Functional implications of quasi-equivalence in a T=3 icosahedral animal virus established by cryo-electron microscopy and X-ray crystallography

Abstract

Background Studies of simple RNA animal viruses show that cell attachment, particle destabilization and cell entry are complex processes requiring a level of capsid sophistication that is difficult to achieve with a shell containing only a single gene product. Nodaviruses [such as Flock House virus (FHV)] are an exception. We have previously determined the structure of FHV at 3 å resolution, and now combine this information with data from cryo-electron microscopy in an attempt to clarify the process by which nodaviruses infect animal cells.

Results A difference map was computed in which electron density at 22 å resolution, derived from the 3.0 å resolution X-ray model of the FHV capsid protein, was subtracted from the electron density derived from the cryo-electron microscopy reconstruction of FHV at 22 å resolution. Comparisons of this density with the X-ray model showed that quasi-equivalent regions of identical polypeptide sequences have markedly different interactions with the bulk RNA density. Previously reported biphasic kinetics of particle maturation and the requirement of subunit cleavage for particle infectivity are consistent with these results.

Conclusions On the basis of this study we propose a model for nodavirus infection that is conceptually similar to that proposed for poliovirus but differs from it in detail. The constraints of a single protein type in the capsid lead to a noteworthy use of quasi-symmetry not only to control the binding of a ‘pocket factor’ but also to modulate maturation cleavage and to release a pentameric helical bundle (with genomic RNA attached) that may further interact with the cell membrane.

Introduction

Genetic economy in viruses is evident from the prevalence of icosahedral particles used to transport many different viral genomes. A shell formed by 60 identical gene products arranged with the symmetry of the icosahedron provides the largest container in which all subunits are in identical environments. The principles by which icosahedral virus shells are constructed with more than 60 subunits was first explained by the concept of quasi-equivalence [1], [2]. Quasi-equivalent shells are denoted by the triangulation number, T, which, for most simple viruses, specifies the number of chemically identical subunits (gene products) in each of the 60 icosahedral asymmetric units. The total number of subunits in the capsid is 60 T. Each subunit within the icosahedral asymmetric unit must occupy a slightly different environment and in those T=3 shells, where structures are known to atomic or near atomic resolution, the quasi-equivalent states of the subunits are determined by a switching mechanism that is directed by the protein subunit itself or the packaged RNA or both [2], [3].

Capsids with T=3 symmetry are common among RNA plant viruses, but there are few examples of quasi-equivalence among the simple RNA viruses that infect animals. The dominant capsid form in the latter category of viruses is the P=3 (pseudo T=3) picornavirus shell. The T=3 and P=3 shells are clearly related (Figure 1), but the latter contains three different gene products while the former contains one. The trapezoids labeled A, B and C in Figure 1 correspond to the same gene product located in slightly different environments within one icosahedral asymmetric unit (the central triangle). The tertiary folds of A, B and C are virtually identical, but the quasi- equivalent C–C ₂ contact and A–B ₅ contact are flat and bent respectively (side views shown in Figure 1b), although the same protein surfaces are juxtaposed. A protein polypeptide (‘arm’ in Figure 1b) and a portion of duplex RNA (dsRNA) are ordered only at the C–C ₂ contact and serve as a wedge to prevent bending. These structures are disordered at the A–B ₅ interface and the contact is bent about an axial hinge that is conserved in both flat and bent contacts. Viral protein 1 (VP1), VP2 and VP3 are different gene products in the picornavirus capsid and therefore the pseudo-equivalent VP2– VP2 interface and VP1–VP3 interface have different protein surfaces juxtaposed and there is no need for a molecular switch as observed in the T=3 shell. In both capsid types, the tertiary structure of the subunit is almost always an eight- stranded, antiparallel β -barrel with a jelly roll topology (Figure 1d) [3].

Fundamental differences which accompany the infection of plant and animal cells may skew the distribution of capsid types for viruses infecting the different hosts. First, higher animals are protected by a circulating immune system which may exert selective pressure for a P=3 capsid. The P=3 capsid favors the formation of escape mutations because, with three gene products in the icosahedral asymmetric unit instead of one, the combinatorial possibility for altering epitopes is much greater. Furthermore, a single mutation in the T=3 gene generates three changes in the icosahedral asymmetric unit, thereby increasing the probability that a function of the capsid will be affected by the mutation [4]. Second, viruses infecting an animal host enter the cell through a receptor- mediated path. Considerable progress has been made recently in understanding the process by which rhinovirus [5] and poliovirus [6] bind to receptors and enter cells. In these viruses it is clear that there are distinct roles for VP1 compared with VP2 and VP3 (Figure 1), suggesting that the multiple gene products in the capsid are important to achieve the level of sophistication needed for cell entry.

