Advanced Cantilever-Based Techniques for Virus Research

Humans have endured many severe viral outbreaks in the last 100 years. These viral outbreaks included the Spanish flu and other similar influenza epidemics, Ebola, MERS, SARS-CoV, HIV, and the recent coronavirus pandemic—SARS-CoV-2.

During this time, science has made considerable advancements in interpreting the many different aspects of viral outbreaks, such as viral infectivity, virulence, pathogenicity, transmission, and the tendency to cause a pandemic or epidemic.

Contemporary methods such as infectivity assays, cell culture, immunoassays, polymerase chain reactions, electron microscopy, and fluorescence microscopy have allowed people to gain a deeper insight into the viral replication, virus life cycle, and also the interaction between the virus and the host.

Today, people have a better understanding of the function and structure of the viral genome, viral particle, immune response, virus-host cell interaction, and DNA or RNA expression levels. Such advances have led to the development of drugs and vaccines for treating many different kinds of viruses.

One of the latest methods available for virus research is atomic force microscopy or AFM. The AFM is a cantilever-based method that uses a sharp tip to examine surfaces at resolutions well within the optical diffraction limit.

In addition to imaging, AFM is also a robust tool for nano-manipulation and nano-mechanical probing. AFM offers a major benefit—it can work on samples submerged in liquid. This aspect empowers experiments performed on living cells at physiologically applicable conditions. Scientists have used AFM imaging to analyze live virus particles and to study the morphology, dimension, and packaging of viral genomic material.

Besides imaging, the AFM technique has been used to manipulate single viruses through force spectroscopy to analyze the early events of interactions between the virus and the host. This article summarizes two comparatively new cantilever-based methods for studying the interactions between the virus and host at the single-cell level.

In the first case, a hollow cantilever was used to deposit the virus particles on a single cell, and the response of the host cell following virus infection was subsequently analyzed. In the second case, a virus was used to infect single cells attached to a cantilever and the impact of infection on the cell mass was tracked over several hours.

Cooperative Vaccinia Infection

The objective of the first study is to investigate the cooperativity of viral particles in cell infection. To put this in simple terms, the aim was to determine how the likelihood of virus infection relies on the quantity of virus particles attacking a cell.

To analyze the cooperativity of virus infection, precise control is required on the timing of infection as well as the number of virions to which cells are exposed.

The Institute of Microbiology of the ETH Zurich and the Laboratory of Biosensors and Bioelectronics, which are both pioneering laboratories, used a cantilever-based manipulation tool called FluidFM® to accurately send a defined number of viral particles onto single cells. This tool can be integrated into an AFM and utilizes hollow cantilevers to aspire or locally deposit the material (1). Figure 1 shows a schematic diagram of the FluidFM® technology.

Schematic of AFM with FluidFM®. A hollow cantilever featuring an opening at its free end is filled with liquid from a reservoir. By applying a positive or negative pressure liquid can be secreted out of or aspired into the cantilever to locally manipulate a sample. The AFM handles accurate force control and the complete system is integrated on an inverted, optical microscope to provide optical access to the sample.

Figure 1. Schematic of AFM with FluidFM®. A hollow cantilever featuring an opening at its free end is filled with liquid from a reservoir. By applying a positive or negative pressure liquid can be secreted out of or aspired into the cantilever to locally manipulate a sample. The AFM handles accurate force control and the complete system is integrated on an inverted, optical microscope to provide optical access to the sample. Image Credit: Nanosurf AG

In this analysis, an AFM equipped with FluidFM® functionality was incorporated with a fluorescence microscope to analyze the response of single cells following exposure to varied numbers of viral particles. The research team from ETH created a new protocol to utilize the FluidFM® technology to deposit 1 to 12 mature vaccinia virions on single cells (2). Figure 2 describes the process of virus deposition.

