Viruses: Packing up the genome

Nucleotide and force-dependent mechanisms control how the viral genome of lambda bacteriophage is inserted into capsids.
  1. Bálint Kiss
  2. Miklós Kellermayer  Is a corresponding author
  1. Department of Biophysics and Radiation Biology, Semmelweis University, Hungary

In order to survive, viruses must be able to copy themselves to make more viral particles. They do this by passing their genetic material to a host cell that can replicate their DNA and produce the components required to assemble more viruses. While all viruses have evolved remarkable mechanisms to ensure the successful completion of their life cycle, viruses containing double-stranded DNA display some of the most ingenious molecular tricks nature has to offer.

Imagine double-stranded DNA as a stiff, pencil-thick piece of yarn the length of a football pitch that needs to be quickly threaded into a protein capsule the size of a football, known as the capsid. During an infection, the packaged DNA strands must then be rapidly ejected from the viral capsid so they can be transmitted to another host cell. How a linear chain, that also displays random conformations, passes through the narrow opening of the capsid during viral genome packaging or subsequent ejection puzzled scientists for decades (Kasianowicz et al., 2002). But little more than twenty years ago, the fascinating mechanical features of viral DNA packaging were discovered (Smith et al., 2001).

Packaging is accomplished by a motor enzyme composed of five circularly arranged oligomers made up of large subunits (known as TerL) and small subunits (TerS) that work together in a coordinated manner (Chemla et al., 2005; Rao and Feiss, 2015). The motor converts energy (generated by breaking down the nucleotide ATP into ADP and inorganic phosphate) into movements that can rapidly thread the entire viral genome into the capsid without ever needing to dissociate and rebind to the DNA (Schliwa and Woehlke, 2003).

Previous work on a virus that infects bacteria called T4 bacteriophage showed that DNA can partially slip out of the capsid during packaging (Ordyan et al., 2018). To prevent this from occurring, the motor has a grip mechanism that controls this process, and an end clamp that stops the viral DNA from completely exiting the capsid once packaging has started. Yet, how the different subunits of the motor contribute to these processes, and how these processes differ in structurally distinct viruses, remained unknown. Now, in eLife, Douglas Smith (University of California San Diego), Carlos Catalano (University of Colorado) and colleagues – including Brandon Rawson as first author – report experiments investigating the mechanical properties of the motor complex in another virus, known as lambda bacteriophage (Rawson et al., 2023).

During packaging, the lambda motor complex frequently transitions between gripping the viral DNA in place and loosening its grip, resulting in the DNA slipping out of the capsid. To better understand the molecular interactions behind this, Rawson et al. employed optical tweezers – a tool that uses focused laser beams to create gentle forces – to pull on the free end of a partially packaged DNA molecule and to measure the direction and magnitude of DNA displacement. These experiments were conducted in a microfluidic chamber so that the virus-DNA assembly could be exposed to different biochemical environments.

To assess the role of ATP in this transition between slipping and gripping, the motor complex was studied in a nucleotide-free state, in the presence of either ADP, or a version of ATP that is not hydrolyzed into ADP. Rawson et al. found that in the nucleotide-free state, rapid DNA slippage occurred, but friction between the DNA and the motor and an end-clamp mechanism prevented complete escape of the DNA molecule. At times, slippage was randomly halted by gripping stages (Figure 1A). In the ADP condition, the frequency and duration of the DNA-gripped state increased, leading to significantly reduced DNA slippage. DNA slippage was essentially halted in non-hydrolyzable ATP conditions, with the DNA being gripped almost continuously (Figure 1B).

Mechanical control of genome packaging in the lambda bacteriophage.

(A) The genome of the lambda bacteriophage virus consists of double stranded DNA (red) which is threaded into a viral capsid (hexagon; black line) by a complex made up of one large (TerL; blue) and two small (TerS; green) motor subunits. For simplicity, only one out of the five molecules in the motor complex is represented schematically. In the absence of the nucleotide ATP, the lambda-phage genome is under a slight grip that prevents the DNA molecule from escaping the capsid despite exposure to experimental pulling forces. Occasionally, however, rapid slipping bursts may occur. (B) In the presence of ADP or non-hydrolyzable ATP analogs (ATP*), the grip tightens and slipping bursts become slower and less frequent. (C) Complete departure of the DNA from the capsid is prevented by an end-clamp mechanism.

Applying force to the free end of the viral DNA using optical tweezers tilted the balance between slipping and gripping states towards slippage. This suggests that the motor of the lambda bacteriophage is regulated by both nucleotides and mechanical forces.