In this report we identify some structural features in the T=3 nodaviruses [as exemplified by the Flock House virus (FHV), an insect nodavirus] that are different from any other T=3 structures reported and which probably relate to early events in cell entry. We suggest that FHV has a mechanism of entry related to that observed in the polio picornavirus system, and that quasi- equivalent interactions facilitate this mechanism with just a single gene product.

The nodaviruses are an excellent model system for investigating animal virus assembly, maturation, and factors that affect viral stability and infectivity. They are single-stranded RNA viruses with 180 identical gene products (M _r = 44 kDa) forming their capsids. Nodaviruses infect insects [7], mammals [8] and fish [9], and they are among the simplest biological replicating systems. The nodavirus genome contains only three genes located on two RNA molecules which are both packaged in the same particle. The capsid proteins undergo a post assembly cleavage:

This cleavage is required for infectivity [10], [11].

We have determined the structures of black beetle virus (BBV) at 2.8 å [12] and FHV at 3.2 å [13] resolution by X-ray crystallography. The structure of FHV has been subsequently refined to 3.0 å resolution (V Reddy, A Fisher and J Johnson, unpublished data). The FHV and BBV atomic structures showed that duplex RNA contributes to the formation of the T=3 capsid (Figure 1a), with 10 well-ordered base pairs packed against the protein shell near each icosahedral two-fold axis. This highly ordered RNA accounts for ∼20 % of the genome. These studies also showed that the subunit cleavage site (Asn363/Ala364) is on the interior of the protein shell and inaccessible to cellular enzymes. Residues 364–407 (the γ -peptide) are on the capsid interior with amino acids 364–379 in an amphipathic α -helical configuration. The 28 carboxy-terminal residues (380–407) are not visible in the X-ray electron density map.

The three subunits in the icosahedral asymmetric unit have nearly identical tertiary structures with their γ -helices bound to the interior surface of the β -barrel by hydrophobic interactions. However, in the assembled particle, the γ -helices are located in dramatically different environments because of the quasi- equivalent contacts in the shell. The γ -helices in the A subunits (γ _A ) form a pentameric helical bundle that is stabilized by interactions among the hydrophilic surfaces of the helices. In contrast, the γ _C -helices interact with the ordered duplex RNA and with the γ _B -helices in pairs about the icosahedral three-fold axes. The γ _B - and γ _C -helices are the innermost protein features in the X-ray electron density map and hydrophilic residues of these helices interact with each other, point towards the particle interior, or (in γ _C ) interact with the ordered RNA. Although the primary structure of the γ -peptides is the same in all subunits, the presence or absence of RNA dramatically changes the relative positions of the γ -peptides near the symmetry axes. The relative disposition of the γ -helices and ordered duplex RNA are displayed in Figure 2.

The differences in the surroundings of the γ _A, γ _B, and γ _C -peptides with respect to each other, the ordered RNA, and the bulk RNA [which is invisible in the X-ray map because it is not icosahedrally ordered to a high enough resolution (15– 3.0 å )] suggests that the roles of these peptides depend on the differences in the quasi-equivalent environments. The data from the X-ray analysis alone did not allow us to confirm that these helices had different interactions with the bulk RNA. Such interactions were significant at two levels. First, kinetic studies suggest that 120 subunits cleave at a faster rate than the remaining 60 subunits [10]. Based on the subunit tertiary structures, the environments at the cleavage points of the A, B and C proteins appeared to be identical. We wanted to see if the bulk RNA affected the cleavage site in a way that could explain the kinetic result. Second, a hypothesis that we developed for the role of the γ _A -helices in the early interaction of the virus with the cell surface predicts that these helices will have a different interaction with the bulk RNA than the γ _B - and γ _C -helices. Based on an analogy with the picornaviruses [14], [15], the γ _A pentameric helical bundle may be released upon interaction of the virion with the cellular receptor, or with the membrane, or both. This hypothesis predicts minimal interaction of the γ _A -helices with the RNA in order to facilitate release of the cleaved γ -peptides along the pentamer axis.

We investigated features of the protein–RNA interface in FHV by combining data from the X-ray and the cryo-electron microscopy (cryoEM) experiments in a manner similar to that used to study complexes between viruses and Fab fragments [16], [17], whole antibodies [18], and receptor molecules [5]. The fidelity of the procedure used to visualize the RNA density was tested with two control experiments that demonstrated the extraordinary level of detail available from the electron microscopy data when density for the protein and RNA in the virus can be assigned on the basis of the X-ray model.