Liquid Biomolecular Condensates and Viral Lifecycles: Review and Perspectives

Abstract

Viruses are highly dependent on the host they infect. Their dependence triggers processes of virus-host co-adaptation, enabling viruses to explore host resources whilst escaping immunity. Scientists have tackled viral-host interplay at differing levels of complexity-in individual hosts, organs, tissues and cells-and seminal studies advanced our understanding about viral lifecycles, intra- or inter-species transmission, and means to control infections. Recently, it emerged as important to address the physical properties of the materials in biological systems; membrane-bound organelles are only one of many ways to separate molecules from the cellular milieu. By achieving a type of compartmentalization lacking membranes known as biomolecular condensates, biological systems developed alternative mechanisms of controlling reactions. The identification that many biological condensates display liquid properties led to the proposal that liquid-liquid phase separation (LLPS) drives their formation. The concept of LLPS is a paradigm shift in cellular structure and organization. There is an unprecedented momentum to revisit long-standing questions in virology and to explore novel antiviral strategies. In the first part of this review, we focus on the state-of-the-art about biomolecular condensates. In the second part, we capture what is known about RNA virus-phase biology and discuss future perspectives of this emerging field in virology.

Keywords: HIV; LLPS; Measles; Rabies; SARS-CoV-2; Vesicular Stomatitis virus; biomolecular condensates; influenza A virus; liquid organelles; viral factories; viruses.

Figure 1

The principles of liquid–liquid phase separation (LLPS) in a binary system. Graphical representation of a phase diagram of a binary system (A) and how changes in energy drive the stability/instability of thermodynamic systems (B). The conditions leading to LLPS for a binary system composed of molecule A and solvent B (even if the solvent is in itself a mixture such as the cytosol) are depicted in the phase diagram in A. A phase diagram is a graphical representation of conditions (temperature, fraction of concentration of components, pressure, volume, pH, partition coefficient, inverse interaction strength) at which physical states of matter are thermodynamically stable. At very low concentration, A dissolves in B, mixing. As the concentration of molecule A increases, it reaches a point where the solubility of A in B saturates (at CAsat, concentration of saturation of A). Crossing this point, condensates start to form as follows: Molecules A and B can demix into two liquid phases when the binodal or coexistence line is reached. However, for this to happen, in the region between spinodal and binodal lines, a trigger or nucleation event must occur, and a metastable structure is obtained. As the concentration of molecule A increases, and the spinodal line is reached, the trigger is no longer necessary, as the mixture is unstable and spontaneously separates into two coexisting liquids or phases by a process called spinodal decomposition. A further increase in A, CAdense (dense phase concentration of A), passes the upper limit of the binodal line [48]. The mixture is energetically favorable, this time predominant in A. The tie line in the graph connects points of equal chemical potentials. In this line, the size of the droplets increases, but the concentration within the droplet is the same, because the partial molar free energy is equal. To change the molar fractions of CAsat and CAdense, the thermodynamic parameter must be changed. The lower the Csat (concentration of saturation), the stronger the driving forces of phase separation. At the critical point, the mixture is more favorable than the two existing phases [16,48,49]. Abbreviations: temp–temperature, PTM–post-translational modifications.

Figure 2

The consequences of LLPS in biological systems. CA, concentration of molecule A; CB, concentration of molecule B.

Figure 3

Phase diagrams with liquid, gels, and crystals. Biomolecular condensates can display a range of material properties from liquids with high dynamics to gels, hydrogels, and glasses that are less dynamic and to inert crystals or fibers as observed in neurodegenerative disorders. Phase transitions are possible by manipulating either the concentration, PTMs, valency, and strength of interactions (as happens in mutations) or by changing thermodynamic parameters such as ionic strength, temperature, pH, etc.

Figure 4

Viral infections reorganize cellular structure and organization. Seminal studies have shown several examples of viruses that re-shape membrane-bound organelles to facilitate their replication, which are depicted in the left side of the figure. Recent reports suggest that viruses may also induce the de novo formation of other types of cellular compartments, which lack delimiting membranes and display the properties of liquids, and that will be discussed in the next sections of this review. Interestingly, several viruses such as reovirus, influenza A virus (IAV), human immunodeficiency virus-1 (HIV), and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) show an intimate association between the two types of cellular organelles, and the membrane interface has been shown to actively modulate protein LLPS in several ways. It has been shown to lower Csat for protein LLPS by restricting the movement of drivers and clients and even by operating as platforms for multivalent interactions. Additionally, it can build special environments to enable function; it can amplify signal transduction and even facilitate the fusion and fission of condensates, increasing their dynamics and avoiding that they reach equilibrium.

Figure 5

Graphical depiction of the genomic organization shared among the viral order of Mononegavirales. N, nucleoprotein; P, phosphoprotein; M, matrix protein; G, glycoprotein; L, Large protein.

Figure 6

In IAV infection, viral ribonucleoproteins (vRNPs) concentrate in liquid compartments devoid of delimiting membranes—see correlative light and electron microscopy (CLEM) image—prior to be packaged into budding virions. CLEM also reveals membranes (inside) IAV liquid inclusions and electron dense material that resembles vRNPs in budding virions. Top right shows a cross-section of virions with eight vRNPs arranged as seven segments surrounding a central piece (reviewed in [190]).

Figure 7

Liquid organelles can play different roles in viral infections. Despite being early days, liquid organelles have already been hypothesized to play many different functions in viral infections.

Figure 8

Graphical summary of the steps of the lifecycle of selected viruses, highlighted in the yellow box, with the inclusion, in blue, of the steps taking place in liquid organelles. Abbreviations mean transcription (TXN), measles virus (MeV), rabies virus (RABV), vesicular stomatitis virus (VSV), severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), influenza A virus (IAV), and human immunodeficiency virus (HIV). The graphical representation is inspired by adverse output pathways models.