A Multistep, ATP-dependent Pathway for Assembly of Human Immunodeficiency Virus Capsids in a Cell-free System

To understand the mechanism by which human immunodeficiency virus type 1 (HIV) capsids are formed, we have reconstituted the assembly of immature HIV capsids de novo in a cell-free system. Capsid authenticity is established by multiple biochemical and morphologic criteria. Known features of the assembly process are closely reproduced, indicating the fidelity of the cell-free reaction. Assembly is separated into co- and posttranslational phases, and three independent posttranslational requirements are demonstrated: (a) ATP, (b) a detergent-sensitive host factor, and (c) a detergent-insensitive host subcellular fraction that can be depleted and reconstituted. Assembly appears to proceed by way of multiple intermediates whose conversion to completed capsids can be blocked by either ATP depletion or treatment with nondenaturing detergent. Specific subsets of these intermediates accumulate upon expression of various assembly-defective Gag mutants in the cell-free system, suggesting that each mutant is blocked at a particular step in assembly. Furthermore, the accumulation of complexes of similar sizes in cells expressing the corresponding mutants suggests that comparable intermediates may exist in vivo. From these data, we propose a multi-step pathway for the biogenesis of HIV capsids, in which the assembly process can be disrupted at a number of discrete points.     

Generation of Affinity-Tagged Fluoromycobacteriophages by Mixed Assembly of Phage Capsids

Addition of affinity tags to bacteriophage particles facilitates a variety of applications, including vaccine construction and diagnosis of bacterial infections. Addition of tags to phage capsids is desirable, as modification of the tails can lead to poor adsorption and loss of infectivity. Although tags can readily be included as fusions to head decoration proteins, many phages do not have decoration proteins as virion components. The addition of a small (10-amino-acid) Strep-tag II (STAG II) to the mycobacteriophage TM4 capsid subunit, gp9, was not tolerated as a genetically homogenous recombinant phage but could be incorporated into the head by growth of wild-type phage on a host expressing the capsid-STAG fusion. Particles with capsids composed of wild-type and STAG-tagged subunit mixtures could be grown to high titers, showed good infectivities, and could be used to isolate phage-bacterium complexes. Preparation of a STAG-labeled fluoromycobacteriophage enabled capture of bacterial complexes and identification of infected bacteria by fluorescence.     

Unclosed HIV-1 Capsids Suggest a Curled Sheet Model of Assembly

The RNA genome of retroviruses is encased within a protein capsid. To gather insight into the assembly and function of this capsid, we used electron cryotomography to image human immunodeficiency virus (HIV) and equine infectious anemia virus (EIAV) particles. While the majority of viral cores appeared closed, a variety of unclosed structures including rolled sheets, extra flaps, and cores with holes in the tip were also seen. Simulations of nonequilibrium growth of elastic sheets recapitulated each of these aberrations and further predicted the occasional presence of seams, for which tentative evidence was also found within the cryotomograms. To test the integrity of viral capsids in vivo, we observed that ~ 25% of cytoplasmic HIV complexes captured by TRIM5α had holes large enough to allow internal green fluorescent protein (GFP) molecules to escape. Together, these findings suggest that HIV assembly at least sometimes involves the union in space of two edges of a curling sheet and results in a substantial number of unclosed forms.     

Nuclear Entry of Hepatitis B Virus Capsids Involves Disintegration to Protein Dimers followed by Nuclear Reassociation to Capsids

Assembly and disassembly of viral capsids are essential steps in the viral life cycle. Studies on their kinetics are mostly performed in vitro, allowing application of biochemical, biophysical and visualizing techniques. In vivo kinetics are poorly understood and the transferability of the in vitro models to the cellular environment remains speculative. We analyzed capsid disassembly of the hepatitis B virus in digitonin-permeabilized cells which support nuclear capsid entry and subsequent genome release. Using gradient centrifugation, size exclusion chromatography and immune fluorescence microscopy of digitonin-permeabilized cells, we showed that capsids open and close reversibly. In the absence of RNA, capsid re-assembly slows down; the capsids remain disintegrated and enter the nucleus as protein dimers or irregular polymers. Upon the presence of cellular RNA, capsids re-assemble in the nucleus. We conclude that reversible genome release from hepatitis B virus capsids is a unique strategy different from that of other viruses, which employs irreversible capsid destruction for genome release. The results allowed us to propose a model of HBV genome release in which the unique environment of the nuclear pore favors HBV capsid disassembly reaction, while both cytoplasm and nucleus favor capsid assembly.     

