Virus capsids are made from subunits

Buller, R. Characterization of adenovirus-associated virus-induced polypeptides in KB cells. McPherson, R. Human cytomegalovirus completely helps adeno-associated virus replication.

Virology , — Weindler, F. Weitzman, M. Adeno-associated virus biology. Methods Mol. Wistuba, A. Intermediates of adeno-associated virus type 2 assembly: identification of soluble complexes containing Rep and Cap proteins.

Sonntag, F. A viral assembly factor promotes AAV2 capsid formation in the nucleolus. Natl Acad. USA , — Ogden, P. AAV capsid fitness landscape reveals a viral gene and enables machine-guided design. Science , — Zinn, E. Adeno-associated virus: fit to serve. PubMed Article Google Scholar. Gao, G. Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. USA 99 , — Clades of adeno-associated viruses are widely disseminated in human tissues.

Wu, Z. Adeno-associated virus serotypes: vector toolkit for human gene therapy. Russell, S. Efficacy and safety of voretigene neparvovec AAV2-hRPE65v2 in patients with RPEmediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial.

Lancet , — Hoy, S. Onasemnogene abeparvovec: first global approval. Drugs 79 , — Endgame: glybera finally recommended for approval as the first gene therapy drug in the European union. Wang, D. Adeno-associated virus vector as a platform for gene therapy delivery.

Drug Discov. Naso, M. Adeno-associated virus AAV as a vector for gene therapy. BioDrugs 31 , — Kimura, T. Production of adeno-associated virus vectors for in vitro and in vivo applications.

Chapman, M. Srivastava, A. Nucleotide sequence and organization of the adeno-associated virus 2 genome. Agbandje-McKenna, M. Tse, L. Structure-guided evolution of antigenically distinct adeno-associated virus variants for immune evasion. USA , E—E Judd, J. Random insertion of mcherry into VP3 domain of adeno-associated virus yields fluorescent capsids with no loss of infectivity. Google Scholar.

Eichhoff, A. Nanobody-enhanced targeting of AAV gene therapy vectors. CAS Google Scholar. Johnson, F. Rose, J. Snijder, J. Defining the stoichiometry and cargo load of viral and bacterial nanoparticles by orbitrap mass spectrometry. Mietzsch, M. Twenty-five years of structural parvovirology. Venkatakrishnan, B. Structure and dynamics of adeno-associated virus serotype 1 VP1-unique N-terminal domain and its role in capsid trafficking. Padron, E. Structure of adeno-associated virus type 4.

Kronenberg, S. Electron cryo-microscopy and image reconstruction of adeno-associated virus type 2 empty capsids. EMBO Rep. In the adeno-associated virus type 2 capsid leads to the exposure of hidden VP1 N termini. Similarly to how the size of the virus particle varies significantly, the genome size can also vary greatly from virus to virus.

A typical virus genome falls in the range of —20, base pairs bp 7—20 kilobase pairs kb. Smaller-sized virions will naturally be able to hold less nucleic acid than larger virions, but large viruses do not necessarily have large genomes.

While most viruses do not contain much nucleic acid, some dsDNA viruses have very large genomes: herpesviruses have genomes that are — kb in total, and the very large pandoraviruses mentioned previously have the largest genomes: up to 2. In comparison, eukaryotic cells have much larger genomes: a red alga has the smallest known eukaryotic genome, at 8 million base pairs; a human cell contains over 3 billion nucleotides in its hereditary material; the largest genome yet sequenced, at over 22 billion base pairs, is that of the loblolly pine tree.

The infectious virus particle must be released from the host cell to infect other cells and individuals. In the extracellular environment, the virus will be exposed to enzymes that could break down or degrade nucleic acid. Physical stresses, such as the flow of air or liquid, could also shear the nucleic acid strands into pieces. In addition, viral genomes are susceptible to damage by ultraviolet radiation or radioactivity, much in the same way that our DNA is. If the nucleic acid genome of the virus is damaged, then it will be unable to produce progeny virions.

This repeating structure forms a strong but slightly flexible capsid. Combined with its small size, the capsid is physically very difficult to break open and sufficiently protects the nucleic acid inside of it. Together, the nucleic acid and the capsid form the nucleocapsid of the virion Fig. Viral capsid proteins protect the fragile genome, composed of nucleic acid, from the harsh environment.

