Virus crystallisation

Virus crystallisation is the re-arrangement of viral components into solid crystal particles. The crystals are composed of thousands of inactive forms of a particular virus arranged in the shape of a prism. The inactive nature of virus crystals provide advantages for immunologists to effectively analyze the structure and function behind viruses. Understanding of such characteristics have been enhanced thanks to the enhancement and diversity in crystallisation technologies. Virus crystals have a deep history of being widely applied in epidemiology and virology, and still to this day remains a catalyst for studying viral patterns to mitigate potential disease outbreaks.

Historical background

Pre-20th century Virus crystals originate back to the late 19th century where the first protein crystallisation discoveries were made by German biologists Ritthausen and Osborne, mainly for hemoglobin in worms and fishes. These early observations were primarily regarded as laboratory curiosities. What began as mere curiosities evolved into the need for purification and isolation of proteins for clearer visualisation, thus leading to protein crystallisation. Protein crystallisation techniques were ultimately introduced in virology after the rise of the Tobacco Mosaic Viruses (TMV), which were the first ever viruses to be discovered.

1930s Achieving clear visualisation of viruses using limited technology, such as microscopy was difficult due to their relatively miniature size, with the smallest of viruses measuring in at roughly 20 nm in diameter. Microscopy was therefore a relatively challenging field, with alternative methods of observation in high demand. TMV viruses were first crystallised by Wendell Stanley, who demonstrated that TMV viruses retained its infectivity even in crystal form. It was during this time when researchers discovered that crystallised viruses (much like proteins and other organic molecules) could diffract X-rays, implying a complex structural mechanism in viral bodies. This breakthrough served as the basis for the expansion of virology into X-ray crystallography.

1950s, 1960s

X-ray crystallography was developed during the mid 20th century by scientists' efforts to study the characteristics of crystallised viruses in laboratory investigations. Amongst them was Dorothy Hodgkin, an expert in molecular microbiology, who determined TMV structure through virus crystals that could diffract X-ray. This discovery served as a basis to continuous refinement in methods of virus crystallography, which later led to the determination of numerous other virus structures, including the poliovirus, rhinovirus, and Human retrovirus (HIV). Such advancements provided valuable insights into the mechanisms of viral infection and replication, thus facilitating the development of antiviral drugs and vaccines heading into the late 20th century.

1990s–Today It was towards the end of the 20th century when scientists realized viruses surrounded with thick lipid membranes were unable to form ordered crystals. Such viruses made it difficult to properly obtain X-ray diffraction results. In response to this, cryogenic electron microscopy (cryo-EM) emerged as a new, alternative method for studying virus structures. Cryo-EM enables scientists to visualise viruses at near-atomic resolution without crystallisation. Combination of both X-ray crystallography and cryo-EM have contributed towards the field of virus morphology and behaviour in the immune system. Such advancements in technology have not only shed light on viral characteristics, but has revolutionized virology as a whole, and continue to be subject to heavy focus to this day.

Viral structure and behaviour Viruses are defined as "obligate intracellular parasites" that contain DNA or RNA in the viral genome core, and are encased by a protective protein coat. Generally, the core is encased in capsid proteins in a single or double-layered structure. Some viruses, such as some Coronaviruses, also develop a large lipid membrane known as the envelope when found in particular hosts. This membrane is composed of a lipid bilayer surrounding a layer of membrane-bound proteins, with either surface glycoproteins or spike proteins protruding from the extracellular aspect. Such viral envelope is usually acquired when travelling through the plasma or intracellular matrices of the host organism and may vary in composition depending on the host cell's membrane lipid content and host cell proteins. The structure of note for crystallisation and identification is the capsid protein structure. Viruses are majorly icosahedral in structure, with the second most common organisation being a helical, spring-like, structure. Viral capsid structures are organised in such a way as to maximise the efficiency of carrying its specific length of RNA or DNA chain. The kinetics of the capsid proteins may also play a role in its organisation, though this has not yet been fully elucidated. The symmetry and geometry of viruses is facilitated by the crystallisation of viruses (and more specifically their capsid protein subunits) in order to study protein-protein interactions; a proxy for the capsids' properties and functions.

Helical capsid structure

The helical capsid structure is majorly dependent on the length of the viral RNA or DNA genome. Due to the nature of packing identical asymmetric proteins with no rotational symmetry in order to minimise disturbance to protein-protein bonds at specific binding and receptor sites, capsid protein structures composed of a repetition of identical protein subunits necessarily arranges itself into a lattice that folds to encase its contents in a helical structure, much like the naturally occurring helical structure seen in DNA. This resultant helical structure is the case due to the geometric limitations and symmetrical nature necessitated by the protein sub-assembly array and its protein-protein interactions. The Tobacco Mosaic Virus studied by Caspar and Klug in their 1962 crystallisation study was discovered to be composed of a '2 to 5 capsid protein subunit aggregate', arranged in a helical capsid structure.

Icosahedral capsid structure

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