- Email: [email protected]
Assembly of macromolecular complexes in bacterial and baculovirus expression systems
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Assembly of macromolecular complexes in bacterial and baculovirus expression systems Polly Roy*t$ and lan Jones*§ Many proteins exist normally as oligomers or complexes with other proteins. Recent advances in vector design have allowed this aspect of protein function to be mimicked in recombinant expression systems. Examples of the ordered oligomerization of a single protein through to the assembly of eight different proteins have been documented in recombinant Escherichia coil and recombinant baculovirus systems.
Addresses *Institute of Virology, Mansfield Road, Oxford OX1 3SR, UK t Department of International Public Health, University of Alabama at Birmingham, 303 Tidwell Hall, 720 20th Street South, UAB Station, Birmingham, Alabama 35294, USA ~-Department of Biophysics, University of Oxford, The Rex Richards Building, South Parks Road, Oxford OX1 3QU, UK; e-mail: [email protected] § e-mail: [email protected],uk Current Opinion in Structural Biology 1996, 6:157-161 © Current Biology Ltd ISSN 0959-440X
Abbreviations BTM bluetongue virus CLP core-bikeparticles FHV flock house virus FMDV foot and mouth disease virus HSV herpes simplex virus MPMV Mason-Pfizer monkey virus TMV tobaccomosaic virus VLP virus-like particle VP virus protein
Introduction
Despite the increasing recognition of the role of specific proteins (chaperones) in the folding and assembly of macromolecular structures [1], it is, nonetheless, generally true that in many cases, complex protein structures will assemble spontaneously provided that the level of interacting proteins is sufficiently high, that all components are present in the correct molar ratios and that, in some cases, a scaffolding (e.g. a membrane or cytoskeletal component) is provided for the process of assembly. With the advent of expression systems that produce high levels of protein, therefore, there has been an increasing number of reports of the assembly of complex protein structures directly following the expression of the components concerned. 'Assembly' may range from the relatively simple oligomerization of higher order structures from a single gene product to the assembly of enzyme complexes or virus-like particles in which several proteins participate. Coupled with improved methods for the visualization of complex structures (e.g. cryoelectron microscopy), the opportunity to study and understand macromolecular assemblies has never been greater. In this review, we
describe two systems that are widely used for protein expression and some examples of the macromolecular assemblies that have been observed following their use. The different expression systems Escherichia coil and recombinant baculoviruses are widely
used for the production of recombinant proteins. Both systems are renowned for the high yield of protein that is possible and, for both systems, vectors have been developed that allow the expression of one or more open reading frames. In what respects do these systems differ? T h e E. coil expression system represents one of the first described systems for the production of a recombinant protein and currently encompasses a plethora of different vectors and methods. In general, vectors are designed to express the desired protein transiently following the addition of an inducer (chemical or heat). T h e sudden burst of protein synthesis that ensues often provides high yields of recombinant protein, but can sometimes lead to the production of inclusion bodies in which the protein is present in a largely denatured and aggregated form. In such cases, the likelihood of the generation of a higher-order assembly of structure is small, although it is still possible (see, for example, [2*]). In many cases, however, the soluble protein is expressed at high levels and oligomerization, or co-assembly with a second (or more) protein(s), can occur. E. coli has no mechanism that enables it to carry out the addition of carbohydrate to proteins and although some secondary modifications can bc engineered to occur (e.g. myristoylation [3]), the level of secondary protein modification that can be achieved in this system is limited. Thus, if the assembly of protein complexes following expression requires a modification that is specific to eukaryotic cells, it may be impossible to achieve without in vitro modification prior to attempted in vitro assembly. In these cases, whilst E. coil expression is undoubtedly enabling, complex formation per se does not occur within the bacterial cell. Expression in recombinant baculoviruses, on the other hand, allows proteins to be modified in the same way as in the majority of higher eukaryotic cells and the yield of protein is often as high or higher than that achievable in E. coli. Proteins are expressed late in the viral life cycle over a two or three day period and, consequently, the frequency of inclusion body formation is low and most expressed proteins are soluble and available to engage in protein-protein interactions. Vectors have been developed to allow the expression of one, two, three or four proteins simultaneously 14"] and, as co-infection is relatively efficient, there is the possibility of expressing up to eight proteins in the same cell [5]. Recently it was
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also shown that the level of misfolded proteins could be minimized by co-expressing proteins with chaperone-like activity (see, for example, [6"1). Accordingly, there are more examples of heterologous macromolecular assembly in the baculovirus expression system than in the E. coil expression system, particularly when the genes expressed are of eukaryotic origin.