The capsid and nucleic acid together are known as the nucleocapsid. Remember that the genomes of most viruses are very small. Genes encode the instructions to make proteins, so small genomes cannot encode many proteins. It is for this reason that the capsid of the virion is composed of one or only a few proteins that repeat over and over again to form the structure. The nucleic acid of the virus would be physically too large to fit inside the capsid if it were composed of more than just a few proteins.

In the same way that a roll of magnets will spontaneously assemble together, capsid proteins also exhibit self-assembly. The first to show this were H.

Fraenkel-Conrat and Robley Williams in They separated the RNA genome from the protein subunits of tobacco mosaic virus, and when they put them back together in a test tube, infectious virions formed automatically. This indicated that no additional information is necessary to assemble a virus: the physical components will assemble spontaneously, primarily held together by electrostatic and hydrophobic forces. Most viruses also have an envelope surrounding the capsid.

Virus capsids are held together by some of the same bonds that are found in living organisms. Rarely are covalent bonds found in capsids; these are the strongest of bonds that are formed when atoms share electrons with each other.

Hydrogen bonds are also weak electrostatic forces that occur between slightly charged atoms, usually between hydrogen slightly positively charged and another atom that is partially negatively charged, such as oxygen. Van der Waals forces are weak interactions that occur when an atom becomes slightly charged due to random asymmetry of its electrons.

The properties of water also contribute to virus assembly and attachment to cells. Water is a polar molecule, meaning that the molecule has two distinct ends, much like a battery or magnet has a positive and a negative end.

Molecules that do not have distinct ends are termed nonpolar. Other polar molecules are attracted to water, since water is polar too. This explains the phenomenon of oil nonpolar not mixing with water polar. These viruses often have proteins, called matrix proteins , that function to connect the envelope to the capsid inside. A virus that lacks an envelope is known as a nonenveloped or naked virus Fig. Each virus also possesses a virus attachment protein embedded in its outer-most layer.

This will be found in the capsid, in the case of a naked virus, or the envelope, in the case of an enveloped virus. The virus attachment protein is the viral protein that facilitates the docking of the virus to the plasma membrane of the host cell, the first step in gaining entry into a cell. The capsid of an enveloped virion is wrapped with a lipid membrane derived from the cell. Virus attachment proteins located in the capsid or envelope facilitate binding of the virus to its host cell.

Each virus possesses a protein capsid to protect its nucleic acid genome from the harsh environment. Virus capsids predominantly come in two shapes: helical and icosahedral. The helix plural: helices is a spiral shape that curves cylindrically around an axis.

It is also a common biological structure: many proteins have sections that have a helical shape, and DNA is a double-helix of nucleotides. In the case of a helical virus, the viral nucleic acid coils into a helical shape and the capsid proteins wind around the inside or outside of the nucleic acid, forming a long tube or rod-like structure Fig.

The nucleic acid and capsid constitute the nucleocapsid. In fact, the protein that winds around the nucleic acid is often called the nucleocapsid protein. Once in the cell, the helical nucleocapsid uncoils and the nucleic acid becomes accessible. A Viral capsid proteins wind around the nucleic acid, forming a helical nucleocapsid. B Helical structure of tobacco mosaic virus. Graph , 12, —44 using a 2xea PDB assembly J. There are several perceived advantages to forming a helical capsid. First, only one type of capsid protein is required.

This protein subunit is repeated over and over again to form the capsid. This structure is simple and requires less free energy to assemble than a capsid composed of multiple proteins. In addition, having only one nucleocapsid protein means that only one gene is required instead of several, thereby reducing the length of nucleic acid required.

Because the helical structure can continue indefinitely, there are also no constraints on how much nucleic acid can be packaged into the virion: the capsid length will be the size of the coiled nucleic acid. Helical viruses can be enveloped or naked. The first virus described, tobacco mosaic virus, is a naked helical virus. In fact, most plant viruses are helical, and it is very uncommon that a helical plant virus is enveloped.

In contrast, all helical animal viruses are enveloped. These include well-known viruses such as influenza virus, measles virus, mumps virus, rabies virus, and Ebola virus Fig.

