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capsid

Capsids and nucleocapsids

17 March 2022 by Vincent Racaniello

One aspect of virology that is confusing to students is the concepts of capsids and nucleocapsids. These two terms describe two different ways that viral nucleic acids and proteins are arranged in virus particles.

The capsid (from the Latin capsa for box) is the protein shell surrounding the nucleic acid genome. Below are two different types of capsid, constructed with helical (left) and icosahedral symmetry. In both cases the nucleic acid is covered with a protein shell:

A nucleocapsid is defined as the nucleic acid-protein assembly within the virus particle. This term is used when the assembly is a discrete substructure in the particle. The term ‘substructure’ seems to confuse students. What exactly constitutes a substructure?

Let’s define substructure with examples. If we take the two capsids shown above, and add a membrane, then the nucleic acid-protein assembly (formerly a capsid) becomes a nucleocapsid:

The nucleic acid-protein assembly within the particle is a nucleocapsid because it is a substructure.

If we remove the protein capsids from both viruses, we no longer have a substructure and there is no longer a nucleocapsid.

The SARS-CoV-2 virus particle has a nucleocapsid, as illustrated below. The RNA genome is present in the enveloped particle as an RNA-protein complex (the proteins are shown as green circles):

Adding a membrane is not the only way to make a nucleocapsid. If we add protein to a genome within a naked icosahedral capsid, we have a nucleocapsid:

Recently it was found that the DNA genome of icosahedrally ordered Mimivirus is elegantly wrapped in a 30 nanometer protein shield. This arrangement gives this non-enveloped virus a nucleocapsid.

The concept of a nucleocapsid substructure is not difficult to grasp in the context of multiple examples. And to make things even easier, the word nucleocapsid should remind you of the nucleus, which is a substructure in the cell.

Images created with BioRender

Filed Under: Basic virology Tagged With: capsid, genome, icosahedral, membrane, nucleocapsid, viral, virology, virus, viruses

Going off on an Arc Tangent

3 February 2022 by Gertrud U. Rey

by Gertrud U. Rey

Memories are formed through the recurrent activities of extensive networks of neural circuits, which consist of neuronal cells that transmit signals to each other at sites called synapses. Synaptic strength can change in intensity over time, a phenomenon known as “synaptic plasticity,” which may involve changes in synapse size, development of new synapses, or removal of existing ones. Synaptic plasticity is thought to be crucial for learning and memory.

At this point you may wonder what this topic has to do with virology! It turns out that one of the key regulators of synaptic plasticity is a gene coding for Activity-regulated cytoskeleton-associated protein (Arc). Arc is a member of a family of mammalian genes thought to have originated from an ancient retrotransposon family that also includes the Gag proteins of HIV and other retroviruses. Retrotransposons are mobile genetic elements that can relocate from one genomic site to another by converting their DNA into RNA and back into a new DNA copy that is then re-inserted at a different place in the host genome. Gag protein, which is essential and sufficient for the formation of viral capsids, consists of three domains: 1) the matrix protein, which mediates virus assembly and steers newly formed virions to the cell surface; 2) the nucleocapsid protein, which binds viral RNA for incorporation into the capsid; and 3) the capsid protein, which forms the inner shell of the virion enclosing the RNA. The amino acid sequences and structures of both the mammalian and Drosophila (i.e., fruit fly) Arc proteins are very similar to those of the capsid domain of Gag protein.

Following his lifelong interest in Arc, Jason Shepherd, a neurobiologist at the University of Utah, accidentally stumbled into the world of virology when he carried out a series of experiments to determine whether Arc’s sequence similarity to Gag is reflected in its function. Shepherd and colleagues purified Arc protein produced in bacterial cells and visualized it by electron microscopy. The resulting images revealed double-shelled soccer ball-like structures with a 32 nanometer diameter that resembled HIV particles, which upon further analysis were not artifacts or contaminants, but were in fact, virus-like capsids.

