Packaging signals in single-stranded RNA viruses: nature’s alternative to a purely electrostatic assembly mechanism

The formation of a protective protein container is an essential step in the life-cycle of most viruses. In the case of single-stranded (ss)RNA viruses, this step occurs in parallel with genome packaging in a co-assembly process. Previously, it had been thought that this process can be explained entirely by electrostatics. Inspired by recent single-molecule fluorescence experiments that recapitulate the RNA packaging specificity seen in vivo for two model viruses, we present an alternative theory, which recognizes the important cooperative roles played by RNA–coat protein interactions, at sites we have termed packaging signals. The hypothesis is that multiple copies of packaging signals, repeated according to capsid symmetry, aid formation of the required capsid protein conformers at defined positions, resulting in significantly enhanced assembly efficiency. The precise mechanistic roles of packaging signal interactions may vary between viruses, as we have demonstrated for MS2 and STNV. We quantify the impact of packaging signals on capsid assembly efficiency using a dodecahedral model system, showing that heterogeneous affinity distributions of packaging signals for capsid protein out-compete those of homogeneous affinities. These insights pave the way to a new anti-viral therapy, reducing capsid assembly efficiency by targeting of the vital roles of the packaging signals, and opens up new avenues for the efficient construction of protein nanocontainers in bionanotechnology.     

Genome packaging within icosahedral capsids and large-scale segmentation in viral genomic sequences

The assembly and maturation of viruses with icosahedral capsids must be coordinated with icosahedral symmetry. The icosahedral symmetry imposes also the restrictions on the cooperative specific interactions between genomic RNA/DNA and coat proteins that should be reflected in quasi-regular segmentation of viral genomic sequences. Combining discrete direct and double Fourier transforms, we studied the quasi-regular large-scale segmentation in genomic sequences of different ssRNA, ssDNA, and dsDNA viruses. The particular representatives included satellite tobacco mosaic virus (STMV) and the strains of satellite tobacco necrosis virus (STNV), STNV-C, STNV-1, STNV-2, Escherichia phages MS2, ?X174, α3, and HK97, and Simian virus 40. In all their genomes, we found the significant quasi-regular segmentation of genomic sequences related to the virion assembly and the genome packaging within icosahedral capsid. We also found good correspondence between our results and available cryo-electron microscopy data on capsid structures and genome packaging in these viruses. Fourier analysis of genomic sequences provides the additional insight into mechanisms of hierarchical genome packaging and may be used for verification of the concepts of 3-fold or 5-fold intermediates in virion assembly. The results of sequence analysis should be taken into account at the choice of models and data interpretation. They also may be helpful for the development of antiviral drugs.     

Simple rules for efficient assembly predict the layout of a packaged viral RNA

Single-stranded RNA (ssRNA) viruses, which include major human pathogens, package their genomes as they assemble their capsids. We show here that the organization of the viral genomes within the capsids provides intriguing insights into the highly cooperative nature of the assembly process. A recent cryo-electron microscopy structure of bacteriophage MS2, determined with only 5-fold symmetry averaging, has revealed the asymmetric distribution of its encapsidated genome. Here we show that this RNA distribution is consistent with an assembly mechanism that follows two simple rules derived from experiment: (1) the binding of the MS2 maturation protein to the RNA constrains its conformation into a loop, and (2) the capsid must be built in an energetically favorable way. These results provide a new level of insight into the factors that drive efficient assembly of ssRNA viruses in vivo.     

