Physical Chemistry of Viruses

Virus particles can be as simple as a molecule of RNA or DNA inside a spherical shell – the “capsid” – made up of multiple copies of a single protein. Further, because viruses only “become alive” when they are inside their hosts, it is possible to study them -- outside their hosts, in a test tube -- as physical objects, i.e., to do the same controlled experiments (and theory) on them that one routinely does with more familiar polymer and colloidal systems.

This approach is best illustrated with the example of one of the simplest viruses we work on – cowpea chlorotic mottle virus (CCMV) – a plant virus with a single-stranded RNA genome. Each particle of CCMV is a one-molecule-thick spherical shell consisting of (exactly!) 180 copies of a single protein, surrounding and protecting single copies of RNA genes of the virus. The 180 capsid proteins are organized into groups of 12 pentamers and 20 hexamers, involving 3 sets of inequivalent positions.

Most remarkably, this icosahedrally-symmetric (soccer-ball-like/”Bucky-ball”-like) structure can form spontaneously: one simply has to mix the purified RNA molecules and purified capsid proteins under the right conditions of pH and ionic strength and a virus forms that is infectious and indistinguishable from those found in infected plants. By changing these solution conditions, and replacing the viral RNA by non-viral RNA (and even by synthetic anionic polymers) and replacing the wildtype capsid protein by genetically- and/or chemically- modified forms of it, we are able to learn a lot about the fundamental physical chemistry of this biological process – the formation of an infectious virus, and also to prepare well-characterized and effective gene delivery systems.

3D Cryo-Electron Microscopy (CryoEM) Reconstructions of RNA Inside Viral Capsids

We use high-resolution cryo-electron microscopy – in particular, asymmetric reconstruction methods, in which no symmetry is assumed – to investigate the structure of single-stranded RNA genes inside a multipartite virus, Brome Mosaic Virus (BMV). Multipartite here refers to the fact that the RNA genome consists of two or more (three in this case) molecules, each of which is packaged in a different, otherwise-identical, capsid. We study separately each of the three kinds of virions involved and find that the RNA is highly disordered, suggesting a different method of assembly for multipartite viruses compared to the monopartite viruses MS2 and Qβ, which have been shown to have highly ordered genomes within their capsids. This can be understood in the context of their differing viral life-cycles, as BMV must package separately each of several different RNA molecules and has been shown to replicate and package them in replication-factories, while MS2 and Qβ depend on “packaging signals” throughout the viral RNA to selectively package their monopartite genomes.

In collaboration with Prof. Hong Zhou's group (MIMG, UCLA) and Prof. A. L. N. Rao's group (Plant Pathology, UC Riverside)

Distribution of RNA Ends in Multiplet-Capsid Structures

It is a remarkable and convenient fact that purified capsid proteins (CPs) of some viruses like Cowpea Chlorotic Mottle Virus (CCMV) and Brome Mosaic Virus (BMV) can spontaneously self-assemble into virus-like particles (VLPs) upon being mixed with purified single-stranded RNA (ssRNA) of different sequences and lengths. Earlier work in our group showed, however, that if the length of the RNA exceeds that of the longest viral gene (~3200 nt) by more than a thousand nucleotides (nts), single molecules of RNA are shared between two or more T=3 or T=4 capsids (multiplets) [1]. Also, theoretical work suggests that in (thousands-of-nts-) long ssRNAs the separation between the 5' and 3' ends of the molecules is small (2-3nm) and constant, independent of their particular sequence and length [2]. We are performing experiments to investigate both the nature of multiplet packaging (e.g., are the ends of over-long RNA in the same or in different capsids?), and the separation between the ends of un-packaged ssRNAs, by conjugating their ends with small (<3nm) gold nanoparticles, thereby allowing them to be observed directly by cryo-electron microscopy imaging.

[1] Garmann, R. F., Comas-Garcia, M., Knobler, C. M., & Gelbart, W. M. (2015). Physical principles in the self-assembly of a simple spherical virus. Accounts of chemical research, 49, 48-55.

