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PubMed1997

Biochemical characterization of a smaller form of recombinant Norwalk virus capsids assembled in insect cells.

The expression of the single capsid protein of Norwalk virus (NV) in Spodoptera frugiperda (Sf9) insect cells infected with recombinant baculovirus results in the assembly of virus-like particles (VLPs) of two sizes, the predominant 38-nm, or virion-size VLPs, and smaller, 23-nm VLPs. Here we describe the purification and biochemical characterization of the 23-nm VLPs. The 23-nm VLPs were purified to 95% homogeneity from the medium of Sf9 cultures by isopycnic CsCl gradient centrifugation followed by rate-zonal centrifugation in sucrose gradients. The compositions of the purified 23- and 38-nm VLPs were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein immunoblots. VLPs of both sizes showed a doublet at 58 kDa, the size of the full-length capsid protein. Upon alkaline treatment, the 23-nm VLPs underwent dissociation into soluble intermediates that were able to reassemble into 23- and 38-nm VLPs upon dialysis, suggesting that the assembly of both types of structures has a common pathway. Antigenic and biochemical properties of the 38- and 23-nm VLPs were examined and found to be conserved. Immunoprecipitation assays using polyclonal and monoclonal antibodies indicated that immunodominant epitopes on the capsid protein as well as conformational epitopes are conserved in the two types of particles. The trypsin cleavage site at residue 227 was protected in the assembled particles of both sizes but exposed after alkaline dissociation. These results, and the conservation of the binding activity of both forms of recombinant NV VLPs to cultured cells (L. J. White, J. M. Ball, M. E. Hardy, T. N. Tanaka, N. Kitamoto, and M. K. Estes, J. Virol. 70:6589-6597, 1996), suggest that the tertiary folding of the capsid protein responsible for these properties is conserved in the two structures. We hypothesize that the 23-nm VLPs are formed when 60 units of the NV capsid protein assembles into a structure with T=1 symmetry.

Animals↗
PubMed1997

The making and breaking of symmetry in virus capsid assembly: glimpses of capsid biology from cryoelectron microscopy.

Virus capsids constitute a diverse and versatile family of protein-bound containers and compartments ranging in diameter from approximately 200 A (mass approximately 1 MDa) to >1500 A (mass>250 MDa). Cryoelectron microscopy of capsids, now attaining resolutions down to 10 A, is disclosing novel structural motifs, assembly mechanisms, and the precise locations of major epitopes. Capsids are essentially symmetric structures, and icosahedral surface lattices have proved to be widespread. However, many capsid proteins exhibit a remarkable propensity for symmetry breaking, whereby chemically identical subunits in distinct lattice sites have markedly different structures and packing relationships. Temporal differences in the conformation of a given subunit are also manifested in the large-scale conformational changes that accompany capsid maturation. Larger and more complex capsids, such as DNA bacteriophages and herpes simplex virus, are formed not by simple self-assembly, but under the control of tightly regulated programs that may include the involvement of viral scaffolding proteins and cellular chaperonins, maturational proteolysis, and conformational changes on an epic scale. In addition to its significance for virology, capsid-related research has implications for biology in general, relating to the still largely obscure assembly processes of macromolecular complexes that perform many important cellular functions.

Antigens, Viral↗
PubMed2006

Irreversible growth model for virus capsid assembly.

We model the spontaneous assembly of a capsid (a virus' closed outer shell) from many copies of identical units, using entirely irreversible steps and only information local to the growing edge. Our model is formulated in terms of (i) an elastic Hamiltonian with stretching and bending stiffness and a spontaneous curvature, and (ii) a set of rate constants for the addition of new units or bonds. An ensemble of highly irregular capsids is generated, unlike the well-known icosahedrally symmetric viruses, but (we argue) plausible as a way to model the irregular capsids of retroviruses such as HIV. We found that (i) the probability of successful capsid completion decays exponentially with capsid size; (ii) capsid size depends strongly on spontaneous curvature and weakly on the ratio of the bending and stretching elastic stiffnesses of the shell; (iii) the degree of localization of Gaussian curvature (a measure of facetedness) depends heavily on the ratio of elastic stiffnesses.

Capsid↗
PubMed2006

Mathematical virology: a novel approach to the structure and assembly of viruses.

