Structure of the dodecahedral penton particle from human adenovirus type 3.

The sub-viral dodecahedral particle of human adenovirus type 3, composed of the viral penton base and fiber proteins, shares an important characteristic of the entire virus: it can attach to cells and penetrate them. Structure determination of the fiberless dodecahedron by cryo-electron microscopy to 9 Angstroms resolution reveals tightly bound pentamer subunits, with only minimal interfaces between penton bases stabilizing the fragile dodecahedron. The internal cavity of the dodecahedron is approximately 80 Angstroms in diameter, and the interior surface is accessible to solvent through perforations of approximately 20 Angstroms diameter between the pentamer towers. We observe weak density beneath pentamers that we attribute to a penton base peptide including residues 38-48. The intact amino-terminal domain appears to interfere with pentamer-pentamer interactions and its absence by mutation or proteolysis is essential for dodecamer assembly. Differences between the 9 Angstroms dodecahedron structure and the adenovirus serotype 2 (Ad2) crystallographic model correlate closely with differences in sequence. The 3D structure of the dodecahedron including fibers at 16 Angstroms resolution reveals extra density on the top of the penton base that can be attributed to the fiber N terminus. The fiber itself exhibits striations that correlate with features of the atomic structure of the partial Ad2 fiber and that represent a repeat motif present in the amino acid sequence. These new observations offer important insights into particle assembly and stability, as well as the practicality of using the dodecahedron in targeted drug delivery. The structural work provides a sound basis for manipulating the properties of this particle and thereby enhancing its value for such therapeutic use.

Functional determinants of the Epstein-Barr virus protease.

Herpesvirus proteases are essential for the production of progeny virus. They cleave the assembly protein that fills the immature capsid in order to make place for the viral DNA. The recombinant protease of the human gamma-herpesvirus Epstein-Barr virus (EBV) was expressed in Escherichia coli and purified. Circular dichroism indicated that the protein was properly folded with a secondary structure content similar to that of other herpesvirus proteases. Gel filtration and sedimentation analysis indicated a fast monomer-dimer equilibrium of the protease with a K(d) of about 60 microM. This value was not influenced by glycerol but was lowered to 1.7 microM in the presence of 0.5 M sodium citrate. We also developed an HPLC-based enzymatic assay using a 20 amino acid residue synthetic peptide substrate derived from one of the viral target sequences for the protease. We found that conditions that stabilised the dimer also led to a higher enzymatic activity. Through sequential deletion of amino acid residues from either side of the cleavage site, the minimal peptide substrate for the protease was determined as P5-P2'. This minimal sequence is shorter than that for other herpesvirus proteases. The implications of our findings are discussed with reference to the viral life-cycle. These results are the first ever published on the EBV protease and represent a first step towards the development of protease inhibitors.

Characterization of a novel complex from halophilic archaebacteria, which displays chaperone-like activities in vitro.

We isolated a protein, P45, from the extreme halophilic archaeon Haloarcula marismortui, which displays molecular chaperone activities in vitro. P45 is a weak ATPase that assembles into a large ring-shaped oligomeric complex comprising about 10 subunits. The protein shows no significant homology to any known protein. P45 forms complexes with halophilic malate dehydrogenase during its salt-dependent denaturation/renaturation and decreases the rate of deactivation of the enzyme in an ATP-dependent manner. Compared with other halophilic proteins, the P45 complex appears to be much less dependent on salt for its various activities or stability. In vivo experiments showed that P45 accumulates when cells are exposed to a low salt environment. We suggest, therefore, that P45 could protect halophilic proteins against denaturation under conditions of cellular hyposaline stress.

Membrane association induces a conformational change in the Ebola virus matrix protein.

The matrix protein VP40 from Ebola virus is targeted to the plasma membrane, where it is thought to induce assembly and budding of virions through its association with the lipid bilayer. Ebola virus VP40 is expressed as a monomeric molecule in solution, consisting of two loosely associated domains. Here we show that a C-terminal truncation of seven residues destabilizes the monomeric closed conformation and induces spontaneous hexamerization in solution, as indicated by chemical cross-linking and electron microscopy. Three-dimensional reconstruction of electron microscopy images shows ring-like structures consisting of the N-terminal domain along with evidence for flexibly attached C-terminal domains. In vitro destabilization of the monomer by urea treatment results in similar hexameric molecules in solution. In addition, we demonstrate that membrane association of wild-type VP40 also induces the conformational switch from monomeric to hexameric molecules that may form the building blocks for initiation of virus assembly and budding. Such a conformational change induced by bilayer targeting may be a common feature of many viral matrix proteins and its potential inhibition may result in new anti-viral therapies.