Thermodynamic characterization of viral procapsid expansion into a functional capsid shell

The assembly of "complex" DNA viruses such as the herpesviruses and many tailed bacteriophages includes a DNA packaging step where the viral genome is inserted into a preformed procapsid shell. Packaging triggers a remarkable capsid expansion transition that results in thinning of the shell and an increase in capsid volume to accept the full-length genome. This transition is considered irreversible; however, here we demonstrate that the phage λ procapsid can be expanded with urea in vitro and that the transition is fully reversible. This provides an unprecedented opportunity to evaluate the thermodynamic features of this fascinating and essential step in virus assembly. We show that urea-triggered expansion is highly cooperative and strongly temperature dependent. Thermodynamic analysis indicates that the free energy of expansion is influenced by magnesium concentration (3-13?kcal/mol in the presence of 0.2-10?mM Mg(2+)) and that significant hydrophobic surface area is exposed in the expanded shell. Conversely, Mg(2+) drives the expanded shell back to the procapsid conformation in a highly cooperative transition that is also temperature dependent and strongly influenced by urea. We demonstrate that the gpD decoration protein adds to the urea-expanded capsid, presumably at hydrophobic patches exposed at the 3-fold axes of the expanded capsid lattice. The decorated capsid is biologically active and sponsors packaging of the viral genome in vitro. The roles of divalent metal and hydrophobic interactions in controlling packaging-triggered expansion of the procapsid shell are discussed in relation to a general mechanism for DNA-triggered procapsid expansion in the complex double-stranded DNA viruses.     

On the origin of order in the genome organization of ssRNA viruses

Single-stranded RNA (ssRNA) viruses form a major class that includes important human, animal, and plant pathogens. While the principles underlying the structures of their protein capsids are generally well understood, much less is known about the organization of their encapsulated genomic RNAs. Cryo-electron microscopy and x-ray crystallography have revealed striking evidence of order in the packaged genomes of a number of ssRNA viruses. The physical determinants of such order, however, are largely unknown. We study here the relative effect of different energetic contributions, as well as the role of confinement, on the genome packaging of a representative ssRNA virus, the bacteriophage MS2, via a series of biomolecular simulations in which different energy terms are systematically switched off. We show that the bimodal radial density profile of the packaged genome is a consequence of RNA self-repulsion in confinement, suggesting that it should be similar for all ssRNA viruses with a comparable ratio of capsid size/genome length. In contrast, the detailed structure of the outer shell of the RNA density depends crucially on steric contributions from the capsid inner surface topography, implying that the various different polyhedral RNA cages observed in experiment are largely due to differences in the inner surface topography of the capsid.     

Nucleic acid packaging in viruses

We review recent literature describing protein nucleic acid interactions and nucleic acid organization in viruses. The nature of the viral genome determines its overall organization and its interactions with the capsid protein. Genomes composed of single strand (ss) RNA and DNA are highly flexible and, in some cases, adapt to the symmetry of the particle-forming protein to show repeated, sequence independent, nucleoprotein interactions. Genomes composed of double-stranded (ds) DNA do not interact strongly with the container due to their intrinsic stiffness, but form well-organized layers in virions. Assembly of virions with ssDNA and ssRNA genomes usually occurs through a cooperative condensation of the protein and genome, while dsDNA viruses usually pump the genome into a preformed capsid with a strong, virally encoded, molecular motor complex. We present data that suggest the packing density of ss genomes and ds genomes are comparable, but the latter exhibit far higher pressures due to their stiffness.     

A promiscuous DNA packaging machine from bacteriophage T4

Complex viruses are assembled from simple protein subunits by sequential and irreversible assembly. During genome packaging in bacteriophages, a powerful molecular motor assembles at the special portal vertex of an empty prohead to initiate packaging. The capsid expands after about 10%-25% of the genome is packaged. When the head is full, the motor cuts the concatemeric DNA and dissociates from the head. Conformational changes, particularly in the portal, are thought to drive these sequential transitions. We found that the phage T4 packaging machine is highly promiscuous, translocating DNA into finished phage heads as well as into proheads. Optical tweezers experiments show that single motors can force exogenous DNA into phage heads at the same rate as into proheads. Single molecule fluorescence measurements demonstrate that phage heads undergo repeated initiations, packaging multiple DNA molecules into the same head. These results suggest that the phage DNA packaging machine has unusual conformational plasticity, powering DNA into an apparently passive capsid receptacle, including the highly stable virus shell, until it is full. These features probably led to the evolution of viral genomes that fit capsid volume, a strikingly common phenomenon in double-stranded DNA viruses, and will potentially allow design of a novel class of nanocapsid delivery vehicles.     