Assembly of complex structures in E. coli Despite the limitations of E. co/i expression, a number of examples of complex structure formation have been reported. In common with the baculovirus system outlined below, the expression of virally encoded proteins has given notable results. T h e replication strategy of many viruses has evolved to maximize both the number of progeny virions and the most economical usage of coding capacity. As a consequence, self assembly is a common property of a number of virus capsids and has been observed in an increasing number of viruses since early observations on tobacco mosaic virus (TMV) assembly [7]. T h e expression of genes from bacterial viruses has, in a number of cases, given rise to subviral structures, allowing detailed progress to be made in understanding the assemblages and assembly pathways being made. A few of the well studied cases include phi 29, MS2 and the lambda-like phages. In an analysis of phage HK97, for example, the different intermediates of assembly have been identified through the use of temperature-sensitive mutations that block the assembly process and allow the build up of part-assembled structures [8]. From this analysis, genes deduced to be responsible for the assembly of the phage head structure were subsequently expressed in isolation to confirm that they do, indeed, assemble into head structures [9"]. A similar analysis has been carried out on the Bacillus subtilis phage phi29, where expression of three phage-encoded proteins in E. coil are necessary for the production of active phage proheads [10 °] whilst the expression of only one or two proteins led to only partial or aberrant prohead formation. One of the proteins required is a scaffold protein that is necessary to enable the assembly of the other two, but is not itself a component of the prohead. These data illustrate well how E. coil can supply a suitable background for the assembly of quite complex structures of non-E, coli origin, provided that all the necessary components, including non-structural components, are present in sufficient yield. An analysis of the E.coli phage MS2 coat protein has delineated a role in assembly and also in the specific incorporation of the RNA genome to such a degree that the coat protein-RNA interaction is understood at atomic level [11,12]. Moreover, in a practical application of assembly studies, the MS2 coat protein gene has been engineered to express multiple foreign peptides on the surface of an RNA-free particle in positions that are known to elicit a strong antibody response [13]. Such applications demonstrate the value of dissecting complex
structure formation by recombinant means and suggest the use of viral structures for a variety of biotechnological applications in the future. Virus assembly following expression in E. coil has not been limited to phage systems. The capsids of Mason-Pfizer monkey virus (MPMV; a type D retrovirus) assemble within inclusion bodies after expression in E. co/i, although capsids of the related HIV (a type C retrovirus) do not [2°]. Interestingly, in normal infections, assembly of type D retroviruses occurs in the cytoplasm, whereas that of type C retroviruses occurs at the plasma membrane concomitant with virus budding. It is possible, therefore, that the specific requirement for co-budding at the membrane is the cause of the failure of type C capsids to assemble in E. coll. The TMV coat protein has been shown to assemble in E. coli to form pseudovirions [14] as has the 70S empty capsid of foot and mouth disease virus (FMDV) [15]. For FMDV, many proteins are necessary for the assembly of the virus particle and all are encoded in a large precursor, polyprotein Pl. Rather than express each protein directly, Lewis et al. [15] made only two expression plasmids, one encoding the Pl precursor and the other the 3C protease responsible for Pl cleavage. Upon co-expression, Pl was correctly processed and 70S particles were assembled. As with the phi 29 scaffold protein, therefore (above), it was necessary to provide an enabling protein function that was not itself directly involved in the final structure. The assembly of complex structures is not restricted to those of viral origin. T h e oligomerization domain of the HIV transmembrane protein gp41 assembles in E, co/i [16"] as do a number of multicomponent enzymes derived from prokaryotic [17] or mammalian origin [18].