A Vesicular stomatitis virus forms bullet-shaped helical nucleocapsids. Fred A. B Tobacco mosaic virus forms long helical tubes. C The helical Ebola virus forms long threads that can extend over nm in length. Of the two major capsid structures, the icosahedron is by far more prevalent than the helical architecture. In comparison to a helical virus where the capsid proteins wind around the nucleic acid, the genomes of icosahedral viruses are packaged completely within an icosahedral capsid that acts as a protein shell.

Section Chapter Questions. Problem 1ACQ. Problem 2ACQ. Problem 3ACQ. Problem 4RQ. Problem 5RQ. Problem 6RQ. Problem 7RQ. Problem 8RQ. Problem 9RQ. Problem 10RQ. Problem 11RQ. Problem 12RQ. Problem 13RQ. Problem 14RQ. Problem 15RQ. Problem 16CTQ. Problem 17CTQ. Problem 18CTQ. Problem 19CTQ. Problem 20CTQ. Problem 21CTQ. Problem 22CTQ.

Chapter 17, Problem 1ACQ. Textbook Problem. Summary Introduction. Introduction: Viruses have diversity in terms of the structure, the method of replication, host and target cells. Answer to Problem 1ACQ. Correct answer: The correct answer is option d Glycoproteins help the virus attach to the host cell. Epitope valency of the e-antigen was also studied, using a sandwich SPR assay where e-antigen was captured with one mab and probed with a second.

This model, however, is incorrect as recombinant e-antigen is a stable dimer and its apparent monovalency is due to steric blockage. This was proven by the formation of a Fab e6:e-antigen complex. These results suggest new approaches for the isolation of the authentic e-antigen, its biological assay, and its stabilization as an immune complex for structural studies.

Hepatitis B virus HBV causes - million chronic infections and approximately 1 million deaths annually. HBcAg refers to the viral nucleocapsid which is seldom found in non-enveloped form outside of infected cells.

HBeAg is a soluble protein that is secreted into the circulation and is thought to promote chronic infection. The full-length HBcAg polypeptide is residues long; however, the amino-terminal residues are fully competent to form dimers that can assemble to form capsids. Capsids are assembled from either 90 or such dimers, with the four-helix bundles projecting as spikes.

Despite this close sequence similarity, HBcAg and HBeAg differ in solubility and in their assembly properties, 14 in their B cell and T cell responses, 7 in the antibodies they are recognized by and in the kinetics they exhibit during infection, 15 and in having different functions. Fortunately, since the time that these studies were done the structure of HBcAg has been determined at high resolution 8 and the epitopes of seven HBcAg-specific Mabs have been identified by cryoelectron microscopy and image reconstruction.

The goal of the present study was to reassess and clarify the serological relationship between HBcAg and HBeAg using surface plasmon resonance. This technique is not only very sensitive but also less error prone than the previously employed plate-based assays and it was therefore used to measure the affinities of a panel of six historically well-documented monoclonal antibodies for a set of highly-purified and well-characterized recombinant forms of HBcAg and HBeAg.

Mabs , , , and are the original monoclonal antibodies from the Mayumi group that define the four primary HBcAg and HBeAg determinants. For HBcAg, we used capsids of the full-length residue protein Cp that contain bacterial nucleic acid substituting for the viral pregenome in authentic HBcAg and capsids composed of the residue protein Cp The latter are free of nucleic acid and, unlike Cp capsids, may be disassembled in vitro to dimers.

For HBeAg, we expressed in E. Cp extended at its amino-terminus by a residue peptide corresponding to the residual propeptide viz. Cp In order to distinguish assembly-dependent properties from conformation-dependent properties of these proteins, we also determined conditions to form novel capsid-like structures from Cp , and exploited a point mutation that greatly reduces the propensity of both core- and e-antigen dimers to assemble into capsids, and included these reagents in the SPR assays.

Cp c , and Cp d. Schematic representation of the recombinant HBV proteins used in this study. The three capsid-related proteins Cp correspond to those listed in Table 1 and in greater detail in Fig. Cp is expressed in E. Such capsids are very stable and cannot be dissociated into subunits without protein denaturation. In Cp the arginine rich carboxy-terminal domain has been deleted and when this protein is expressed in E.

The red line indicates an intermolecular disulfide bond [CC61]. The Cp or e-antigen is expressed in E. The protein can be extracted with 2 - 3 M urea at pH 9.

Purified dimers can be induced to form capsids white fenestrated structure. The reversibility of this has not been fully explored. The red lines indicate intramolecular disulfide bonds [C -7 — C61].