Additional experiments by the Shepherd group showed that not only does Arc protein spontaneously assemble into capsids inside neuronal cells, but the capsids also bind and encapsulate Arc mRNA, are released in extracellular vesicles that form when the cell membrane extends into an outward bubble and pinches off from the cell, and traffic that RNA to the next neuronal cell in a putative mechanism of intercellular communication. The authors observed a higher number of completely formed capsids when there was also RNA present, regardless of whether the RNA encoded Arc protein. This suggested that the presence of RNA enhances the likelihood of capsid assembly, in agreement with what is observed with HIV Gag. They also showed that the ability of target neuronal cells to take in Arc capsids is likely mediated by endocytosis, a mechanism in which cells absorb and engulf external material. Following entry of capsids into neurons, there was an increase in Arc protein levels in the branched extensions of neurons (i.e., the “dendrites”), which did not occur in the presence of a drug that inhibits protein synthesis, thus suggesting that the Arc mRNA contained in the capsids was actually translated inside these dendrites. Work done by a different group showed that each of these functions was also manifested by the corresponding Drosophila gene, even though this gene is thought to have evolved independently of the mammalian Arc gene.

Shepherd is primarily interested in studying the persistence of memory and a process called “memory consolidation,” in which short-term recollections are converted into stable, long-term images. In his research, he discovered that mice that don’t express Arc exhibit some short-term learning potential, but seem to have impaired long-term memory, presumably because they can’t store and consolidate memory. Shepherd has also noted that rats exhibit increased expression of Arc following a learning activity. Based on these observations, Shepherd hypothesizes that because consolidation and storage of memory depend on renewed protein synthesis, Arc may mediate a molecular mechanism that converts short-term memory into long-term memory, possibly by shuttling specific mRNAs between neuronal cells.

In an opinion article published last year, Shepherd and colleagues speculated on various aspects of the Arc “life cycle” based on known features of Gag protein functions. They hypothesized that learning activities promote transcription of the Arc gene, followed by transport of the resulting mRNA molecules to active dendrites, where they are translated into Arc proteins that assemble into capsids and capture/encapsidate surrounding mRNA. The formation of capsids and their interactions with the cell membrane to enable exit from the cell may be regulated by modifications like phosphorylation and/or palmitoylation. Phosphorylation involves the attachment of phosphoryl groups to certain amino acids in a protein, and this modification may regulate capsid assembly. There is evidence suggesting that mice carrying a mutation that blocks phosphorylation of Arc capsids have impaired synaptic plasticity and memory. Palmitoylation involves the attachment of fatty acids – which may induce Arc to interact with the plasma membrane, allowing capsids to exit the cell inside extracellular vesicles. An extracellular vesicle carrying a capsid then likely travels to a nearby neuron, where it attaches to specific receptors and enters the cell via endocytosis. The capsids are subsequently dismantled inside the cytoplasm and the released mRNA is translated into protein. Shepherd and colleagues are currently in the process of determining whether these hypotheses are correct and what happens after translation of the delivered mRNA.

Because viruses can serve as vectors for delivering vaccines, therapeutic drugs, and healthy copies of defective genes to specific target cells, Arc may just come in handy in these applications. Shepherd argues that Arc is likely not immunogenic because it is also expressed in peripheral dendritic cells, which are an important element of the immune system. If that is true, Arc capsids could potentially deliver their cargo to their target cells without being detected by the immune system, making them suitable as delivery vectors. 

The human genome encodes at least 100 retrovirus-derived gag-like genes, few of which have been studied. It is possible that at least some of these genes encode additional proteins that can form virus-like particles. In fact, a recent publication shows that another retrotransposon-derived protein called PEG10 with sequence similarity to Gag protein also binds and encapsidates mRNA to transport it out of cells in extracellular vesicles, and it could potentially be used to selectively deliver specific mRNAs for therapeutic purposes. There are also other mammalian genes of retroviral origin whose functions are just beginning to be defined. For example, a recent preprint suggests that a non-coding RNA of retroviral origin regulates the expression of the myelin gene, which encodes a protein critical for central nervous system function. The endogenous retroviral envelope gene Syncytin-1 also likely evolved to mediate the cell-cell fusion needed for the development of the human placenta. It will be interesting to follow new developments in this field as more data emerge.