Assembly of complex structures in recombinant baculovirus infected cells In common with the findings in recombinant E. co/i (above), there are many examples in the literature of the assembly of viruses and subviral particles following expression using recombinant baculoviruses. The assembly of a single viral capsid protein, p55 Gag, into a virus-like particle (VLP) was observed for HIV as early as 1989 [19,20]. Several other retroviral Gag proteins have also been shown to assemble into VLPs following baculovirus expression (e.g. simian immunodeficiency virus [21], feline immunodeficiency virus [221, bovine immunodeficiency virus [23], MPMV [241 and feline leukaemia vires [25]). For HIV in particular, these observations have spawned a detailed analysis of the sequence requirements for VLP formation and the essential domains within the Gag protein have been delineated in each of the subdomains of the molecule: the N-terminal matrix domain [26°,27°], the central capsid domain [28] and the C-terminal nucleocapsid domain [29]. T h e abundant quantities of Gag VLP that are synthesized from this system coupled with the uniform dimensions of the VLP produced have facilitated an examination of higher order Gag structure
Assembly of macromolecular complexes in bacterial and baculovirus expression systems Roy and Jones
by high-resolution electron microscopy and a tentative model of the Gag shell has been suggested [30]. T h e expression of other single viral proteins has also resulted in the assembly of VLPs. For Norwalk virus, the use of baculoviruses to express the single 58 kDa capsid protein resulted in a source of VLPs that proved uniform and suitable for cryoelectron microscopy and permitted an image reconstruction of the vition shell to a resolution of 22/~, [31"]. Similarly, the expression of a single rotavirus gene encoding the virus protein (VP)2 led to the production of a single-shell particle with icosahedral symmetry discernible by electron microscopy analysis [32]. T h e expression of the VP1 protein of polyomavirus led to the production of single-shell structures resembling the authentic virus [33] and expression of the flock house virus (FHV) coat protein resulted in the synthesis of FHV particles that were indistinguishable from the authentic virions [34]. Interestingly, single point mutations in the FHV coat protein led to altered morphologies including half shells and shells with holes, emphasizing the fact that the three-dimensional representation of the VLP is encoded within the single protein expressed and can be separated from the ability to assemble per se. A mutant virion form of FHV was subsequently crystallized and shown to diffract to 3.3 ,~, suggesting that the uniform particles produced by this system are suitable for crystallographic analysis [35]. T h e r e have been numerous examples of the assembly of papillomavirus L1 protein into VLPs (see, for example [36-38]) and, in some cases, these reports also document the incorporation of the second capsid protein VP2. T h e first report of the expression of two proteins that interacted to form a distinct VLP was that describing bluetongue virus (BTV) [39], in which the expression of structural proteins VP3 and VP7 was shown to result in the synthesis of BTV core-like particles (CLP) that were similar to authentic BTV cores. T h e C L P were shown by cryoelectron microscopy to be faithful mimics of authentic BTV cores [40] and other work established that a double-shell structure could be formed by the additional synthesis of the outer capsid proteins VP5 and VP2 [41]. Cryoelectron microscope studies of these particles has shown them to be near exact mimics of the complete BTV virion [42]. In separate studies the co-expression of the an internal BTV protein, VP1, with either the C L P or VLP led to incorporation of VP1 into the virus-like structures [43]. Thus, although VP1 was not necessary for the synthesis of either C L P or VLP, it could be incorporated into the assembling particle if present, suggesting that it will ultimately be possible to reconstruct a variety of BTV assembly intermediates by co-expression of a select number of BTV proteins. Similar to the exploitation of MS2 phage outlined earlier, the BTV CLPs have been engineered to express new epitopes on their surface with a view to novel vaccine
159
applications [44]. CLPs themselves are exceptionally potent vaccines [45], so there is a precedent to suggest that chimeric CLPs will be equally effective. Similar, though less extensive, studies on the assembly of multicomponent viral components have been reported for the hepatitis B virus core and surface antigens [46], for papillomavirus VP1 and VP2 capsid proteins [38] and for rabies virus N and M1 proteins [47]. T h a t more complex viral assemblies are also possible has been demonstrated by the assembly of thc herpes simplex virus (HSV) capsid in insect cells following the expression of six HSV proteins. An outer icosahcdral shell composed of four proteins and an inner scaffolding layer composed of a further two proteins were assembled [48]. Subsequently, the role of the two scaffold proteins encoded by HSV genes UL26 and UL26.5 was further investigated and the proteins were observed to assemble in their own right to produce intermediate capsids. T h e products of both genes are normally processed during assembly, although the role of the processing event was unclear. Capsid assembly from discrete mutated proteins, however, clearly demonstrated that assembly precedes processing [49"]. These studies, and others like them, bode well for the use of these systems to unravel assembly pathways as well as to achieve an understanding of the assembled structure itself. The assembly of poliovirus capsids following the expression of the entire polyprotein in a recombinant baculovirus has been dcscribed [50], suggesting the possibility that this type of system can be extended to viruses where, despite a cloned genome being available, no productive tissue-culture system for the derivation of large numbers of virions for structural studies exists (e.g. hepatitis C virus). As with E. coli expression, although the instances of known complex assembly concern predominantly structures of viral origin, other multicomponent systems have been shown to assemble. When a class I MHC heavy chain was expressed in conjunction with 132-microglobulin, about 10% of the total class 1 chains were in the form of a heterodimer whose assembly was driven by peptide binding to the complex [51] and similar reports have documented the assembly of antibody heavy and light chains following their co-expression [6"]. T h e assembly of the rodent Na+/K+-ATPase has been reported following the expression of ctl and 131 subunits in insect cells to produce a detergent-resistant functional complex [52] and similarly, the subunits of the bovine GABAA receptor assemble to form ion channels with distinctive physiological behavior I53].