Understanding the structure and life cycle of viruses is a fascinating challenge with a crucial impact on the public health sector. In the early 1960s, Caspar & Klug (Caspar & Klug 1962 Cold Spring Harbor Symp. Quant. Biol. 27, 1-24) established a theory for the prediction of the surface structures of the protein shells, called viral capsids, which encapsulate and hence provide protection for the viral genome. It is of fundamental importance in virology, with a broad spectrum of applications ranging from the image analysis and classification of experimental data to the construction of assembly models. However, experimental results have provided evidence for the fact that it is incomplete and, in particular, cannot account for the structures of Papovaviridae, which are of particular interest because they contain cancer-causing viruses. This gap has recently been closed by the viral tiling theory, which describes the locations of the protein subunits and inter-subunit bonds in viral capsids based on mathematical tools from the area of quasicrystals. The predictions and various recent applications of the new theory are presented, and it is discussed how further research along these lines may lead to new insights in virology and the design of anti-viral therapeutics.

Capsid↗
PubMed1980

Scaffolding proteins and the genetic control of virus shell assembly.

Historically a gap has existed between the study of the one-dimensional organization of hereditary information in genes, and of the three-dimensional organization of macromolecules in biological structures. In this article we describe progress in closing this gap through the genetic and biochemical analysis of the assembly of the icosahedral shells of spherical viruses, a class of subcellular structures whose subunit organization is relatively well understood. The genes specifying the proteins required for capsid assembly have been identified for many bacterial viruses. By using mutants defective in these genes, it has been possible to identify intermediates in shell morphogenesis and DNA condensation, and to unravel the different levels of the genetic control of macromolecular assembly processes. In general, a precursor shell or procapsid is first constructed, and the DNA is subsequently coiled within it. The construction of a closed shell poses as difficult a problem for a virus as for an architect. In the well-studied bacteriophage P22 of Salmonella typhimurium, the construction of the procapsid requires the interaction of about 200 molecules of the gene-8 scaffolding protein with 420 molecules of the gene-5 coat protein, forming a double-shelled structure with the scaffolding protein on the inside. Once completed, procapsids undergo substantial alteration in the course of encapsulating the viral DNA. In P22, the initiation of DNA packaging triggers the exit of all of the scaffolding molecules from within the capsid, probably through the coat-protein lattice. These released molecules are re-utilized, interacting with newly synthesized coat subunits to form further procapsids. Thus, the scaffolding protein functions catalytically in capsid assembly. All of the well-studied DNA phages require a scaffolding protein species for procapsid assembly, though their properties vary. Purified coat and scaffolding subunits by themselves show little tendency to polymerize, and are stable as monomers in solution. Upon mixing together under the appropriate conditions, however, the proteins copolymerize into double shells. Their interaction with each other appears to be critical for efficient assembly; this interaction probably occurs on the edges of growing shells, and not among subunits in solution. We have termed this kind of process, which we previously described in T4 tail morphogenesis, self-regulated assembly. The subunits are synthesized in a nonreactive form and are activated, not in solution, but upon incorporation into the growing substrate structure. A number of further transformations of the capsid subunits occur only within the organized structure and not as free subunits. Thus, aspects of the genetic information controlling the assembly process are not fully expressed at the level of the properties of protein subunits, but become manifest only through interactions with other proteins, or at a higher level, after completion of the correct organized structure.

Capsid↗
PubMed2004

Nuclear translocation of papillomavirus minor capsid protein L2 requires Hsc70.

Minor capsid protein L2 of papillomaviruses plays an essential role in virus assembly by recruiting viral components to PML bodies, the proposed sites of virus morphogenesis. We demonstrate here that the function of L2 in virus assembly requires the chaperone Hsc70. Hsc70 was found dispersed in naturally infected keratinocytes and cultured cells. A dramatic relocation of Hsc70 from the cytoplasm to PML bodies was induced in these cells by L2 expression. Hsc70-L2 complex formation was confirmed by coimmunoprecipitation. The complex was modulated by the cochaperones Hip and Bag-1, which stabilize and destabilize Hsc70-substrate complexes, respectively. Cytoplasmic depletion of Hsc70 caused retention of wild-type and N-terminally truncated L2, but not of C-terminally truncated L2, in the cytoplasm. This retention was partially reversed by overexpression of Hsc70 fused to green fluorescent protein but not by ATPase-negative Hsc70. Hsc70 associated with L1-L2 virus-like particles (VLPs) but not with VLPs composed either of L1 alone or of L1 and C-terminally truncated L2. Moreover, displacement of Hsc70 from L1-L2 VLPs by encapsidation of DNA, generating pseudovirions, was found. These data indicate that Hsc70 transiently associates with viral capsids during the integration of L2, possibly via the L2 C terminus. Completion of virus assembly results in displacement of Hsc70 from virions.