[2] Yoffe, A. M., Prinsen, P., Gelbart, W. M., & Ben-Shaul, A. (2010). The ends of a large RNA molecule are necessarily close. Nucleic acids research, 39, 292-299.

In Vitro Synthesis of Enveloped Virus-Like Particles (EVLPs)

Most mammalian viruses are enveloped by a lipid membrane, which enhances their stability and facilitates their entry into host cells. On the other hand, the plant viruses like CCMV and BMV that we have been studying and utilizing for gene delivery are unenveloped – their RNA genomes are enclosed and protected by a protein shell only. In order to enhance the biocompatibility and targeting of a new delivery system of VLPs, we are wrapping these particles with a lipid bilayer membrane to create enveloped virus like particles (EVLPs). In addition to driving the wrapping by introducing oppositely-charged lipid, we use NHS-ester and maleimide lipids to “seed” the nucleation of bilayer envelope through interaction with lysines and cysteines on the VLP exteriors.

RNA Homopolymer Packaging by CCMV - the Role of RNA Secondary Structure on Self-Assembly

We investigated the effect of RNA secondary structure on CCMV spontaneous self-assembly by looking at how CCMV capsid protein (CP) self-assembles around polyadenylic acid (polyA), a homopolymer that has no self-complementarity but exhibits strong base-stacking and helical structure. Consistent with earlier work in our group with polyU [1], we find that – instead of T=3/180-subunit capsids (26 nm) arising from viral RNA or from heterologous RNA with “normal composition” (i.e., comparable numbers of A, U, G, and C bases) – the virus-like particles that form have T=2/120-subunit size (22 nm) [2]. These results point up the extent to which the spontaneous/preferred curvature of capsid protein is not an intrinsic property of the protein but rather one that depends on the kind of RNA with which it interacts (e.g., with or without normal composition and hence secondary structure formation). Further, unlike polyU, the stiffness of poly A resulting from its base-stacking and helical ordering makes it much less efficiently packaged by capsid protein.

[1] Beren, C., Dreesens, L. L., Liu, K. N., Knobler, C. M., & Gelbart, W. M. (2017). The effect of RNA secondary structure on the self-assembly of viral capsids. Biophysical journal, 113, 339-347.

[2] Thurm, A. R., Beren, C., Duran-Meza, A. L., Knobler, C. M., Gelbart, W. M. (2019) RNA homopolymers form higher-curvature virus-like particles than do normal-composition RNAs. Biophysical Journal, 117 (7), 1331-41.

Catching the Ends of Fluctuating RNA Outside Intact Capsids

We hypothesize that a positive-sense RNA virus does not necessarily have to disassemble (“uncoat”) in its host cell cytoplasm in order for translation of its genome to begin. Instead, we consider the possibility that translation is initiated while the protective capsid is still intact, so that the RNA genome is handed over directly from the protection of its capsid to protection of the ribosomal machinery. This is in fact the scenario suggested by the “co-translational delivery” mechanism suggested many years ago by T. A. M. Wilson, in the context of plant viruses like TCV, and pursued later by A. Helenius in the context of mammalian viruses like Sindbis. More explicitly, we are trying to demonstrate that the 5’ end of the genome fluctuates outside the capsid – through one of the many 1.5-to-2nm-sized holes between capsomers – and is bound by translation initiation factors like eIF4E which then engage it with the ribosomal subunits. To this end we are carrying out several experiments in parallel. One involves incubation of BMV virions with eIF4E proteins that are either fluorescently labeled or bound to beads coated with anti-eiF4E antibodies. Another involves biotinylating the 5’ end of fluorescently-labeld viral RNA, in vitro packaging it into virions, and them incubating them over a streptavidin coated surface and measuring the linear-with-time increase in bound virions (see figure). In another, related, experiment, we prepare “cherry bomb” constructs [1] that consist of BMV VLPs with one end of their RNA stuck outside of the capsid because of it being hybridized to a long enough complementary DNA oligo before in vitro packaging; we then catch this “stem” with a magnetic bead and measure the force-versus-distance profile as we pull on it in a magnetic tweezer apparatus.