Adenovirus type 7 penton purification of soluble pentamers from Escherichia coli and development of an integrin-dependent gene delivery system.

Adenoviral gene therapy vectors suffer from the disadvantages of toxicity and immunogenicity associated with the expression of adenoviral genes from the vector backbone. We report here an alternative strategy for gene delivery that utilizes a single component of the adenoviral type 7 capsid, the penton base (Ad7PB). The Ad7PB gene was sequenced and its amino-acid composition was deduced from its nucleotide sequence. The penton was expressed in Escherichia coli as a soluble C-terminal fusion with glutathione S-transferase (GST-Ad7PB) and was purified by single-step affinity chromatography. Both GST-Ad7PB and cleaved (GST-free) Ad7PB retained the ability to fold into pentamers as observed by electron microscopy. GST-Ad7PB was able to bind a synthetic peptide (FK20) derived from the Ad type 7 fiber and retard DNA through a polylysine chain present at the C-terminus of this linker peptide. GST-Ad7PB was an effective cell transfecting agent when assayed on 293 cells. Transfection was not dependent upon the presence of lysosomotropic agents indicating efficient endosome escape capability. Excess of an RGD-containing peptide derived from Ad7PB was able to inhibit transfection indicating specific integrin-mediated uptake of the GST-Ad7PB-FK20-DNA complexes. We propose that Ad7 pentons can be developed into integrin-specific gene delivery agents.

Structural characterization and membrane binding properties of the matrix protein VP40 of Ebola virus.

The matrix protein VP40 of Ebola virus is believed to play a central role in viral assembly as it targets the plasma membrane of infected cells and subsequently forms a tightly packed layer on the inner side of the viral envelope. Expression of VP40 in Escherichia coli and subsequent proteolysis yielded two structural variants differing by a C-terminal truncation 114 amino acid residues long. As indicated by chemical cross-linking studies and electron microscopy, the larger polypeptide was present in a monomeric form, whereas the truncated one formed hexamers. When analyzed for their in vitro binding properties, both constructs showed that only monomeric VP40 efficiently associated with membranes containing negatively charged lipids. Membrane association of truncated, hexameric VP40 was inefficient, indicating a membrane-recognition role for the C-terminal part. Based on these observations we propose that assembly of Ebola virus involves the formation of VP40 hexamers that is mediated by the N-terminal part of the polypeptide.

A peptide from the adenovirus fiber shaft forms amyloid-type fibrils.

The fiber protein of adenovirus consists of a C-terminal globular head, a shaft and a short N-terminal tail. The crystal structure of a stable domain comprising the head plus a part of the shaft of human adenovirus type 2 fiber has recently been solved at 2.4 A resolution [van Raaij et al. (1999) Nature 401, 935-938]. A peptide corresponding to the portion of the shaft immediately adjacent to the head (residues 355-396) has been synthesized chemically. The peptide failed to assemble correctly and instead formed amyloid-type fibrils as assessed by electron microscopy, Congo red binding and X-ray diffraction. Peptides corresponding to the fiber shaft could provide a model system to study mechanisms of amyloid fibril formation.

Membrane interaction of influenza virus M1 protein.

The M1 protein of influenza virus is thought to make contact with the cytoplasmic tails of the glycoprotein spikes, lipid molecules in the viral membrane, and the internal ribonucleoprotein particles. Here we show electron micrographs of negatively stained virus particles in which M1 is visualized as a 60-A-long rod that touches the membrane but apparently is not membrane inserted. Photolabeling with a membrane restricted reagent resulted in labeling of the transmembrane region of haemagglutinin but not of M1, also suggesting that most of M1 is not embedded into the hydrophobic core of the viral membrane. Finally, in vitro reconstitution experiments using soluble M1 protein and synthetic liposomes or Madin-Darby canine kidney cell membranes suggest that M1 can bind to negatively charged liposomes and to the cellular membranes and that this binding can be prevented under high-salt conditions. Although none of these experiments prove that there does not exist a minor fraction of M1 that is membrane inserted, it appears that most of M1 in the virus is membrane associated through electrostatic interactions.