Conclusions T h e use of recombinant systems to study multicomponent assembly is a growing area and the expression systems we have outlined here play a key role. Whilst there are clearly limits to the type of interaction that can occur within bacteria, the examples we have described provide ample evidence to suggest a continuing role for E. co/i expression in the analysis of complex structures. An area wherc E. co/i has much to offer is in the combined
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use of multiple expression with bacterial genetics. T h e examples we have highlighted largely concern phage genetics but we see no reason why, given the assembly of a complex with a recognized phenotype, in E. coil, genetic studies should not allow the complete dissection of other assembly processes. Such studies are also possible in recombinant baculoviruses but the advantages of eukaryotic expression are tempered by the slower rate of analysis. Furthermore, baculovirus development should see the construction of 'moving windows' of assembly, representing different stages in a complex assembly process, by the expression of select combinations of proteins. That complex structures can assemble is well demonstrated but the order of many assembly pathways remains unclear. In both systems, there is also the exciting possibility of chimeric complex formation to provide multicomponent structures with new and novel properties of biotransformation or immunogenicity.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: ,, of special interest • e of outstanding interest 1.
Wynn RM, Davie JR, Cox RP, Chuang DT: Molecular chaperones: heat-shock proteins, foldases, and matchmakers. J Lab C/in Med 1994, 124:31-36.
2. •
Klikova M, Rhee SS, Hunter E, Rural T: Efficient in vivo and in vitro assembly of retroviral capsids from Gag precursor proteins expressed in bacteria. J Virol 1995, 69:1093-1098. This paper gives details of the first demonstration of retrovirus Gag assembly in a prokaryote. The particles formed within inclusion bodies but could be isolated by differential centrifugation. They lacked membrane and so were particularly suitable for cryoelectron microscopy. 3.
Duronio RJ, Jackson-Machelski E, Heuckeroth RO, Olins PO, Devine CS, Yonemoto W, Slice LW, Taylor SS, Gordon Jh Protein N-myristoylaflon in Escherichie co//: reconstitution of a eukaryotic protein modification in bacteria. Proc Nat/Acacl Sci USA 1990, 87:1506-1510.
4. •
Belyaev AS, Hails RS, Roy P: High-level expression of five foreign genes by a single recombinant beculovirus. Gene 1995, 156:229-233. This paper describes the construction of a novel baculovirus expression vector with the ability to express four different genes at the same time. When this vector was used to create a recombinant virus which already contained one foreign gene, a virus expressing five proteins in the same cell was isolated. Dual infections would allow 10 proteins to be expressed together, although this was not directly demonstrated in this paper. 5.
Roy P: Orbivirus structure and assembly. Virology 1996, 215:1-11.
Hsu TA, Eiden JJ, Bourgarel P, Meo T, Betenbaugh MJ: Effects of co-expressing chaperone BiP on functional antibody production in the baculovirus system. Protein Expr Purif 1994, 5:595-603. The correct folding of proteins, especially after expression at high level, represents a grey area in the application of expression systems. In this report, chaperonins are co-expressed with antibody heavy chains and shown to be beneficial for the amount of functional protein made. The study suggests that folding pathways may be engineered to maximize the recovery of fully functional product.