[1] Garmann, R. F., Sportsman, R., Beren, C., Manoharan, V. N., Knobler, C. M., & Gelbart, W. M. (2015). A simple RNA-DNA scaffold templates the assembly of monofunctional virus-like particles. Journal of the American Chemical Society, 137, 7584-7587.

In collaboration with Prof. Jim Bowie's group (Chemistry & Biochemistry, UCLA) and Prof. David Bensimon's group (Chemistry & Biochemistry, UCLA and Physics, ENS Paris)

Characterization of Assembly Mechanism through Cryo-Electron Tomography (CryoET) of Individual RNA-Capsid Protein Assembly Intermediates

For many unenveloped viruses whose genomes are single-stranded RNA, infectious particles – nucleic acid inside a protein shell (capsid) – form spontaneously from their purified components. There are two prevailing scenarios for this assembly process: (i) nucleation and growth; and (ii) adsorption and rearrangement. According to the nucleation and growth mechanism, viral capsid proteins (CPs) first form an ordered critical nucleus on the genome – a partial capsid – and then grow by sequential addition of CPs from solution until the spherical/icosahedrally-symmetric capsid shell is completed. In the adsorption and rearrangement mechanism, on the other hand, CPs first adsorb onto the genome in a saturating and disordered fashion and then collectively rearrange to form the ordered capsid without further addition of CPs from solution. Molecular dynamics simulations predict that the assembly mechanism can be tuned by solution conditions that alter the relative strengths of CP-CP and CP-genome interactions [1]. We aim to test this idea experimentally for the case of a particularly well-characterized virus, Brome Mosaic Virus (BMV). Specifically, we want to demonstrate a cross-over in assembly mechanism by changing solution conditions – namely, ionic strength and pH – and directly imaging protein-RNA intermediates using Cryo-Electron Tomography (CryoET). We will collect tomography data on our samples in order to time-resolve the structures of single protein-RNA complexes, thereby generating 3D reconstructions of individual assembly intermediates. In the nucleation and growth pathway, we should see partial capsids appearing in CP-RNA complexes after a lag time, quickly evolving into complete capsids. By contrast, when the adsorption and rearrangement mechanism becomes operative we should see saturated but disordered adsorption of CPs on RNA followed by ordering into the icosahedral capsid. This direct visualization study would demonstrate how relative strengths of CP-CP and CP-RNA interactions can be tuned by solution conditions to determine the mechanism of assembling viruses.

[1] Perlmutter, J. D., Perkett, M. R., & Hagan, M. F. (2014). Pathways for virus assembly around nucleic acids. Journal of Molecular Biology, 426 (18), 3148-65.

In collaboration with Prof. Jose Rodriguez (Chemistry & Biochemistry, UCLA) and Prof. Michael Hagan (Physics, Brandeis)

Atomistic Molecular Dynamics Studies of Protein-RNA Interactions

Our goal is to determine the role of the positively charged N-terminal tail for Cowpea Chlorotic Mottle Virus (CCMV) in the binding of capsid protein to RNA. We use atomistic molecular dynamics simulations and various free energy calculation methods to determine binding energies for the N-terminal tail to different RNAs and RNA motifs. In particular, we want to explain the drastically different protein-RNA interactions and packaging scenarios observed experimentally for "normal-composition" RNAs with extensive duplex formation and single-stranded loops, versus for single-letter RNAs like polyU, for which no intramolecular base-pairing is possible [1, 2]. Ultimately, we would like to develop a theory that can predict how RNA sequence and consequent secondary structure affects the encapsidation process.

[1] Beren, C., Dreesens, L. L., Liu, K. N., Knobler, C. M., & Gelbart, W. M. (2017). The effect of RNA secondary structure on the self-assembly of viral capsids. Biophysical journal, 113, 339-347.

[2] Thurm, A. R., Beren, C., Duran-Meza, A. L., Knobler, C. M., Gelbart, W. M. (2019) RNA homopolymers form higher-curvature virus-like particles than do normal-composition RNAs. Biophysical Journal, 117 (7), 1331-41.

In collaboration with Prof. Robijn Bruinsma's group (Physics, UCLA)