On the domain structure and the polymerization state of the sendai virus P protein.

The phosphoproteins (P) of paramyxoviruses and rhabdoviruses are cofactors of the viral polymerase (L) and chaperones of soluble nucleoprotein preventing its polymerization and nonspecific binding to cellular RNA. The primary sequences of six paramyxovirus P proteins were compared, and although there was virtually no sequence similarity, there were two regions with similar secondary structure predictions in the C-terminal part of P: the predicted multimerization domain and the X-protein, the sequence that binds to N in the N:RNA template. The C-terminal part of the Sendai virus P protein, the multimerization domain including the binding site for the polymerase, and the X-protein were expressed in Escherichia coli. All three polypeptides folded with secondary structures similar to those predicted. The C-terminal part of P is a very elongated molecule with most of its length encompassing the multimerization domain. Both the multimerization domain and the C-terminal part of P were found to form tetramers, whereas the X-protein was monomeric.

Unfolding studies of human adenovirus type 2 fibre trimers. Evidence for a stable domain.

Adenovirus fibres are trimeric proteins that protrude from the 12 fivefold vertices of the virion and are the cell attachment organelle of the virus. They consist of three segments: an N-terminal tail, which is noncovalently attached to the penton base, a thin shaft carrying 15 amino acid pseudo repeats, and a C-terminal globular head (or knob) which recognizes the primary cell receptor. Due to their exceptional stability, which allows easy distinction of native trimers from unfolded forms and folding intermediates, adenovirus fibres are a very good model system for studying folding in vivo and in vitro. To understand the folding and stability of the trimeric fibres, the unfolding pathway of adenovirus 2 fibres induced by SDS and temperature has been investigated. Unfolding starts from the N-terminus and a stable intermediate accumulates that has the C-terminal head and part of the shaft structure (shown by electron microscopy). The unfolded part can be digested away using limited proteolysis, and the precise digestion sites have been determined. The remaining structured fragment is recognized by monoclonal antibodies that are specific for the trimeric globular head and therefore retains a native trimeric structure. Taken together, our results indicate that adenovirus fibres carry a stable C-terminal domain, consisting of the knob with five shaft-repeats.

Rabies virus-induced membrane fusion.

Rabies virus is a member of the rhabdovirus family. It enters cells by a process of receptor mediated endocytosis. Following this step, the viral envelope fuses with the endosomal membrane to allow release of the viral nucleocapsid into the cytoplasm. Fusion is induced by the low pH of the endosomal compartment and is mediated by the single viral glycoprotein G, a homotrimeric integral membrane protein. Rabies virus fusion properties are related to different conformational states of G. By different biochemical and biophysical approaches, it has been demonstrated that G can assume at least three different states: the native (N) state detected at the viral surface above pH 7, the activated (A) hydrophobic state which interacts with the target membrane as a first step of the fusion process, and the fusion inactive (I) conformation. Differently from other fusogenic viruses for which low pH-induced conformational changes are irreversible, there is a pH dependent equilibrium between these states, the equilibrium being shifted toward the I-state at low pH. The objective of this review is to detail recent findings on rhabdovirus-induced membrane fusion and to underline the differences that exist between this viral family and influenza virus which is the best known fusogenic virus. These differences have to be taken into consideration if one wants to have a global understanding of virus-induced membrane fusion.

Human herpesvirus 8 and Epstein Barr-virus in a cutaneous B-cell lymphoma and a malignant cell line established from the blood of an AIDS patient.

Human Herpesvirus 8 (HHV-8) has been consistently associated with Primary Effusion Lymphoma (PEL or body-cavity-based lymphoma) but not with other lymphomas. This paper reports on an AIDS patient without obvious malignant effusion in body cavities but with a cutaneous lymphoma where HHV-8 and Epstein-Barr virus (EBV) were detected by PCR and electron microscopy. Both viruses were also detected in all the cells of a malignant cell line (BBG1) established from the patient's peripheral blood mononuclear cells. As in PEL and PEL-derived cell lines, both the tumor and the lines lacked B-antigen expression in immunological studies but were of the same B origin as shown by clonal immunoglobulin gene rearrangements. In contrast to other co-infected cell lines, BBG1 and subclones spontaneously expressed the HHV-8 lytic antigens p40, p27, p60 and the EBV transforming latent antigen EBNA2. These data suggest that the clinical and biological features of HHV-8-and EBV-associated lymphomas could be wider than has been described to date in PEL particularly with the in vivo presence of circulating malignant dually-infected cells engaged in a spontaneous HHV-8 lytic infection.