Mechanical and Assembly Units of Viral Capsids Identified via Quasi-Rigid Domain Decomposition

Key steps in a viral life-cycle, such as self-assembly of a protective protein container or in some cases also subsequent maturation events, are governed by the interplay of physico-chemical mechanisms involving various spatial and temporal scales. These salient aspects of a viral life cycle are hence well described and rationalised from a mesoscopic perspective. Accordingly, various experimental and computational efforts have been directed towards identifying the fundamental building blocks that are instrumental for the mechanical response, or constitute the assembly units, of a few specific viral shells. Motivated by these earlier studies we introduce and apply a general and efficient computational scheme for identifying the stable domains of a given viral capsid. The method is based on elastic network models and quasi-rigid domain decomposition. It is first applied to a heterogeneous set of well-characterized viruses (CCMV, MS2, STNV, STMV) for which the known mechanical or assembly domains are correctly identified. The validated method is next applied to other viral particles such as L-A, Pariacoto and polyoma viruses, whose fundamental functional domains are still unknown or debated and for which we formulate verifiable predictions. The numerical code implementing the domain decomposition strategy is made freely available.     

Periodic Table of Virus Capsids: Implications for Natural Selection and Design

Background

For survival, most natural viruses depend upon the existence of spherical capsids: protective shells of various sizes composed of protein subunits. So far, general evolutionary pressures shaping capsid design have remained elusive, even though an understanding of such properties may help in rationally impeding the virus life cycle and designing efficient nano-assemblies.

Principal Findings

This report uncovers an unprecedented and species-independent evolutionary pressure on virus capsids, based on the the notion that the simplest capsid designs (or those capsids with the lowest “hexamer complexity”, ) are the fittest, which was shown to be true for all available virus capsids. The theories result in a physically meaningful periodic table of virus capsids that uncovers strong and overarching evolutionary pressures, while also offering geometric explanations to other capsid properties (rigidity, pleomorphy, auxiliary requirements, etc.) that were previously considered to be unrelatable properties of the individual virus.

Significance

Apart from describing a universal rule for virus capsid evolution, our work (especially the periodic table) provides a language with which highly diverse virus capsids, unified only by geometry, may be described and related to each other. Finally, the available virus structure databases and other published data reiterate the predicted geometry-derived rules, reinforcing the role of geometry in the natural selection and design of virus capsids.     

Rational Engineering of Recombinant Picornavirus Capsids to Produce Safe,Protective Vaccine Antigen

Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals.     

Microtubule-mediated Transport of Incoming Herpes Simplex Virus 1 Capsids to the Nucleus

Herpes simplex virus 1 fuses with the plasma membrane of a host cell, and the incoming capsids are efficiently and rapidly transported across the cytosol to the nuclear pore complexes, where the viral DNA genomes are released into the nucleoplasm. Using biochemical assays, immunofluorescence, and immunoelectron microscopy in the presence and absence of microtubule depolymerizing agents, it was shown that the cytosolic capsid transport in Vero cells was mediated by microtubules. Antibody labeling revealed the attachment of dynein, a minus end–directed, microtubule-dependent motor, to the viral capsids. We propose that the incoming capsids bind to microtubules and use dynein to propel them from the cell periphery to the nucleus.     

Incorporation of Antigens into Viral Capsids Augments Immunogenicity of Adeno-Associated Virus Vector-Based Vaccines

Genetic modification of adeno-associated virus (AAV) capsids has previously been exploited to redirect viral tropism. Here we demonstrate that engineering of AAV capsids as scaffolds for antigen display augments antigen-specific immunogenicity. Combining antigen display with vector-mediated overexpression resulted in a single-shot prime-boost vaccine. This new class of vaccines induced immune responses significantly faster and an IgG antibody pool of higher avidity than conventional vectors, highlighting the potency of capsid modification in vaccine development.     