[The ability of Arc to spontaneously form capsid-like particles was previously discussed on TWiV 477 and TWiN 4]. 

Filed Under: Basic virology, Gertrud Rey Tagged With: capsid, extracellular vesicle, gag, synapse

A Viral Heist

6 January 2022 by Gertrud U. Rey

by Gertrud U. Rey

Viruses that require access to the host cell nucleus typically get there by exploiting cellular motor proteins for their own transportation purposes. For example, herpesviruses use two cellular proteins called dynein and kinesin to shuttle their cargo to and from the nucleus. However, the exact mechanism by which these viruses engage the motor proteins was unclear until recently.

Dynein and kinesin traffic around the cell on long cylindrical structures called microtubules, which not only serve as a “railway” system for a variety of motor proteins that transport cargo to support various cellular functions, but also provide a framework for the cytoskeleton. Microtubules have a well-defined polarity that is integral to their biological function: one end is designated the plus end, while the other end is the minus end. Dynein moves from the plus end of the microtubule towards the minus end in the direction of the nucleus, while kinesin moves in the opposite direction, typically leading to the cell periphery.

Herpes simplex virus type 1 (HSV-1) initially infects epithelial cells of the skin and mucous membranes. During this infection, viral particles fuse with the host cell membrane and release the protein shell that encompasses the viral genome (the “capsid”), into the cytoplasm. A virally encoded and capsid-bound protein called pUL36 then binds dynein, allowing the capsid to travel along a microtubule to the nucleus, where the viral genome is injected through a nuclear pore. When newly assembled capsids carrying newly replicated viral genomes exit from the nucleus at a later time, they engage kinesin to motor along the microtubules toward the cell membrane. Newly formed virions emerging from the cell membrane subsequently infect nearby sensory neurons, where they establish a lifelong, persistent infection.

Studies have shown that deletion of the portion of pUL36 that tethers it to the viral capsid results in its ability to move along microtubules in both directions, suggesting that in addition to binding dynein, pUL36 also binds kinesin. Subsequent follow-up studies exploring the kinetics of this presumed interaction revealed that pUL36 does indeed bind kinesin through a series of tryptophan-aspartate (WD) motifs. Two of these motifs – WD3 and WD4 – appear to be particularly important: WD3 improves pUL36-kinesin binding and WD4 is essential for the interaction.

In an effort to more clearly define the role of kinesin during herpesvirus infection, Caitlin Pegg, Sofia Zaichick and co-authors of a recent study, carried out a series of experiments using HSV-1 and pseudorabiesvirus (PRV), a herpesvirus in the same subfamily as HSV-1, which is often used as a model to study the basic processes of herpesvirus infection. To determine whether WD3 and WD4 are needed for PRV replication and viability, the authors introduced mutations in the DNA sequences encoding these motifs in PRV. Plaque assays done using cells infected with the respective modified viruses revealed that viruses with a mutation in WD3 produced smaller plaques, suggesting that this motif enhanced viral replication. Viruses with a mutation in WD4 produced almost no plaques, suggesting that this motif was essential for viral replication. These results complemented the data obtained from the pUL36-kinesin binding studies, implying that an interaction between pUL36 and kinesin is needed for viral replication.