Variation in ATP requirement during influenza virus transcription.

The ATP requirement of influenza A virus RNA-dependent RNA polymerase was studied during in vitro transcription reactions. In complete transcription reactions, the Km for ATP was 10-fold higher than the Km values for the other NTPs. However, during transcription elongation the Km for ATP was as low as the Km values for the other NTPs, suggesting a special requirement for ATP during transcription initiation. Gel analysis of RNA products of transcription initiation reactions showed that the incorporation of AMP into nascent RNA was more efficient at positions 4, 6 and 7 relative to the template RNA than at position 5. The polymerase produced short, abortive transcripts with lengths corresponding to positions 3 and 4 relative to the template but never to position 5 or longer. These results suggest that incorporation of AMP at position 5 induces the influenza A virus polymerase to go through a transition from a transcription initiation to an elongation complex. This functional change of the polymerase complex rather than a requirement for ATP beta-gamma bond hydrolysis is the most likely reason for the particularly high Km for ATP during the early phase of transcription. This conclusion is supported by the fact that the ATP analogue ATPgammaS [adenosine 5'-O-(3-thiotriphosphate)] can efficiently replace ATP in in vitro transcription reactions and shows a comparable drop of Km between transcription initiation and elongation.

Adenovirus dodecahedron, a new vector for human gene transfer.

Recombinant adenovirus is one of most efficient delivery vehicles for gene therapy. However, the initial enthusiasm for the use of recombinant adenovirus for gene therapy has been tempered by strong immune responses that develop to the virus and virus-infected cells. Even though recombinant adenoviruses are replication-defective, they introduce into the recipient cell, together with the gene of interest, viral genetes that might lead to fortuitous recombination if the recipient is infected by wild-type adenovirus. We propose the use of a dodecahedron made of adenovirus pentons or penton bases as an alternative vector for human gene therapy. The penton is a complex of two oligomeric proteins, a penton base and fiber, involved in the cell attachment, internalization, and liberation of virus into the cytoplasm. The dodecahedron retains many of the advantages of adenovirus for gene transfer such as efficiency of entry, efficient release of DNA from endosomes, and wide range of cell and tissue targets. Because it consists of only one or two adenovirus proteins instead of the 11 contained in an adenovirus virion and it does not contain the viral genome, it is potentially a safer alternative to recombinant adenovirus.

Identification of amino acids controlling the low-pH-induced conformational change of rabies virus glycoprotein.

The glycoprotein (G) of rabies virus assumes at least three different conformations: the native state detected at the viral surface above pH 7, the activated state involved in the first step of the fusion process, and the fusion-inactive conformation (I). A new category of monoclonal antibodies (MAbs) which recognized specifically the I conformation at the viral surface has recently been described. These MAbs (17A4 and 29EC2) became neutralizing when the virus was preincubated at acidic pH to induce the conformational change toward the I state of G. Mutants escaping neutralization were then selected. In this study, we have investigated the fusion and the low-pH-induced fusion inactivation properties of these mutants. All of these mutants have fusion properties similar to those of the CVS parental strain, but five mutants (E282K, M44I, M44V, V392G, and M396T) were considerably slowed in their conformational change leading to the I state. These mutants allow us to define regions that control this conformational change. These results also reinforce the idea that structural transition toward the I state is irrelevant to the fusion process. Other mutations in amino acids 10, 13, and 15 are probably located in the epitopes of selecting MAbs. Furthermore, in electron microscopy, we observed a hexagonal lattice of glycoproteins at the viral surface of mutants M44I and V392G as well as strong cooperativity in the conformational change toward the I state. This finding demonstrates the existence of lateral interactions between the spikes of a rhabdovirus.

The avian adenovirus penton: two fibres and one base.

The penton capsomer of mammalian adenoviruses consists of a trimeric, long and thin fibre inserted into a pentameric base. The avian adenoviruses possess a penton which presents another symmetry mismatch: each pentameric base is associated with two fibres. Here we have studied the morphology of the penton of CELO virus, an avian adenovirus, and we have determined the sequence of both fibres, one long and one short. The short fibre is probably associated with the base in the same way as the mammalian viral fibres and we will discuss how the long fibre could be attached. The shafts of all known adenovirus fibres consist of a series of 15-residue repeats. The avian virus fibres show a more complicated and less regular shaft repeat structure with single, double and triple repeats. The sequences of the receptor binding (head) domains of both fibres are very different from all other known fibre head domains and very different from each other, suggesting that the two fibres might bind to different receptors. The genome organization of the sequenced region is rather different from that in human adenoviruses. In particular, a region homologous to the human virus E3 region was not found at the position where it normally occurs in the human virus genome.