Enhancement of Adeno-Associated Virus Infection by Mobilizing Capsids into and Out of the Nucleolus

Adeno-associated virus (AAV) serotypes are being tailored for numerous therapeutic applications, but the parameters governing the subcellular fate of even the most highly characterized serotype, AAV2, remain unclear. To understand how cellular conditions control capsid trafficking, we have tracked the subcellular fate of recombinant AAV2 (rAAV2) vectors using confocal immunofluorescence, three-dimensional infection analysis, and subcellular fractionation. Here we report that a population of rAAV2 virions enters the nucleus and accumulates in the nucleolus after infection, whereas empty capsids are excluded from nuclear entry. Remarkably, after subcellular fractionation, virions accumulating in nucleoli were found to retain infectivity in secondary infections. Proteasome inhibitors known to enhance transduction were found to potentiate nucleolar accumulation. In contrast, hydroxyurea, which also increases transduction, mobilized virions into the nucleoplasm, suggesting that two separate pathways influence vector delivery in the nucleus. Using a small interfering RNA (siRNA) approach, we then evaluated whether nucleolar proteins B23/nucleophosmin and nucleolin, previously shown to interact with AAV2 capsids, affect trafficking and transduction efficiency. Similar to effects observed with proteasome inhibition, siRNA-mediated knockdown of nucleophosmin potentiated nucleolar accumulation and increased transduction 5- to 15-fold. Parallel to effects from hydroxyurea, knockdown of nucleolin mobilized capsids to the nucleoplasm and increased transduction 10- to 30-fold. Moreover, affecting both pathways simultaneously using drug and siRNA combinations was synergistic and increased transduction over 50-fold. Taken together, these results support the hypothesis that rAAV2 virions enter the nucleus intact and can be sequestered in the nucleolus in stable form. Mobilization from the nucleolus to nucleoplasmic sites likely permits uncoating and subsequent gene expression or genome degradation. In summary, with these studies we have refined our understanding of AAV2 trafficking dynamics and have identified cellular parameters that mobilize virions in the nucleus and significantly influence AAV infection.Adeno-associated virus (AAV) is classified as a dependovirus because it requires the presence of a helper virus, such as adenovirus or herpesvirus, in order to enter into a productive lytic cycle (6). Because of its nonpathogenicity and ability to promote sustained, long-term transgene expression in a wide variety of tissues such as the brain, liver, muscle, retina, and vasculature (51), several recombinant AAV (rAAV) serotypes are emerging as attractive vectors for gene therapy. Despite many advances in AAV vector design, barriers such as a preexisting immune response and off-target binding have necessitated administration of high viral titers to achieve efficient transduction (24, 51).Beyond the barriers of the immune response (9, 42) and cell surface targeting (52), researchers are becoming increasingly aware that subcellular processing is a significant barrier to infection (16, 29, 52). Subcellular processing may include conformational changes within the endosome or similar compartments, endosomal escape, nuclear targeting, and uncoating, but the factors that control these events are not well defined. Understanding how cellular conditions affect subcellular processing of virions will lead to improved gene delivery through exploitation of these parameters and promote better vector design.Given that the virion is an icosahedral particle only 25 nm in diameter, rAAV must contain all of the molecular components required to navigate through the subcellular environment in a remarkably small structure. Wild-type AAV is a nonenveloped parvovirus that packages a single-stranded DNA genome of approximately 4.7 kb in length. The viral genome is flanked by two inverted terminal repeats and contains two open reading frames, one that codes for replication proteins and another that codes for capsid proteins. Three capsid proteins (VP1, VP2, and VP3) are encoded in the second overlapping reading frame, each beginning with a different start codon but sharing a common C terminus and stop codon. Capsids are comprised of 60 copies of V1, VP2, and VP3 in a