Active Transport, Cell Nucleus↗
PubMed1996

Influenza virus hemagglutinin and neuraminidase glycoproteins stimulate the membrane association of the matrix protein.

We have analyzed the mechanism by which the matrix (M1) protein associates with cellular membranes during influenza A virus assembly. Interaction of the M1 protein with the viral hemagglutinin (HA) or neuraminidase (NA) glycoprotein was extensively analyzed by using wild-type and transfectant influenza viruses as well as recombinant vaccinia viruses expressing the M1 protein, HA, or NA. Membrane binding of the M1 protein was significantly stimulated at the late stage of virus infection. Using recombinant vaccinia viruses, we found that a relatively small fraction (20 to 40%) of the cytoplasmic M1 protein associated with cellular membranes in the absence of other viral proteins, while coexpression of the HA and the NA stimulated membrane binding of the M1 protein. The stimulatory effect of the NA (>90%) was significant and higher than that of the HA (>60%). Introduction of mutations into the cytoplasmic tail of the NA interfered with its stimulatory effect. Meanwhile, the HA may complement the defective NA and facilitate virus assembly in cells infected with the NA/TAIL(-) transfectant. In conclusion, the highly conserved cytoplasmic tails of the HA and NA play an important role in virus assembly.

Hemagglutinins, Viral↗
PubMed1976

Assembly of tobacco mosaic virus.

The assembly of tobacco mosaic virus requires the presence of a particular protein aggregate, the disk. During the nucleation, a specific region of the RNA interacts with a single disk, to bring about a necessarily cooperative transition from the paired two-layer structure to a short segment of nucleo-protein helix. There is a high selectivity for this region of the TMV RNA, because of the many nucleotides bound at once, and other nucleotide sequences appear only to bind by a different mechanism. Elongation of the nucleated rods can continue with either further disks or the less aggregated 'A-protein' as the protein source, but the continued cooperativity inherent with disks would have some advantages. The rates of the two processes have been separately determined and growth is faster when disks are still present. New experiments show that the breakdown of disks to yield A-protein is relatively slow and it is concluded that virus growth from disks could not proceed through a prior breakdown in solution, but must involve the direct interaction of the disk with the growing nucleoprotein rod. The detailed mechanism of disk addition is not understood but it may involve a directed breakdown, since there is also evidence for the existence of a non-equilibrium form of A-protein which has aggregation kinetics distinct from those of equilibrium A-protein. Some implications for the general assembly pathways of viruses both of the specificity and of the assembly/disassembly cycle during the viral infection are considered.

Kinetics↗
PubMed2002

Rafts promote assembly and atypical targeting of a nonenveloped virus, rotavirus, in Caco-2 cells.

Rotavirus follows an atypical pathway to the apical membrane of intestinal cells that bypasses the Golgi. The involvement of rafts in this process was explored here. VP4 is the most peripheral protein of the triple-layered structure of this nonenveloped virus. High proportions of VP4 associated with rafts within the cell as early as 3 h postinfection. In the meantime a significant part of VP4 was targeted to the Triton X-100-resistant microdomains of the apical membrane, suggesting that this protein possesses an autonomous signal for its targeting. At a later stage the other structural rotavirus proteins were also found in rafts within the cells together with NSP4, a nonstructural protein required for the final stage of virus assembly. Rafts purified from infected cells were shown to contain infectious particles. Finally purified VP4 and mature virus were shown to interact with cholesterol- and sphingolipid-enriched model lipid membranes that changed their phase preference from inverted hexagonal to lamellar structures. Together these results indicate that a direct interaction of VP4 with rafts promotes assembly and atypical targeting of rotavirus in intestinal cells.

Caco-2 Cells↗
PubMed2006

Foamy virus capsid assembly occurs at a pericentriolar region through a cytoplasmic targeting/retention signal in Gag.

Foamy viruses (FV) are unusual retroviruses that differ in many aspects of their life cycle from the orthoretroviruses such as human immunodeficiency virus. Similar to Mason-Pfizer monkey virus (MPMV), FV assemble into capsids intracellularly. The capsids are then transported to a cellular membrane for acquisition of envelope (Env) glycoproteins and budding. However, unlike MPMV, budding of FV is dependent upon the presence of Env. Previous work suggested that FV Env proteins are localized to the endoplasmic reticulum (ER) where budding takes place. However, very little was known about the details of FV assembly. We have used immunofluorescence and electron microscopy to visualize the intracellular location of FV assembly and budding. We have found that, as in the case of MPMV, FV capsids assemble at a pericentriolar site in the cytoplasm. Surprisingly, FV Env is mostly absent from this site and, contrary to expectations, FV capsid structural protein (Gag) is absent from the ER. Gag and Env only co-localize at the trans-Golgi network, suggesting that Env-Gag interactions that are required for viral egress from the cell, occurs at this site. Finally, inhibitor studies suggest an important role of microtubule networks for foamy viral assembly and budding.