Biological function of the low-pH, fusion-inactive conformation of rabies virus glycoprotein (G): G is transported in a fusion-inactive state-like conformation.

The glycoprotein (G) of rabies virus can assume at least three different conformations: the native (N) state detected at the viral surface above pH 7; the activated (A) hydrophobic state, which is probably involved in the first steps of the fusion process; and the fusion-inactive (I) conformation. There is a pH-dependent equilibrium between these states, the equilibrium being shifted towards the I state at low pH. It has been supposed that the transition from the N to the I state mediates membrane fusion. By using a lipid-mixing assay, we studied the kinetics of fusion and fusion inactivation for two rabies virus strains, PV and CVS. In addition, by using electron microscopy and a trypsin sensitivity assay, we analyzed the kinetics of the conformational change towards the I state for both strains. Although the PV strain fuses faster, inactivation and the conformational change of PV G occur more slowly than for the CVS strain. This suggests that the structural transition towards the I state is irrelevant to the fusion process. Immunofluorescence and immunoprecipitation experiments performed with infected cells and two different monoclonal antibodies, one specific for the N form of G and one which recognizes both the N and the I states, suggest that G is transported in an I state-like conformation through the Golgi apparatus and acquires its N structure only near or at the cell surface. We propose that the role of the I state is to avoid unspecific fusion during transport of G in the acidic Golgi vesicles.

Photolabeling identifies a putative fusion domain in the envelope glycoprotein of rabies and vesicular stomatitis viruses.

Vesicular stomatitis and rabies viruses enter cells through receptor-mediated endocytosis, followed by fusion of the viral with the endosomal membrane. The latter step is catalyzed by the viral envelope glycoprotein, which, in the low pH environment of the endosome, undergoes a conformational transition to a fusion-competent state. To investigate whether fusion competence involves the low pH exposure of a hydrophobic fusion region(s), we have applied hydrophobic photolabeling using the recently developed phospholipid analogue 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3H- diazirin-3-yl)benzyl]oxy]carbonyl] nonanoyl]-sn-glycero-3-phosphocholine ([125I]TID-PC/16) (Weber, T., and Brunner, J. (1995) J. Am. Chem. Soc. 117, 3084-3095). Rosettes of rabies virus glycoprotein, whole rabies virus, or vesicular stomatitis virus were incubated with large unilamellar vesicles containing [125I]TID-PC/16. Following reagent activation, the labeled glycoprotein was isolated and analyzed. In all cases, labeling of the glycoprotein strongly increased as the pH was lowered from 7.0 to 6.0, suggesting the exposure at acidic pH of a domain capable of interacting with membranes. To identify the labeled region(s), CNBr fragments were generated and analyzed by SDS-polyacrylamide followed by autoradiography. In rabies glycoprotein, the labeled segment was found to be contained within fragment RCr5 (residues 103-179). Glycoprotein from vesicular stomatitis virus was labeled within fragment VCr1 (residues 59-221). These results demonstrate that rhabdovirus glycoprotein contains a domain that at low pH is capable of interacting with a target membrane in a hydrophobic manner. This domain may play a role similar to that of the fusion peptide found in many other viral fusion proteins.

Structure of influenza virus ribonucleoprotein particles. II. Purified RNA-free influenza virus ribonucleoprotein forms structures that are indistinguishable from the intact influenza virus ribonucleoprotein particles.

Influenza virus nucleoprotein (NP) was purified from the virus and found to be virtually free from RNA. The morphology of the negatively stained NP was studied using electron microscopy. The monomer protein was found to be a small rod with dimensions of 62 by 35 A. However, most of the protein was found to exist in polymeric forms ranging from trimers to large structures that were morphologically indistinguishable from the intact viral RNP. Each monomer has two sites for NP-NP contacts at one extremity of the rod. The consequences of this finding for crystallization studies, for in vitro studies on NP-RNA interactions and the possible consequences for in vivo ribonucleoprotein particle assembly are discussed.