Sequential packaging of RNA genomic segments during the assembly of Bluetongue virus

Bluetongue virus (BTV), a member of the Orbivirus genus within the Reoviridae family, has a genome of 10 double-stranded RNA segments, with three distinct size classes. Although the packaging of the viral genome is evidently highly specific such that every virus particle contains a set of 10 RNA segments, the order and mechanism of packaging are not understood. In this study we have combined the use of a cell-free in vitro assembly system with a novel RNA–RNA interaction assay to investigate the mechanism of single-stranded (ss) RNAs packaging during nascent capsid assembly. Exclusion of single or multiple ssRNA segments in the packaging reaction or their addition in different order significantly altered the outcome and suggested a particular role for the smallest segment, S10. Our data suggests that genome packaging probably initiates with the smallest segment which triggers RNA–RNA interaction with other smaller segments forming a complex network. Subsequently, the medium to larger size ssRNAs are recruited until the complete genome is packaging into the capsid. The untranslated regions of the smallest RNA segment, S10, is critical for the instigation of this process. We suggest that the selective packaging observed in BTV may also apply to other members of the Reoviridae family.     

Capsid protein synthesis from replicating RNA directs specific packaging of the genome of a multipartite, positive-strand RNA virus

Flock house virus (FHV) is a bipartite, positive-strand RNA insect virus that encapsidates its two genomic RNAs in a single virion. It provides a convenient model system for studying the principles underlying the copackaging of multipartite viral RNA genomes. In this study, we used a baculovirus expression system to determine if the uncoupling of viral protein synthesis from RNA replication affected the packaging of FHV RNAs. We found that neither RNA1 (which encodes the viral replicase) nor RNA2 (which encodes the capsid protein) were packaged efficiently when capsid protein was supplied in trans from nonreplicating RNA. However, capsid protein synthesized in cis from replicating RNA2 packaged RNA2 efficiently in the presence and absence of RNA1. These results demonstrated that capsid protein translation from replicating RNA2 is required for specific packaging of the FHV genome. This type of coupling between genome replication and translation and RNA packaging has not been observed previously. We hypothesize that RNA2 replication and translation must be spatially coordinated in FHV-infected cells to facilitate retrieval of the viral RNAs for encapsidation by newly synthesized capsid protein. Spatial coordination of RNA and capsid protein synthesis may be key to specific genome packaging and assembly in other RNA viruses.     

The DEAD-box RNA helicase DDX6 is required for efficient encapsidation of a retroviral genome

Viruses have to encapsidate their own genomes during the assembly process. For most RNA viruses, there are sequences within the viral RNA and virion proteins needed for high efficiency of genome encapsidation. However, the roles of host proteins in this process are not understood. Here we find that the cellular DEAD-box RNA helicase DDX6 is required for efficient genome packaging of foamy virus, a spumaretrovirus. After infection, a significant amount of DDX6, normally concentrated in P bodies and stress granules, re-localizes to the pericentriolar site where viral RNAs and Gag capsid proteins are concentrated and capsids are assembled. Knockdown of DDX6 by siRNA leads to a decreased level of viral nucleic acids in extracellular particles, although viral protein expression, capsid assembly and release, and accumulation of viral RNA and Gag protein at the assembly site are little affected. DDX6 does not interact stably with Gag proteins nor is it incorporated into particles. However, we find that the ATPase/helicase motif of DDX6 is essential for viral replication. This suggests that the ATP hydrolysis and/or the RNA unwinding activities of DDX6 function in moderating the viral RNA conformation and/or viral RNA-Gag ribonucleoprotein complex in a transient manner to facilitate incorporation of the viral RNA into particles. These results reveal a unique role for a highly conserved cellular protein of RNA metabolism in specifically re-locating to the site of viral assembly for its function as a catalyst in retroviral RNA packaging.     