ratio of approximately 1:1:10, respectively (11, 43). During production, AAV capsids are known to assemble at early time points in the nucleolus (64), a subdomain of the nucleus and one of the oldest known cellular structures. Intact capsids have been shown to interact with nucleolar proteins such as nucleolin (NCL) and B23/nucleophosmin (NPM1) in the context of assembly (8, 46), but how these proteins affect infection or vector delivery is currently unknown.Initial cell surface binding of AAV capsids is mediated by expression of glycoprotein receptors and specified by residues in VP3 (45, 58, 59). After binding receptors on the host cell plasma membrane, AAV serotype 2 (AAV2) is endocytosed from the cell surface in a clathrin- and dynamin-dependent process (3, 5, 19). Following endocytosis, many AAV particles accumulate in late endosomes, lysosomes, or other compartments and do not deliver their genome to the nucleus (17). This impediment to gene delivery is exacerbated when particles lack VP1 or contain specific mutations in the unique N terminus of VP1 (23). The N terminus of VP1 is normally folded inside the capsid, harboring a phospholipase domain and putative nuclear localization signals necessary for infection (13, 23, 74). These regions of VP1 are thought to translocate to the capsid exterior during subcellular processing of the virus (10, 35, 57). Even with proper capsid composition, the vast majority of internalized particles remain clearly outside the nuclear membrane, and although recent evidence suggests that successful infection occurs when the capsid uncoats inside the nucleus (57, 61), whether AAV can enter the nucleus as an intact capsid is still vehemently debated.In general, it has proven difficult to discern whether infectious particles truly cross the nuclear membrane, due to the limitations of fluorescence microscopy (5, 67). In an in vitro setting it has been demonstrated that unmodified AAV capsids are capable of entering purified nuclei (28), yet these conditions do not accurately represent what occurs physiologically, since virus directly microinjected into cytoplasm will not enter the nucleus or efficiently transduce the cell (17, 57). In one instance, single-particle tracking of AAV has been used to follow capsids in a live-cell imaging paradigm and has found that they can be quickly and directly transported to the nucleus (54). However, another recent study has parsed confocal images of green fluorescent protein-tagged AAV2 particles during infection and has reported that few if any particles enter the nucleus during infection (38).Although it is unclear whether capsids enter the nucleus intact, it has been well established that nuclear delivery of the genome is highly inefficient and significantly limits transduction. Several studies have identified agents that surmount subcellular barriers to transduction (20, 22, 69). Two of the most well-documented agents known to improve subcellular processing are proteasome inhibitors and hydroxyurea (HU); however, their mechanisms of action remain unknown. Therefore, we set out to determine what effect, if any, these agents had on subcellular trafficking of rAAV2 in the hope of identifying specific cellular parameters that promote efficient transduction.Here we report that rAAV2 capsids accumulate in the nucleolus during infection. Proteasome inhibitors were found to potentiate nucleolar accumulation, while HU reduced nucleolar accumulation and appeared to mobilize capsids to the nucleoplasm. Acting independently, both proteasome inhibitors and HU increased transduction, and together they were cooperative, which suggests that these treatments operate through separate pathways to improve gene delivery. In addition, we found that small interfering RNA (siRNA) knockdown of nucleolar proteins NCL and NPM1 had effects similar to those of proteasome inhibition or HU and increased transduction. Based on our results, we have proposed a model wherein AAV virions initially enter the nucleus intact and can be sequestered in the nucleolus in stable form. Disruption of the nucleolus subsequently mobilizes virions from the nucleolus to nucleoplasmic sites and likely permits uncoating.     

重组风疹病毒抗原的分离纯化

为了制备临床诊断用的风疹病毒抗原,建立了亲合色谱分离纯化的方法.风疹抗原以可溶性形式在大肠杆菌工程菌中获得高效表达,用GST亲合色谱在不变性的条件下直接从细菌裂解液中分离纯化.纯化的目的蛋白电泳为单一条带,EUISA试验表明,重组抗原与风疹病毒IgM阳性血清能特异反应,而与IgM阴性血清不反应,表明重组蛋白具有良好的抗原性,能满足临床检验要求.