Previous studies have shown the motor complex of the T4 bacteriophage – which lacks the TerS subunits – does not grip when in a nucleotide-free state, in contrast to the lambda motor (Ordyan et al., 2018). This difference between lambda and T4 bacteriophages is likely related to the function of the TerS subunits, which may act as a sliding clamp that controls the level of friction between the motor proteins and DNA. The lambda bacteriophage does, however, display the end-clamp feature observed in T4 phages (Figure 1C), suggesting this is a general mechanism that ensures viral DNA packaging is efficient by avoiding complete genome release and the need to re-initiate the packaging process.

The tour-de-force experiments conducted by Rawson et al. are fundamental for understanding the ever-intricate mechanisms underlying the viral life cycle. A myriad of questions await exploration, however. For instance, how does the motor complex recognize the end of DNA to enable end-clamping? Are the gripping and slipping processes controlled by the same set of protein-DNA interactions? And does DNA ejection rely on similar mechanisms? It also remains to be seen whether these viral tricks could be manipulated to externally control and inhibit viral infections or employed in biotechnology. As questions continue to arise, the elegant viral phenomena will carry on fascinating scientists.

References

Article and author information

Author details

  1. Bálint Kiss

    Bálint Kiss is in the Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary

    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1595-0426
  2. Miklós Kellermayer

    Miklós Kellermayer is in the Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary

    For correspondence
    kellermayer.miklos@med.semmelweis-univ.hu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5553-6553

Publication history

  1. Version of Record published: December 14, 2023 (version 1)

Copyright

© 2023, Kiss and Kellermayer

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 298
    Page views
  • 26
    Downloads
  • 0
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Bálint Kiss
  2. Miklós Kellermayer
(2023)
Viruses: Packing up the genome
eLife 12:e94128.
https://doi.org/10.7554/eLife.94128

Further reading

    1. Microbiology and Infectious Disease
    2. Physics of Living Systems
    Ray Chang, Ari Davydov ... Manu Prakash
    Research Article

    Microsporidia are eukaryotic, obligate intracellular parasites that infect a wide range of hosts, leading to health and economic burdens worldwide. Microsporidia use an unusual invasion organelle called the polar tube (PT), which is ejected from a dormant spore at ultra-fast speeds, to infect host cells. The mechanics of PT ejection are impressive. Anncaliia algerae microsporidia spores (3–4 μm in size) shoot out a 100-nm-wide PT at a speed of 300 μm/s, creating a shear rate of 3000 s-1. The infectious cargo, which contains two nuclei, is shot through this narrow tube for a distance of ∼60–140 μm (Jaroenlak et al, 2020) and into the host cell. Considering the large hydraulic resistance in an extremely thin tube and the low-Reynolds-number nature of the process, it is not known how microsporidia can achieve this ultrafast event. In this study, we use Serial Block-Face Scanning Electron Microscopy to capture 3-dimensional snapshots of A. algerae spores in different states of the PT ejection process. Grounded in these data, we propose a theoretical framework starting with a systematic exploration of possible topological connectivity amongst organelles, and assess the energy requirements of the resulting models. We perform PT firing experiments in media of varying viscosity, and use the results to rank our proposed hypotheses based on their predicted energy requirement. We also present a possible mechanism for cargo translocation, and quantitatively compare our predictions to experimental observations. Our study provides a comprehensive biophysical analysis of the energy dissipation of microsporidian infection process and demonstrates the extreme limits of cellular hydraulics.

    1. Physics of Living Systems
    Yangfan Zhang, George V Lauder
    Research Article

    Many animals moving through fluids exhibit highly coordinated group movement that is thought to reduce the cost of locomotion. However, direct energetic measurements demonstrating the energy-saving benefits of fluid-mediated collective movements remain elusive. By characterizing both aerobic and anaerobic metabolic energy contributions in schools of giant danio (Devario aequipinnatus), we discovered that fish schools have a concave upward shaped metabolism–speed curve, with a minimum metabolic cost at ~1 body length s-1. We demonstrate that fish schools reduce total energy expenditure (TEE) per tail beat by up to 56% compared to solitary fish. When reaching their maximum sustained swimming speed, fish swimming in schools had a 44% higher maximum aerobic performance and used 65% less non-aerobic energy compared to solitary individuals, which lowered the TEE and total cost of transport by up to 53%, near the lowest recorded for any aquatic organism. Fish in schools also recovered from exercise 43% faster than solitary fish. The non-aerobic energetic savings that occur when fish in schools actively swim at high speed can considerably improve both peak and repeated performance which is likely to be beneficial for evading predators. These energetic savings may underlie the prevalence of coordinated group locomotion in fishes.