Mutually-induced Conformational Switching of RNA and Coat Protein Underpins Efficient Assembly of a Viral Capsid

Single-stranded RNA viruses package their genomes into capsids enclosing fixed volumes. We assayed the ability of bacteriophage MS2 coat protein to package large, defined fragments of its genomic, single-stranded RNA. We show that the efficiency of packaging into a T = 3 capsid in vitro is inversely proportional to RNA length, implying that there is a free-energy barrier to be overcome during assembly. All the RNAs examined have greater solution persistence lengths than the internal diameter of the capsid into which they become packaged, suggesting that protein-mediated RNA compaction must occur during assembly. Binding ethidium bromide to one of these RNA fragments, which would be expected to reduce its flexibility, severely inhibited packaging, consistent with this idea. Cryo-EM structures of the capsids assembled in these experiments with the sub-genomic RNAs show a layer of RNA density beneath the coat protein shell but lack density for the inner RNA shell seen in the wild-type virion. The inner layer is restored when full-length virion RNA is used in the assembly reaction, implying that it becomes ordered only when the capsid is filled, presumably because of the effects of steric and/or electrostatic repulsions. The cryo-EM results explain the length dependence of packaging. In addition, they show that for the sub-genomic fragments the strongest ordered RNA density occurs below the coat protein dimers forming the icosahedral 5-fold axes of the capsid. There is little such density beneath the proteins at the 2-fold axes, consistent with our model in which coat protein dimers binding to RNA stem-loops located at sites throughout the genome leads to switching of their preferred conformations, thus regulating the placement of the quasi-conformers needed to build the T = 3 capsid. The data are consistent with mutual chaperoning of both RNA and coat protein conformations, partially explaining the ability of such viruses to assemble so rapidly and accurately.     

Assembly of double-stranded RNA viruses: bacteriophage ø6 and yeast virus L-A

Double-stranded RNA viruses have a virion-associated RNA-dependent RNA polymerase activity which is involved in such critical steps of viral assembly as genome packaging and minus strand synthesis. In vitro studies of a bacterial dsRNA virus, ø6, and a yeast virus, L-A, have shed light on capsid formation as well as on the protein/RNA interactions and packaging of the viral genomes. In the ø6 system, an empty dodecahedral polymerase complex (procapsid) composed of four protein species is formed without the help of other viral proteins or RNA. This particle packages positive sense viral RNA genome segments in an ATP dependent reaction. The presence of all rNTPs allows the synthesis of complementary (-) strands within the particle. Self-assembly of an additional protein shell (composed of protein P8) around this particle takes place in the presence of Ca²⁺ ions. In vivo, these nucleocapsids obtain an envelope while still residing in the cell cytoplasm. L-A, in contrast, is not known to make a prohead structure. The Pol domain of L-A's Gag-Pol fusion protein is necessary for packaging of the (+) strand RNA and probably actually binds to the (+) strand packaging site (a stem-loop with a protruding A) insuring its packaging while the Gag domain primes polymerization of the coat protein. N-Acetylation of Gag by the host MAK3 N-acetyltransferase is necessary for proper assembly, and the ratio of Gag-Pol/Gag, determined by the efficiency of - 1 ribosomal frameshifting, is critical for propagation of the M₁ satellite dsRNA.     

Three-dimensional structure of baculovirus-expressed Norwalk virus capsids.