A thorough analysis of the requirements for assembly of the prohead of phage HK97. In contrast to the data in [10"], prohead formation occurs with a single gene product in the absence of a scaffold protein. Co-expression of the viral protease led to maturation of the prohead and provides a model for understanding the interaction of the protease with the prohead substrata. 10. Lee CS, Guo P: Sequential interactions of structural proteins in • phage phi 29 procapsid assembly. J Viro11995, 69:5024-5032. An elegant study demonstrating the assembly of the prohead of the Bacillus subtilis phage phi29 in E. colt. The work concludes that correct prohead assembly is inherent as long as all necessary components are present and that neither the gene order of the expression system used nor the molar ratios of protein is important as long as they reach the threshold level for assembly to occur.
1 1.
Valeg&rdK, Murray JB, Stockley PG, Stonehouse NJ, Liljas L: Crystal structure of an RNA bacteriophage coat protein-operator complex. Nature 1994, 371:623-626.
12.
Stockley PG, Stonehouse NJ, Valeg&rd K: Molecular mechanism of RNA phage morphogenesis'/nt J Biochem 1994, 26:1249-1 260.
13.
Mastico RA, Talbot S J, Stockley PG: Multiple presentation of foreign peptides on the surface of an RNA-free spherical bacteriophage capsid. J Gen Viro/1993, 74:541-548.
14.
Hwang DJ, Roberts IM, Wilson TM: Expression of tobacco mosaic virus coat protein and assembly of pseudovirus particles in Escherichia coil Prec Nat/Acad Sci USA 1994, 91:906'7-9071.
15.
Lewis SA, Morgan DO, Grubman MJ: Expression, processing, and assembly of foot-and-mouth disease virus capsld structures in heterologous systems: induction of a neutralizing antibody response in guinea pigs. J Viro11991,65:6572-6580.
16. •
Bernstein HB, Tucker SP, Kar SR, McPherson SA, McPherson DT, Dubay JW, Lebowitz J, Compans RW, Hunter E: OIIgomerlzation of the hydrophobic heptad repeat of gp41. J Viro11995, 69:2745-2750. Eukaryotic glycoproteins are seldom well expressed in E. coil but the use of fragments here demonstrates that inherent properties of self assembly can be visualized in E. coIL The rapid analysis of mutants that is possible in E. colt allows an analysis of the residues involved in the oligomerization. 17.
Lessard IA, Perham RN: Expression in Escherichie colt of genes encoding the E1 alpha and E1 beta subunits of the pyruvate dehydrogenase complex of Bacillus stearothermophilus and assembly of a functional E1 component (alpha 2 beta 2) in vitro. J Biol Chem 1994, 269:10378-10383.
18.
Davie JR, Wynn RM, Cox RP, Chuang DT: Expression and assembly of a functional E1 component (alpha 2 beta 2) of mammalian branched-chain alpha-ketoacid dehydrogenase complex in Escherichie colt. J Biol Chem 1992, 267:16601-16606.
19.
Overton HA, Fujii Y, Price I, Jones IM: The protease and Gag gene products of the human immunodefictency virus: authentic cleavage and post-translational modification in an insect cell expression system. Virology 1989, 170:107-116.
20.
Gheysen D, Jacobs E, De-Foresta F, Thiriart C, Francotte M, Thines D, De-Wilde M: Assembly and release of HIV-1 precursor Pr55gag virus-like particles from recombinant baculovirus-infected insect cells, Cell 1989, 59:103-112.
21.
Delchambre M, Gheysen D, Thines D, Thiriart C, Jacobs E, Verdin E, Horth M, Burny A, Bex F: The Gag precursor of simian immunodeficiency virus assembles into virus like particles. EMBO J 1989, 8:2653-2660.
22.
Morikawa S, Booth TF, Bishop DH: Analyses of the requirements for the synthesis of virus-like particles by feline immunodeficiency virus Gag using baculovirus vectors. Virology 1991,183:288-297.
23.
RasmussenL, Battles JK, Ennis WH, Nagashima K, Gonda MA: Characterization of virus-like particles produced by a recombinant baculovirus containing the Gag gene of the bovine immunodeficiency-like virus. Virology 1990, 178:435-451.
24.
Sommen~eltMA, Roberts CR, Hunter E: Expression of simian type D retroviral (Mason-Pfizer monkey virus) capsids in insect cells using recombinant baculovirus" Virology 1993, 192:298-306.