The authors also determined that a mutation in the sequence encoding the acidic amino acids of the WD4 motif led to reduced kinesin binding but did not impair PRV replication. To study the transport mechanism of PRV, they generated a virus with reduced kinesin binding ability (“RKB”) by changing the amino acids in both WD3 and the acidic amino acids in WD4. They further modified the virus by inserting a gene encoding a fluorescent marker next to the capsid genes. The joint expression of the capsid and marker genes in cells would result in fluorescently tagged capsids, allowing one to visualize these particles using a fluorescence microscope. Microscopic analysis of sensory neurons infected with fluorescently tagged RKB viruses revealed that the capsids traveled through the cytoplasm toward the nucleus, but never actually reached the nucleus. This suggests that pUL36 binding to kinesin is important for delivery of viral particles into the neuronal nucleus during infection.

To further substantiate this finding, the authors performed infection experiments using retinal pigment epithelial (RPE) cells that do not express kinesin. Infection of these cells with fluorescently-tagged HSV-1 revealed that viral capsids only advanced to the centrosome, an organelle involved in cell division that is somewhat removed from the nucleus. In contrast, infection of wild type RPE cells having functional kinesin resulted in trafficking of viral capsids right up to the nucleus, confirming that kinesin is needed for transporting capsids from the centrosome to the nucleus.

To determine what happens when virions are produced in the absence of kinesin, the authors infected kinesin-deficient RPE cells with either wild type HSV-1 or wild type PRV, isolated the resulting viral particles and then used those viruses to infect wild type (i.e., kinesin-expressing) RPE cells. Visualization of capsid movement in the wild type RPE cells revealed that the capsids only moved as far as the centrosome, and that centrosome-to-nucleus trafficking was impaired, an effect that was particularly pronounced when the experiment was repeated using wild type sensory neurons. The fact that the virus particles isolated from kinesin-deficient cells were unable to traffic to the nucleus in neuronal cells in spite of the fact that these cells produced kinesin suggests that viruses that do make it to the neuronal nucleus must typically carry kinesin when they enter these cells, presumably inside the virion. However, analysis of wild type cell-originating HSV-1 extracellular virions using anti-kinesin antibodies did not reveal the presence of kinesin in these virions.

In a different approach aimed at identifying kinesin inside HSV-1 virions, the authors generated a DNA construct consisting of kinesin fused to the reporter enzyme β-lactamase and inserted this construct into kinesin-deficient RPE cells. They then infected the cells with HSV-1, extracted newly replicated virions from the cells and mixed the virions with nitrocefin, a molecule that reacts with β-lactamase to produce a color change that can be visually detected. A color change would indicate the presence of β-lactamase, which would mean that kinesin was present as well. This method revealed that the virions isolated from the kinesin/β-lactamase-expressing cells did contain β-lactamase, further suggesting that they also contained kinesin. These results suggested that HSV-1 virions capture kinesin from initially infected epithelial cells and ferry it to subsequently infected neuronal cells to deliver their genome into the nucleus of those cells.

To confirm whether this hypothesis was true, the authors prepared HSV-1 virions expressing kinesin fused to DmrB, a protein that can be induced to dimerize through addition of a drug. When the drug was added to primary sensory neurons infected with HSV-1 carrying the DmrB-kinesin fusion protein, capsid transport from the centrosome to the nucleus was reduced, presumably because DmrB formed a dimer which structurally impaired the motility of kinesin. These results confirmed that kinesin mediates the movement of capsids from the centrosome to the nucleus. And although kinesin-mediated transport of HSV-1 presumably leads the virions to the cell membrane instead of the nucleus, the microtubules radiating out from the centrosome toward the nucleus are oriented such that their minus end is anchored in the centrosome, while the plus end is at the nucleus, consistent with the directional movement of kinesin.

The fact that viruses can capture a cellular protein and incorporate it for later use is a remarkable discovery that the authors refer to as “assimilation.” As more research emerges in this field, it will be interesting to see whether herpesviruses steal other cellular proteins and whether members of other virus families also assimilate cellular proteins to suit their purposes.     