The three-dimensional structure of the baculovirus-expressed Norwalk virus capsid has been determined to a resolution of 2.2 nm using electron cryomicroscopy and computer image processing techniques. The empty capsid, 38.0 nm in diameter, exhibits T = 3 icosahedral symmetry and is composed of 90 dimers of the capsid protein. The striking features of the capsid structure are arch-like capsomeres, at the local and strict 2-fold axes, formed by dimers of the capsid protein and large hollows at the icosahedral 5- and 3-fold axes. Despite its distinctive architecture, the Norwalk virus capsid has several similarities with the structures of T = 3 single-stranded RNA (ssRNA) viruses. The structure of the protein subunit appears to be modular with three distinct domains: the distal globular domain (P2) that appears bilobed, a central stem domain (P1), and a lower shell domain (S). The distal domains of the 2-fold related subunits interact with each other to form the top of the arch. The lower domains of the adjacent subunits associate tightly to form a continuous shell between the radii of 11.0 and 15.0 nm. No significant mass density is observed below the radius of 11.0 mm. It is suspected that the hinge peptide in the adjoining region between the central domain and the shell domain may facilitate the subunits adapting to various quasi-equivalent environments. Architectural similarities between the Norwalk virus capsid and the other ssRNA viruses have suggested a possible domain organization along the primary sequence of the Norwalk virus capsid protein. It is suggested that the N-terminal 250 residues constitute the lower shell domain (S) with an eight-strand beta-barrel structure and that the C-terminal residues beyond 250 constitute the protruding (P1+P2) domains. A lack of an N-terminal basic region and the ability of the Norwalk virus capsid protein to form empty T = 3 shells suggest that the assembly pathway and the RNA packing mechanisms may be different from those proposed for tomato bushy stunt virus and southern bean mosaic virus but similar to that in tymoviruses and comoviruses.     

The icosahedral RNA virus as a grotto: organizing the genome into stalagmites and stalactites

There are two important problems in the assembly of small, icosahedral RNA viruses. First, how does the capsid protein select the viral RNA for packaging, when there are so many other candidate RNA molecules available? Second, what is the mechanism of assembly? With regard to the first question, there are a number of cases where a particular RNA sequence or structure—often one or more stem-loops—either promotes assembly or is required for assembly, but there are others where specific packaging signals are apparently not required. With regard to the assembly pathway, in those cases where stem-loops are involved, the first step is generally believed to be binding of the capsid proteins to these “fingers” of the RNA secondary structure. In the mature virus, the core of the RNA would then occupy the center of the viral particle, and the stem-loops would reach outward, towards the capsid, like stalagmites reaching up from the floor of a grotto towards the ceiling. Those viruses whose assembly does not depend on protein binding to stem-loops could have a different structure, with the core of the RNA lying just under the capsid, and the fingers reaching down into the interior of the virus, like stalactites. We review the literature on these alternative structures, focusing on RNA selectivity and the assembly mechanism, and we propose experiments aimed at determining, in a given virus, which of the two structures actually occurs.     

Interaction with capsid protein alters RNA structure and the pathway for in vitro assembly of cowpea chlorotic mottle virus

Viruses use sophisticated mechanisms to allow the specific packaging of their genome over that of host nucleic acids. We examined the in vitro assembly of the Cowpea chlorotic mottle virus (CCMV) and observed that assembly with viral RNA follows two different mechanisms. Initially, CCMV capsid protein (CP) dimers bind RNA with low cooperativity and form virus-like particles of 90 CP dimers and one copy of RNA. Longer incubation reveals a different assembly path. At a stoichiometry of about ten CP dimers per RNA, the CP slowly folds the RNA into a compact structure that can be bound with high cooperativity by additional CP dimers. This folding process is exclusively a function of CP quaternary structure and is independent of RNA sequence. CP-induced folding is distinct from RNA folding that depends on base-pairing to stabilize tertiary structure. We hypothesize that specific encapsidation of viral RNA is a three-step process: specific binding by a few copies of CP, RNA folding, and then cooperative binding of CP to the "labeled" nucleoprotein complex. This mechanism, observed in a plant virus, may be applicable to other viruses that do not halt synthesis of host nucleic acid, including HIV.     

