Virology Studies

Hodges J., Tang X., Landesman MB., Ruedas JB., Ghimire A., Gudheti MV., Perrault J., Jorgensen EM., Gerton JM., Saffarian S., 2013. Asymmetric Packaging of Polymerases Within Vesicular Stomatitis Virus. Biochemical and Biophysical Research Communications, Volume 440, Issue 2, 271-276. doi.org/10.1016/j.bbrc.2013.09.064.

Using the Vutara SR 200 microscope, Hodges, et al. investigated how different proteins localized throughout the vesicular stomatitis virus (VSV) – an RNA virus with one tapered and one blunt end. SMLM was used to locate the internal proteins within individual virions, proteins were labeled with eGFP, and the envelope of VSV virions was mapped using the same microscopy technique. This enabled the authors to determine the proteins' total fluorescence intensity and the position of their center of fluorescence with respect to the center of the envelope. In doing so, they found that:

• G protein was uniformly distributed on the surface of the virion.
• P and L proteins were packaged asymmetrically at the blunt end of the virion.

Alonas, E., Lifland, A.W., Gudheti, M., Vanover, D., Jung, J., Zurla, C., Kirschman, J., Fiore, V.F., Douglas, A., Barker, T.H., Yi, H., Wright, E.R., Crowe, J.E., Santangelo, P.J., 2014. Combining Single RNA Sensitive Probes with Subdiffraction-Limited and Live-Cell Imaging Enables the Characterization of Virus Dynamics in Cells. ACS Nano 8, 302–315. doi.org/10.1021/nn405998v

The authors developed MTRIPs (multiply labeled tetravalent RNA imaging probes) -- a method to live label hRSV viral genomes. They then used a Vutara SR 200 and Vutara SRX software to study the early infectivity and replication of enveloped viruses. They were able to identify and isolate individual, infectious, fluorescently labeled viral particles, localize them in three dimensions, and quantify virus replication in single cells over an entire cycle of replication. They found that MTRIP labeling of viral RNA:

• Enabled the simultaneous super-resolution imaging of proteins and the viral genome, which is not possible with conventional fluorescence in situ hybridization techniques (FISH).
• Allows determination of the distribution of viral proteins along the viral gRNA. Only single-molecule localization microscopy gives the resolution to image these sub-300 nm particles.
• Has the potential to become a general methodology for the labeling and study of many important RNA viruses.

Tiwari, P.M., Vanover, D., Lindsay, K.E., Bawage, S.S., Kirschman, J.L., Bhosle, S., Lifland, A.W., Zurla, C., Santangelo, P.J., 2018. Engineered mRNA-Expressed Antibodies Prevent Respiratory Syncytial Virus Infection. Nature Communications 9, 1–15. doi.org/10.1038/s41467-018-06508-3

The authors used the Vutara microscope to determine the mechanism of action of the therapeutic antibody, palivizumab, on RSV infections.

• Using the Vutara’s ability to image cultured cells in 3D, the authors were able to visualize viral particles on the cell membrane.
• When the cells expressed palivizumab on their cell surface the virions could be observed adjacent, but outside, of the cell membrane (virion size of ~100-300 nm).
• This suggested the mechanism of action of palivizumab is to prevent infection by stopping fusion and cytosolic uptake of RSV.

Milrot, E., Shimoni, E., Dadosh, T., Rechav, K., Unger, T., Etten, J.L.V., Minsky, A., 2017. Structural Studies Demonstrating a Bacteriophage-like Replication Cycle of the Eukaryote-infecting Paramecium Bursaria Chlorella Virus-1. PLOS Pathogens 13, e1006562. doi.org/10.1371/journal.ppat.1006562

The authors used the Vutara to determine the effects of viral infection on cytoskeletal structure. From this, they were able to determine that the actin cytoskeleton plays a critical role in viral infectivity.

• The authors used super-resolution imaging to monitor how the microtubule and actin cytoskeleton changed over the course of viral infection.
• During infection, the microtubule network became more fragmented and disappeared from the center of the cell.
• During infection, the actin cytoskeleton loses its fine structure at the periphery of the cell and forms a shell around the rounded edge of the cell.
• Pharmacological experiments and other experiments revealed that disruption of the microtubule network had little effect on virion production, while disruption of the actin cytoskeleton reduced virion production.

Akiyama H., Ramirez NGP., Gudheti MV., Gummuluru S., 2015. CD169-Mediated Trafficking of HIV to Plasma Membrane Invaginations in Dendritic Cells Attenuates Efficacy of Anti-gp120 Broadly Neutralizing Antibodies. PLOS Pathogens 11(3): e1004751. doi.org/10.1371/journal.ppat.1004751

Myeloid dendritic cells (DCs) are important to elicit a potent antiviral immunity and can capture HIV-1 via the receptor CD169/Siglec-1 that binds to the ganglioside, GM3, in the virus particle membrane. This research group utilized the Vutara 200 microscope, to investigate the role of CD169 in HIV-1 trafficking and trans-infection. Using super-resolution microscopy, they revealed:

• In a LPS induced cell, CD169 and HIV-1 accumulated in pocket-like compartments.
• LPS and IFN-α can both upregulate CD169 expression.
• HIV virions were closely associated with CD169.