See Figs. S1 and S2 for analytical data on these various proteins. Unlike the very stable Cp c , Cp c can be dissociated into dimers under conditions which do not denature the constituent subunits. Cp d is more soluble than Cp d but can be induced to form capsids by elevating the temperature see Materials and Methods.

The mutation GA also enhances the solubility of Cp d. The two cysteine 61 residues in Cp d form an intermolecular disulfide bond Fig. S1, a whereas in Cp d they form two intramolecular disulfides with the cysteine -7 residues Fig. S1, a 11 ; 12 and for this reason, cysteine 61 was retained in all constructs. However, cysteines 48 and are not involved in disulfide bond formation 8 and were routinely substituted with alanine. The capsid proteins and the various mutants used in this study are listed in Table 1 and schematically represented in Fig.

The assembly state of capsids under native conditions i. S1, b and under non-reducing condition, two major bands corresponding to monomers and dimers Fig. S1, b and lesser amounts of higher order multimers S2 , indicating that the proteins are dimeric at neutral pH. Supplementary Table 1 summarizes the principal antigenic regions on HBcAg and HBeAg reported in the literature, and Table 2 summarizes the epitopes of the antibodies employed in this study.

The locations of the epitopes are also shown in Fig. Locations of the epitopes on HBV capsids and subunits. The cartoon represents three adjacent dimeric subunits on a capsid with the protruding spikes corresponding to the four-helix bundles. The epitopes for Mabs 88, , , , 9c8, and F11A4 are all located on the apices of the spikes whereas the epitope for Mab is located between the spikes and involves residues from two adjacent subunits.

The locations of the epitopes for Mabs and are uncertain, but epitope may be on the spike apex and near the C-terminus see text. The positions of the three carboxy-terminal mutations employed to map the e6 epitope, and which affect capsid assembly, are also indicated.

The binding kinetics of various protein preparations to the panel of Mabs are summarized in Table 3. From a cursory inspection the affinities shown in this table do not appear to be consistent with the published specificities of the antibodies used Table 2. One might expect that HBeAg-specific and HBcAg-specific Mabs would, by definition, have higher affinities for their respective antigens.

How these results can be explained is discussed below. The dimers Cp d and Cp d also bound with moderate to high affinity to all members of the same panel, except Mab For example, the respective bindings of Cp c and Cp d to Mab nominally HBcAg-specific show fairly similar kinetics and affinity Fig. Thus - with the exceptions noted above - these Mabs are largely insensitive to the assembly state of the capsid protein or, for a given assembly state, to the sequences appended at either terminus.

Typical sensograms illustrating the binding of antigen to Mab Antigen was allowed to bind to immobilized antibody. Duplicate, and in some cases triplicate, injections of antigen were done. The concentrations refer to the concentration of antigen, in terms of dimers. The kinetics and affinities of Cp c A and Cp d B for Mab are similar, indicating that the epitope is not affected significantly by the assembly state of the antigen. This is in keeping with its known location on the apex of the spike s.

The failure of Cp d and Cp d to bind Mab is consistent with the identification of its epitope as bridging two subunits on adjacent dimers. The other nominally HBcAg-specific Mabs, and F11A4, bind to the two so-called immunodominant loops located at the apex of the capsid spike. Thus, the binding of these spike-specific antibodies is unaffected by the assembly state of the capsid protein: they all bind strongly to both capsids and dimers Table 3.

We have assumed that the interactions in Table 3 represent interactions between immobilized antibodies and antigens, and furthermore, that these interactions represent affinity rather than avidity, despite the fact that the antibody is bivalent and the antigens are either bi- or polyvalent.

To promote monovalent interactions between antibody and antigen we employed the minimum density of immobilized antibody consistent with reliable measurements densities of , , and 50 RU were tested. The equilibrium constants reported in Table 3 were essentially unaltered over this range of densities.

Hence, at the routinely used density of RU, the contribution to the data of single antigens bound to more than one antibody was considered to be minimal. With regard as to whether the interactions represent affinity or avidity, we considered the dimers to be too small to engage with both antigen-binding domains of a given IgG simultaneously. This was confirmed by experiment, for example, Cp d bound to immobilized Fab and Mab with K d values of 6.

Similar experiments with capsids were not possible because capsids failed to bind to immobilized Fabs, probably due to restricted access.