virion.…”

Section: Subviral Structure Localizationmentioning

confidence: 99%

Superresolution imaging of viral protein trafficking

Colberg-Poley

Patterson

Salka

et al. 2015

Med Microbiol Immunol

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The endoplasmic reticulum (ER) membrane is closely apposed to the outer mitochondrial membrane (OMM), which facilitates communication between these organelles. These contacts, known as mitochondria-associated membranes (MAM), facilitate calcium signaling, lipid transfer, as well as antiviral and stress responses. How cellular proteins traffic to the MAM, are distributed therein, and interact with ER and mitochondrial proteins are subject of great interest. The human cytomegalovirus UL37 exon 1 protein or viral mitochondria-localized inhibitor of apoptosis (vMIA) is crucial for viral growth. Upon synthesis at the ER, vMIA traffics to the MAM and OMM, where it reprograms the organization and function of these compartments. vMIA significantly changes the abundance of cellular proteins at the MAM and OMM, including proteins that regulate calcium homeostasis and cell death. Through the use of superresolution imaging, we have shown that vMIA is distributed at the OMM in nanometer scale clusters. This is similar to the clusters reported for the mitochondrial calcium channel, VDAC, as well as electron transport chain, translocase of the OMM complex, and mitochondrial inner membrane organizing system components. Thus, aside from addressing how vMIA targets the MAM and regulates survival of infected cells, biochemical studies and superresolution imaging of vMIA offer insights into the formation, organization, and functioning of MAM. Here, we discuss these insights into trafficking, function, and organization of vMIA at the MAM and OMM and discuss how the use of superresolution imaging is contributing to the study of the formation and trafficking of viruses.

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“…While the beads retained their height during the AFM scan, the VSV virion has a significantly smaller young's modulus and is significantly deformed in height. In this image the virion was specifically imaged with a stiff cantilever in tapping mode which produced a small xy convolution (used to determine the tip vs blunt end of the virus) and the height difference between the tip and blunt end of the virus is used to detect extra protein density at the blunt end of the virus 19 . …”

Section: Single Virion Super Resolution Imagingmentioning

confidence: 99%

“…The aim has been to deform the virus to the point that the extra protein density within the virus becomes visible as a bump within the AFM image. This method is used to detect the extra protein density within the virus cavity 19 .…”

Section: B In Solution Antibody Attackmentioning

confidence: 99%

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Hodges

Saffarian

2014

JoVE

Self Cite

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Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

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Asymmetric packaging of polymerases within vesicular stomatitis virus

Cited by 17 publications

References 32 publications

Tracking the Fate of Genetically Distinct Vesicular Stomatitis Virus Matrix Proteins Highlights the Role for Late Domains in Assembly

Tracking the Fate of Genetically Distinct Vesicular Stomatitis Virus Matrix Proteins Highlights the Role for Late Domains in Assembly

Superresolution imaging of viral protein trafficking

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

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