Four phases of virus deposition with a hollow cantilever to study single-cell infection. I. A cell is selected using the optical microscope. II. The hollow cantilever is moved over the cell and brought into gentle contact under force control. III. During deposition, the virions exiting the cantilever are monitored. IV. The number of virions on the cell membrane is counted.

Figure 2. Four phases of virus deposition with a hollow cantilever to study single-cell infection. I. A cell is selected using the optical microscope. II. The hollow cantilever is moved over the cell and brought into gentle contact under force control. III. During deposition, the virions exiting the cantilever are monitored. IV. The number of virions on the cell membrane is counted. Image Credit: Nanosurf AG

To prevent the leakage of virus particles into the solution, which otherwise can lead to uncontrolled infection, the FluidFM® hollow cantilever was slightly pushed onto the cell, creating a seal between the cell surface and the cantilever. Since sub-nanonewton force control is provided by the AFM technique, the cells are not impaired at the time of this process.

To track the deposition of virions and the development of infection, the team utilized a recombinant vaccinia virus (VACV) that integrated a fluorescent protein, called mCherry, into the virus core protein A5. Through this fusion protein, the assembled virion particles were traced by fluorescence. This also helped count the number of deposited particles and monitor the assembly of new virions in the late infection phase.

Moreover, the virus-encoded for eGFP under control of a late or early viral promoter. Once the virus penetrated the host cells, the eGFP was expressed and a reporter of the varied infection phases was created, as shown in Figure 3.

Monitoring the stages of the VACV lifecycle microscopically by eGFP and mCherry fluorescence signals. Top row: 7 hours post infection, bottom row: 11.5 hours. The intensity of the EGFP signals on the left clearly shows the entering of the early and late viral gene expression phases. The strong increase in the mCherry intensity at the right between 7 and 11.5 hours indicates the assembly of new virions.

Figure 3. Monitoring the stages of the VACV lifecycle microscopically by eGFP and mCherry fluorescence signals. Top row: 7 hours post-infection, bottom row: 11.5 hours. The intensity of the EGFP signals on the left clearly shows the entering of the early and late viral gene expression phases. The strong increase in the mCherry intensity at the right between 7 and 11.5 hours indicates the assembly of new virions. Image Credit: Nanosurf AG

The study results demonstrate the cooperativity of the virus infection. A cell had just around 10% probability of being infected when attacked by a single virus (n = 73). But this chance increased to around 35% when targeted by two particles (n = 23) and to 90% when attacked by four virus particles (n = 20). A potential reason for this cooperativity is that the antiviral mechanism in host cells is weakened when numerous viruses arrive at the same time.

Effect of Virus Infection on Cell Mass

Back in 2017, scientists from the Biophysics lab of the ETH Zurich demonstrated an inertial pico-balance to quantify the mass of adherent cells in a non-invasive way and at high mass and time resolution (3).

The pico-balance has millisecond time resolution and picogram mass sensitivity. The mass is obtained from the resonance frequency of a cantilever, and this cantilever is made to oscillate with sub-nanometer amplitudes with the help of a laser with varying intensity directed at the bottom of the cantilever.

As a result, the resonance frequency of the cantilever reduces with the binding of the cell, and any additional charges in the cell mass will change the resonance frequency of the cantilever accordingly. Figure 4 shows a schematic diagram of the principles of the pico-balance.

Schematic of pico-balance setup. The cantilever is made to oscillate at about 0.5 nm amplitude using an intensity-modulated laser at the base of the cantilever (Blue). Another laser focused on the free end of the cantilever (red) is used to monitor the resonance frequency of the cantilever. The mass of the cell attached to the cantilever can be derived from the resonance frequency.