Alonas, E., Lifland, A.W., Gudheti, M., Vanover, D., Jung, J., Zurla, C., Kirschman, J., Fiore, V.F., Douglas, A., Barker, T.H., Yi, H., Wright, E.R., Crowe, J.E., Santangelo, P.J., 2014. Combining Single RNA Sensitive Probes with Subdiffraction-Limited and Live-Cell Imaging Enables the Characterization of Virus Dynamics in Cells. ACS Nano 8, 302–315. doi.org/10.1021/nn405998v

The authors developed MTRIPs (multiply labeled tetravalent RNA imaging probes) -- a method to live label hRSV viral genomes. They then used a Vutara SR 200 and Vutara SRX software to study the early infectivity and replication of enveloped viruses. They were able to identify and isolate individual, infectious, fluorescently labeled viral particles, localize them in three dimensions, and quantify virus replication in single cells over an entire cycle of replication. They found that MTRIP labeling of viral RNA:

• Enabled the simultaneous super-resolution imaging of proteins and the viral genome, which is not possible with conventional fluorescence in situ hybridization techniques (FISH).
• Allows determination of the distribution of viral proteins along the viral gRNA. Only single-molecule localization microscopy gives the resolution to image these sub-300 nm particles.
• Has the potential to become a general methodology for the labeling and study of many important RNA viruses.

Yaakov, L.B., Mutsafi, Y., Porat, Z., Dadosh, T. and Minsky, A., 2019. Kinetics of Mimivirus Infection Stages Quantified Using Image Flow Cytometry. Cytometry, 95: 534-548. doi.org/10.1002/cyto.a.23770

In this study, the authors investigated the temporal progression of events in virally infected cells using the Vutara 200 microscope. Motivated by the ability to analyze large populations of infected cells despite the heterogeneity of viruses and their hosts, they utilized flow cytometry to study Mimivirus and Acanthamoeba polyphaga. Their work found:

• The microtubule network became more fragmented and disappeared from the center of the cell.
• The actin cytoskeleton lost its fine structure at the periphery of the cell and formed a shell around the cell edge.

Tiwari, P.M., Vanover, D., Lindsay, K.E. et al. Engineered mRNA-expressed antibodies prevent respiratory syncytial virus infection. Nat Commun 9, 3999 (2018). doi.org/10.1038/s41467-018-06508-3

This research group used the Vutara 352 microscope and Vutara SRX software to evaluate a novel mRNA-based approach to preventing respiratory syncytial virus (hRSV) infection -- and potentially other viral infections -- in the lung. Using the Vutara for both widefield and super-resolution imaging, the authors were able to (1) investigate the expressed antibodies' methods for preventing infection and (2) test the intervention for cellular consequences, like undesirable inflammation, that could present safety concerns for its translational application. They found:

• mRNA-expressed anchored neutralizing antibodies are retained on the plasma membrane of transfected cells.
• mRNA-based expression of neutralizing antibodies can prevent RSV infection in vivo and in vitro.
• Delivery of mRNA-expressed antibodies caused no significant inflammatory response.
• Data suggests that expressing membrane-anchored broadly neutralizing antibodies in the lungs could be a promising pulmonary prophylaxis approach.
• Theoretically, many expressed therapeutic proteins could be anchored and delivered in vitro in this manner.

SMLM microscopy allows labeling and imaging of viral components such as proteins (spike, membrane, envelope, nucleocapsid) and nucleotides (RNA or DNA) at a resolution that allows clear visualization and analysis. EM can achieve a resolution sufficient to resolve viruses, but differentiation of molecule components is not possible. Diffraction-limited techniques such as confocal microscopy allow fluorescent labeling of components but cannot resolve sub-diffraction components. SMLM can.

SMLM allows analysis of a variety of virus-host interactions. SMLM can allow visualization and analysis of specific target cell membrane proteins involved in viral infection. Specific target cell membrane proteins involved in infection can be identified, and functional significance as well as possible therapeutic strategies can be explored. Conversely, SMLM allows investigation of the role of specific viral proteins in the infection process. SMLM allows analysis of how viruses move into target cells, primarily through single particle tracking. SMLM also allows analysis of the effect of viral infection of target cells by imaging at the super-resolution level changes in target cell intracellular structure, such as cytoskeleton.

Challenges to imaging viruses with SMLM are primarily related to developing labeling protocols for imaging viral and target cell components.

SMLM allows imaging and analysis at the molecular level of viral components and target cell components involved in viral infection. As with most biological research, SMLM will not be the only technique used to study viruses and the impact of viral infection on target cells. Other techniques, such as sequencing to determine nucleotide sequences and viruses, as well as assays to evaluate target cells such as proteomics and assays of metabolism, will continue to be employed.

SMLM allows researchers to label and image molecular components of viruses and target cells at the super-resolution level. SMLM has been shown to be useful to determine viral structure, how viruses attach to target cells, how virus material enters cells, and the effect of viral infection on target cells.

SMLM provides a tool for better understanding of the mechanisms of viral infection as well as a tool ideally suited to explore therapy strategies at the molecular level.