25.
ThomsenDR, Meyer AL, Post LE: Expression of feline leukaemia virus gp85 and Gag proteins and assembly into virus-like particles using the baculovirus expression vector system. J Gen Viro/1992, 73:1819-1824.
6.
•
7
FraenkeI-ConratH, Williams RC: Reconstitution of active tobacco mosaic virus from its inactive protein and nucleic acid components. Proc Nat/Acad Sci USA 1955, 41:690-698.
8.
Popa MP, McKelvey TA, Hempel J, Hendrix RW: Bacteriophage HK97 structure: wholesale covalent cross-linking between the major head shell subunits. J Viro/1991, 65:3227-3237.
9. •
Duda RL, Martincic K, Hendrix RW: Genetic basis of bacteriophage HK97 prohead assembly. J Mol Bio11995, 247:636-647.
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26. •
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Chazal N, Gay B, Carriere C, Tournier J, Boulanger P: Human immunodeficiency virus type 1 MA deletion mutants expressed in baculovirus-infected cells: cis and trans effects on the Gag precursor assembly pathway. J Vim/1995, 69:365-375. This paper describes the use of the baculovirus system to map the sites of assembly of the HIV Gag protein into virus-like particles. It also includes an analysis of trans-dominant negative phenotypes which prevent assembly of the wild-type molecule by formation of hetero-oligomeric VLE
39.
FrenchTJ, Roy P: Synthesis of bluetongue virus (BTV) corelike particles by a recombinant baculovirus expressing the two major structural core proteins of BTV. J Viro11990, 64:1530-1536.
40.
27. •
PrasadBV, Yamaguchi S, Roy P: Three-dimensional structure of single-shelled bluetongue virus. J Virol 1992, 66:2135-2142.
41.
FrenchTJ, Marshall JJ, Roy P: Assembly of double-shelled, viruslike particles of bluetongue virus by the simultaneous expression of four structural proteins. J Viro/1990, 64:5695-5700.
42.
Hewat EA, Booth TF, Roy P: Structure of correctly selfassembled bluetongue virus-like particles. J Struct Bio11994, 112:183-191.
43.
Loudon PT, Roy P: Assembly of five bluetongue virus proteins expressed by recombinant baculoviruses: inclusion of the largest protein VP1 in the core and virus-like proteins. Virology 1991, 180:798-802.
44.
Belyaev AS, Roy P: Presentation of hepatitis B virus preS2 epitope on bluetongue virus core-like particles. Virology 1992, 190:840-844.
45.
Roy P, Urakawa T, Van-Dijk AA, Erasmus BJ: Recombinant virus vaccine for bluetongue disease in sheep. J Virol 1990, 64:1998-2003.
46.
TakeharaK, Ireland D, Bishop DH: Co-expression of the hepatitis B surface and core antigens using baculovirus multiple expression vectors. J Gen Viro11988, 69:2763-2777
47
PrehaudC, Nel K, Bishop DH: Baculovirus-expressed rabies virus M1 protein is not phosphorylated: it forms multiple complexes with expressed rabies N protein. Virology 1992, 189:766-770.
48.
ThomsenDR, Roof LL, Homa FL: Assembly of herpes simplex virus (HSV) intermediate capsids in insect cells infected with recombinant baculoviruses expressing HSV capsid proteins. J Viro/1994, 68:2442-2457.
Morikawa Y, Kishi T, Zhang W-H, Nermut MV, Hockley DJ, Jones IM: A molecular determinant of HIV particle assembly located in the matrix antigen pl Z J Viro/1995, 69:4519-4523. This paper constitutes the first analysis of HIV Gag VLPs that includes an interpretation of mutational data in the knowledge of protein structure and shows the clustering of mutations that affect assembly into a single (z helix. 28.
Chazal N, Carriere C, Gay B, Boulanger P: Phenotypic characterization of insertion mutants of the human immunodeficiency virus type 1 Gag precursor expressed in recombinant baculovirus-infected cells. J Viro/1994,
type 33 into virus-like particles and tubular structures in insect cells. Virology 1994, 200:504-512.
68:111-122.
29.
30.