Asymmetric Genome Organization in an RNA Virus Revealed via Graph-Theoretical Analysis of Tomographic Data

Cryo-electron microscopy permits 3-D structures of viral pathogens to be determined in remarkable detail. In particular, the protein containers encapsulating viral genomes have been determined to high resolution using symmetry averaging techniques that exploit the icosahedral architecture seen in many viruses. By contrast, structure determination of asymmetric components remains a challenge, and novel analysis methods are required to reveal such features and characterize their functional roles during infection. Motivated by the important, cooperative roles of viral genomes in the assembly of single-stranded RNA viruses, we have developed a new analysis method that reveals the asymmetric structural organization of viral genomes in proximity to the capsid in such viruses. The method uses geometric constraints on genome organization, formulated based on knowledge of icosahedrally-averaged reconstructions and the roles of the RNA-capsid protein contacts, to analyse cryo-electron tomographic data. We apply this method to the low-resolution tomographic data of a model virus and infer the unique asymmetric organization of its genome in contact with the protein shell of the capsid. This opens unprecedented opportunities to analyse viral genomes, revealing conserved structural features and mechanisms that can be targeted in antiviral drug design.     

The bacteriophage lambda gpNu3 scaffolding protein is an intrinsically disordered and biologically functional procapsid assembly catalyst

Procapsid assembly is a process whereby hundreds of copies of a major capsid protein assemble into an icosahedral protein shell into which the viral genome is packaged. The essential features of procapsid assembly are conserved in both eukaryotic and prokaryotic complex double-stranded DNA viruses. Typically, a portal protein nucleates the co-polymerization of an internal scaffolding protein and the major capsid protein into an icosahedral capsid shell. The scaffolding proteins are essential to procapsid assembly. Here, we describe the solution-based biophysical and functional characterization of the bacteriophage lambda (λ) scaffolding protein gpNu3. The purified protein possesses significant α-helical structure and appears to be partially disordered. Thermally induced denaturation studies indicate that secondary structures are lost in a cooperative, apparent two-state transition (T_m = 40.6 ± 0.3 °C) and that unfolding is, at least in part, reversible. Analysis of the purified protein by size-exclusion chromatography suggests that gpNu3 is highly asymmetric, which contributes to an abnormally large Stokes radius. The size-exclusion chromatography data further indicate that the protein self-associates in a concentration-dependent manner. This was confirmed by analytical ultracentrifugation studies, which reveal a monomer-dimer equilibrium (K_d,app ~ 50 μM) and an asymmetric protein structure at biologically relevant concentrations. Purified gpNu3 promotes the polymerization of gpE, the λ major capsid protein, into virus-like particles that possess a native-like procapsid morphology. The relevance of this work with respect to procapsid assembly in the complex double-stranded DNA viruses is discussed.     

Nonspecific nucleoside triphosphatase P4 of double-stranded RNA bacteriophage phi6 is required for single-stranded RNA packaging and transcription

Bacteriophage phi6 has a segmented double-stranded RNA genome. The genomic single-stranded RNA (ssRNA) precursors are packaged into a preformed protein capsid, the polymerase complex, composed of viral proteins P1, P2, P4, and P7. Packaging of the genomic precursors is an energy-dependent process requiring nucleoside triphosphates. Protein P4, a nonspecific nucleoside triphosphatase, has previously been suggested to be the prime candidate for the viral packaging engine, based on its location at the vertices of the viral capsid and its biochemical characteristics. In this study we were able to obtain stable polymerase complex particles that are completely devoid of P4. Such particles were not able to package ssRNA segments and did not display RNA polymerase (either minus- or plus-strand synthesis) activity. Surprisingly, a mutation in P4, S250Q, which reduced the level of P4 in the particles to about 10% of the wild-type level, did not affect RNA packaging activity or change the kinetics of packaging. Moreover, such particles displayed minus-strand synthesis activity. However, no plus-strand synthesis was observed, suggesting that P4 has a role in the plus-strand synthesis reaction also.