Capsid↗
PubMedDate not supplied

Effects of IFN alpha on late stages of HIV-1 replication cycle.

IFN alpha causes a modest reduction of HIV-1 expression in chronically infected monocytoid U937 cells. However, the ratio between cell-associated and shed viral p24 antigen is altered, being the cell-associated fraction dose-dependently enhanced by IFN. Furthermore, a significant decrease of infectivity of both cell-associated and shed material is observed. Transmission electron microscopy of IFN-treated cells revealed virus assembly being strongly inhibited, with the production of morphologically altered (tear-drop shaped) virus particles. Proteolytic processing of gag proteins appeared to be normal in IFN-treated cultures. However, virions shed from IFN-treated cells showed a markedly reduced incorporation of virus-specific gp120 and cell-derived ICAM-1 by the virus envelope. Additionally, these particles showed a significantly decreased ability to become bound to CD4+ target cells, accounting for, at least in part, the observed decrease of infectivity. Taken together, the data suggest that, in chronically infected cells, IFN alpha can affect late stages of HIV-1 replication, by inhibiting virus assembly and release, and by reducing the infectivity of shed virions. The latter effect seems to be due, at least in part, to altered incorporation of surface glycoproteins and defective particle formation. The relationship between impaired gp120 incorporation and altered morphogenesis of HIV-1 virions is under investigation.

Antiviral Agents↗
PubMed2004

Hepatitis B virus capsid assembly is enhanced by naturally occurring mutation F97L.

In chronic hepatitis B virus (HBV) infections, one of the most common mutations to the virus occurs at amino acid 97 of the core protein, where leucine replaces either phenylalanine or isoleucine, depending on strain. This mutation correlates with changes in viral nucleic acid metabolism and/or secretion. We hypothesize that this phenotype is due in part to altered core assembly, a process required for DNA synthesis. We examined in vitro assembly of empty HBV capsids from wild-type and F97L core protein assembly domains. The mutation enhanced both the rate and extent of assembly relative to those for the wild-type protein. The difference between the two proteins was most obvious in the temperature dependence of assembly, which was dramatically stronger for the mutant protein, indicating a much more positive enthalpy. Since the structures of the mutant and wild-type capsids are essentially the same and the mutation is not involved in the contact between dimers, we suggest that the F97L mutation affects the dynamic behavior of dimer and capsid.

Amino Acid Substitution↗
PubMed2006

Core protein cleavage by signal peptide peptidase is required for hepatitis C virus-like particle assembly.

Hepatitis C virus (HCV) core protein, expressed with a Semliki Forest virus replicon, self-assembles into HCV-like particles (HCV-LP) at the endoplasmic reticulum (ER) membrane, providing an opportunity to study HCV assembly and morphogenesis by electron microscopy. This model was used to investigate whether the processing of the HCV core protein by the signal peptide peptidase (SPP) is required for the HCV-LP assembly. Several mutants were designed as there are conflicting reports concerning the cleavage of mutant proteins by SPP. Production of the only core mutant protein that escaped SPP processing led to the formation of multiple layers of electron-dense ER membrane, with no evidence of HCV-LP assembly. These data shed light on the HCV core residues involved in SPP cleavage and suggest that this cleavage is essential for HCV assembly.

Animals↗
PubMed1995

Assembly of vaccinia virus: incorporation of p14 and p32 into the membrane of the intracellular mature virus.

The cytoplasmic assembly of vaccinia virus begins with the transformation of a two-membraned cisterna derived from the intermediate compartment between the endoplasmic reticulum and the Golgi complex. This cisterna develops into a viral crescent which eventually forms a spherical immature virus (IV) that matures into the intracellular mature virus (IMV). Using immunoelectron microscopy, we determined the subcellular localization of p32 and p14, two membrane-associated proteins of vaccinia virus. p32 was associated with vaccinia virus membranes at all stages of virion assembly, starting with the viral crescents, as well as with the membranes which accumulated during the inhibition of assembly by rifampin. There was also low but significant labelling of membranes of some cellular compartments, especially those in the vicinity of the Golgi complex. In contrast, anti-p14 labelled neither the crescents nor the IV but gave strong labelling of an intermediate form between IV and IMV and was then associated with all later viral forms. This protein was also not significantly detected on identifiable cellular membranes. Both p32 and p14 were abundantly expressed on the surface of intact IMV. Our data are consistent with a model whereby p32 would become inserted into cellular membranes before being incorporated into the crescents whereas p14 would be posttranslationally associated with the viral outer membrane at a specific later stage of the viral life cycle.