[For an in-depth discussion of this paper, please check out TWiV 833]

Filed Under: Basic virology, Gertrud Rey Tagged With: capsid, centrosome, dynein, herpesvirus, HSV-1, kinesin, microtubule, motor protein, neuronal cell, nucleus, plaque assay, pseudorabies virus, pUL36, sensory neurons

Evolution of a bacterial protein into a virus-like, RNA binding capsid

8 July 2021 by Vincent Racaniello

Starting with a bacterial protein, directed evolution in the laboratory has been used to produce a virus-like capsid that binds and protects RNA. This finding has implications for the origins of viruses.

One view of the evolution of life is that viruses were present even before the first cells in the form of self-replicating molecules. These were technically not viruses, because they did not need cells for their replication. The idea is that when cells did arise, the replicators invaded them and then recruited host proteins for the formation of capsids.

The starting point for experiments intended to provide insight into how a protein might become a viral capsid is a bacterial protein, lumazine synthase, that forms 60-subunit particles that have no affinity for nucleic acids. The protein was redesigned and linked to a peptide that binds to an RNA stem-loop called BoxB. It was then further modified for the ability to package an mRNA that encodes the synthase. Packaging was stimulated by adding BoxB tags to the ends of the mRNA. However, only one of 8 particles packaged the mRNA.

To improve the capsid, the bacterial gene was mutagenized and capsids were selected by rounds of incubation with ribonuclease of smaller and smaller size. The idea was that this strategy would select for capsids that not only packaged the mRNA but would protect it from RNAse digestion. The result was a protein that could form a capsid called NC-4 (pictured) which encapsidated mainly the mRNA and protected it from digestion.

The changes that occurred during this evolution process led to the production of what looks very much like a viral capsid. It is composed of 240 protein subunits arranged in pentamers and hexamers with t=4 icosahedral symmetry*. The amino acid changes that led to the formation of this capsid can be readily discerned. The pores on the capsid are very small, explaining its relative nuclease resistance compared with earlier versions. In addition, the RNA appears to play a role in assembly of the capsid. Finally, changes in the mRNA appear to have led to formation of a stem-loop which facilitates packaging of the nucleic acid into the particle.

These results are amazing: we know that arranging proteins with icosahedral symmetry is an efficient way to build a stable virus particle with the least number of subunits, confirmed by the directed evolution of an icosahedrally ordered and stable capsid.

These observations have multiple implications. They show how ancient self-replicating RNA molecules, the precursors of viruses, might have recruited a host protein and driven its evolution into a protective capsid that specifically packages only the viral RNA. They also indicate how stable particles might be designed as alternatives (dare I say improvements?) to viruses for therapeutic purposes, such as gene therapy and vaccination.

*If you do not understand what icosahedral symmetry is or what t=4 means, please watch my lecture on virus structure.

Filed Under: Basic virology, Information Tagged With: capsid, directed evolution, primordial replicator, RNA packaging, RNA world, viral, virology, virus, viruses

TWiV 582: This little virus went to market

12 January 2020 by Vincent Racaniello

TWiV provides updates on the new coronavirus causing respiratory disease in China, the current influenza season, and the epidemic of African swine fever, including determination of the three-dimensional structure of the virus particle.

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Show notes at microbe.tv/twiv

Filed Under: This Week in Virology Tagged With: african swine fever virus, capsid, China, coronavirus, cryo-electron micrography, giant virus, Huanan Fish Market, influenza, influenza excess mortality, influenza like illness, influenza vaccine, pig, swine, three dimensional virus structure, viral, virology, virus, viruses, Wuhan pneumonia, zoonosis

Virus Watch: Buckyball Viruses

13 March 2018 by Vincent Racaniello

In this short video, I show you how to make different types of virus particles using the small magnetic spheres called Buckyballs.

Filed Under: Basic virology, Information Tagged With: Buckyball, capsid, helical symmetry, icosahedral symmetry, video, viral, virology, virus, viruses

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by Vincent Racaniello

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