Figure 4. Schematic of pico-balance setup. The cantilever is made to oscillate at about 0.5 nm amplitude using an intensity-modulated laser at the base of the cantilever (Blue). Another laser-focused on the free end of the cantilever (red) is used to monitor the resonance frequency of the cantilever. The mass of the cell attached to the cantilever can be derived from the resonance frequency. Image Credit: Nanosurf AG

Sub-optimal environmental conditions can easily affect the development of cell mass. To avoid this, a controlled environmental system was ultimately designed to sustain cell viability and track the cell mass for numerous days. The controlled environmental system is fitted with a unique sample holder that regulates CO2, temperature, and humidity, serving as a tiny cell culture incubator. Figure 5 shows a schematic diagram of this setup.

Schematic of the pico-balance setup.

Figure 5. Schematic of the pico-balance setup. Image Credit: Nanosurf AG

The researchers selected HeLa cells as a model system and adhered the cells to collagen I coated cantilevers. To analyze the effect of a virus infection on the cell mass, the researchers allowed a single cantilever-bound HeLa cell to make contact with a vaccinia virus (VACV) infected BSC 40 cells seeded on the base of the incubator cell.

Thus, the cell-bound to the cantilever could be selectively infected. The researchers infected the cells with a recombinant vaccinia virus comprising the eGFP-tagged core protein A4. This was done to track the production of novel virus particles by fluorescence microscopy. Over time, the fluorescence intensity of the infected cells increased, which indicated that novel viral particles were forming in the cells.

Both control cells and infected HeLa cells showed that the mass of the bound cells varied intermittently by a few percent at a timescale in the range of a few seconds. But the mass of the infected cells continued to remain constant over a longer period and did not enter mitosis.

Control cells that did not make contact with the infected cells slowly increased in mass and then divided on the cantilever, as shown in Figure 6.

Long-term cultivation and mass monitoring of control (black trace) and virus-infected HeLa cells (gray trace). Cells were infected by bringing the cell attached to the cantilever in contact with an infected cell in the Petri dish. The dips in the black trace show where mitosis occurred, during which cells rounded up and adhered only weakly to the cantilever.

Figure 6. Long-term cultivation and mass monitoring of control (black trace) and virus-infected HeLa cells (gray trace). Cells were infected by bringing the cell attached to the cantilever in contact with an infected cell in the Petri dish. The dips in the black trace show where mitosis occurred, during which cells rounded up and adhered only weakly to the cantilever. Image Credit: Nanosurf AG

Conclusion

AFM is a robust method that can be used to study and exploit biological samples under physiological conditions. The method can be integrated with sophisticated optical methods, to help correlate the optical characterization of sample cells along with the AFM experiments.

This article demonstrated two studies of the interactions between the host and the virus at the single-cell level. In the first case, the AFM method was utilized as a manipulation tool using FluidFM® technology. In the second case, the mass development of the cells infected by the virus was analyzed following infection.

In the past, virus research had resulted in the development of vaccines and treatments. Novel viruses, like the recent SARS-CoV-2, which has caused the COVID-19 pandemic, underscores the significance of sustained virus research to develop a better and faster response to upcoming outbreaks.

References and Further Reading

  1. Force-controlled manipulation of single cells: from AFM to FluidFM. O. GuillaumeGentil, E. Potthoff, D. Ossola, C.M. Franz, T. Zambelli and J.A. Vorholt. Trends in Biotech. 2014. 32(7), 381–388.
  2. Cooperative Vaccinia Infection Demonstrated at the Single-Cell Level Using FluidFM. P. Stiefel, F.I. Schmidt, P. Dörig, P. Behr, T. Zambelli, J.A. Vorholt, and J. Mercer. Nano Letters. 2012, 12(8), 4219–4227.
  3. Inertial picobalance reveals fast mass fluctuations in mammalian cells. D. Martínez-Martín, G. Fläschner, B. Gaub, S. Martin, R. Newton, C. Beerli, J. Mercer, C. Gerber & D.J. Müller. Nature. 2017, 550, 500–505.

This information has been sourced, reviewed and adapted from materials provided by Nanosurf AG.

For more information on this source, please visit Nanosurf AG.

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