Animals↗
PubMed1984

Coordinated two-disk nucleation, growth and properties, of virus-like particles assembled from tobacco-mosaic-virus capsid protein with poly(A) or oligo(A) of different length.

Assembly of nucleoprotein rods from tobacco mosaic virus (TMV) coat protein and poly(A) depends on the presence of 20S disks in a manner very similar to nucleation and growth of virions in reconstitution with TMV RNA. Products assembled with (A) approximately equal to 5000 appear to have the same buoyant density in CsCl, the same nucleotide/protein ratio and the same nuclease stability, as reconstituted and native TMV. Their rate of formation is very similar to the rate of reconstitution with TMV RNA when high-molecular-mass (A) approximately equal to 5000 is used, but becomes a function of chain length particularly with (A) less than or equal to 185. The composition of assembly products can be described sufficiently with the relation between number of capsid polypeptide monomers/particle, np, to the number of nucleotide residues/chain, nnt, of np = 1/3 (nnt + 50) with two important restrictions: (1) particles of less than four turns of helically arranged capsid subunits are unstable, and (2) particles with about 150 or less nucleotides per chain deviate in structure from mature virus and virus-like (= longer) assembly products. This is indicated by changes in both buoyant density in CsCl and optical properties, while 'dislocation' of the disk to the helical arrangement of capsid subunits ('helicalization') and nuclease stability already become established with chains as short as (A) approximately equal to 58 +/- 20. Consequently, we suggest that assembly proceeds through three distinct phases: (1) nucleation (resulting in helicalization) by interaction of nucleic acid with the first disk; (2) stabilization of the primary (unstable!) nucleation complex by addition of a second disk and formation of a four-turn virus-like and stable nucleoprotein helix, which is then fit for (3) elongation by addition of further disks. The question of what makes the TMV protein disk select specifically TMV RNA during virion assembly is discussed in some detail.

Adenine Nucleotides↗
PubMed1995

A molecular determinant of human immunodeficiency virus particle assembly located in matrix antigen p17.

We report single-point mutations that are located in the matrix protein domain of the gag gene of human immunodeficiency virus type 1 and that prevent Gag particle formation. We show that mutations of p17 that abolish human immunodeficiency virus particle assembly also prevent the dimerization of p17 protein, as measured directly by a protein-protein binding assay. In the three-dimensional structure of p17, mutations that abolish dimerization are located in a single alpha helix that forms part of a fingerlike projection from one side of the molecule. Peptides derived from this region of p17 also reduce the level of p17 dimer when they are added to p17-expressing cells and compete for p17 self-association when present in protein-protein binding assays. We propose that the dimerization of the Gag precursor that occurs by the interdigitation of alpha helices on adjacent matrix molecules is a key stage in virion assembly and that the prevention of such an interaction is the molecular basis of particle misassembly.

Base Sequence↗
PubMed2005

Human cytomegalovirus tegument protein ppUL35 is important for viral replication and particle formation.

The tegument proteins ppUL35 and ppUL82 (pp71) of human cytomegalovirus (HCMV) physically interact and cooperatively activate the major immediate-early transcription. While an HCMV mutant lacking UL82 displayed a multiplicity of infection (MOI)-dependent growth, the biological significance of ppUL35 has not been addressed so far. We generated a mutant virus with a deletion of the UL35 gene. Using an MOI of 0.1, the progeny virus yield of this mutant was reduced by a factor of 1,000; however, when infected at a low MOI (0.01), the gene was essential. Characterization of the replication cycle showed that the mutant virus had two defects: when virus inoculum was standardized by the amount of viral DNA, a reduced immediate-early gene expression was observed, leading to a strongly delayed expression of lytic genes. A second defect was apparent in the virus assembly, as fewer enveloped particles and no dense bodies were present in cells infected with the mutant virus. However, the particles produced by wild-type and mutant viruses did not show significant ultrastructural differences. These results suggest an important role for ppUL35 in immediate-early gene expression and virus assembly.

Base Sequence↗