Carriere C, Gay B, Chazal N, Morin N, Boulanger P: Sequence requirements for encapsidation of deletion mutants and chimeras of human immunodeficiency virus type 1 Gag precursor into retrovirus-like particles. J Virol 1995, 69:2366-237?. NermutMV, Hockley DJ, Jowett JBM, Jones IM, Garreau M, Thomas D: Fullerene-like organisation of HIV Gag-protein shell in virus-like particles produced by recombinant baculoviruses. Virology 1994, 198:288-296.
31.
Prasad BV, Rothnagel R, Jian9 X, Estes MK: Three-dimensional structure of baculovirus-expressed Norwalk virus capsids. J Virol 1994, 68:5117-5125. The authors provide an excellent demonstration of the possibilities of combining baculovirus expression of virus capsid protein with the high-resolution observational power of cryoelectron microscopy. •
32.
Zeng CQ, Labbe M, Cohen J, Prasad BV, Chen D, Ramig RF, Estes MK: Characterization of rotavirus VP2 particles. Virology 1 9 9 4 , 201:55-65.
33.
Montross L, Watkins S, Moreland RB, Maroon H, Caspar DL, Garcea RL: Nuclear assembly of polyomavirus capsids in insect cells expressing the major capsid protein VP1. J Virol 1991,65:4991-4998.
34.
Schneemann A, Dasgupta R, Johnson JE, Ruecked RR: Use of recombinant baculoviruses in synthesis of morphologically distinct viruslike particles of flock house virus, a nodavirus. J Virol 1993, 67:2756-2763.
35.
36.
37.
38.
49.
ThomsenDR, Newcomb WW, Brown JC, Homa FL: Assembly of the herpes simplex virus capsid: requirement for the carboxylterminal twenty-five amino acids of the proteins encoded by the UL26 and UL26.5 genes. J Viro11995, 69:3690-3703. An excellent example of the use of the system described in [48] to dissect stages of the process, including the role of post-translational modifications. •
50.
Brautigam S, Snezhkov E, Bishop DH: Formation of polioviruslike particles by recombinant baculoviruses expressing the individual VPO, VP3, and VP1 proteins by comparison to particles derived from the expressed poliovirus polyprotein. Virology 1993, 192:512-524.
51.
Rose RC, Bonnez W, Reichman RC, Garcea RL: Expression of human papillomavirus type 11 L1 protein in insect cells: in vivo and in vitro assembly of viruslike particles. J Viro/1993, 67:1936-1944.
Godeau F, Casanova JL, Luescher IF, Fairchild KD, Delarbre C, Saucier C, Gachelin G, Kourilsky P: Binding of low concentration of peptide to H-2Kd produced in insect cells requires mouse beta 2-microglobulin co-expression. /nt/mmuno/1992, 4:265-275.
52.
Kirnbauer R, Taub J, Greenstone H, Roden R, Durst M, Gissmann L, Lowy DR, Schiller JT: Efficient self-assembly of human papillomavirus type 16 L1 and L1-L2 into virus-like particles. J Virol 1993, 67:6929-6936.
DeTomasoAW, Xie Z J, Liu G, Mercer RW: Expression, targeting, and assembly of functional Na,K-ATPase polypeptides in baculovirus-infected insect cells. J Bio/Chem 1993, 268:1470-1478.
53.
Atkinson AE, Bermudez I, Darlison MG, Barnard EA, Earley FG, Possee RD, Beadle DJ, King LA: Assembly of functional GABAA receptors in insect cells using baculovirus expression vectors. Neuroreport 1992, 3:597-600.
FisherAJ, McKinney BR, Schneemann A, Rueckert RR, Johnson JE: Crystallization of viruslike particles assembled from flock house virus coat protein expressed in a baculovirus system. J Viro11993, 67:2950-2953.
Volpers C, Schirmacher P, Streeck RE, Sapp M: Assembly of the major and the minor capsid protein of human papillomavirus
Assembly of macromolecular complexes in bacterial and baculovirus expression systems Polly Roy*t$ and lan Jones*§ Many proteins exist normally as oligomers or complexes with other proteins. Recent advances in vector design have allowed this aspect of protein function to be mimicked in recombinant expression systems. Examples of the ordered oligomerization of a single protein through to the assembly of eight different proteins have been documented in recombinant Escherichia coil and recombinant baculovirus systems.
Addresses *Institute of Virology, Mansfield Road, Oxford OX1 3SR, UK t Department of International Public Health, University of Alabama at Birmingham, 303 Tidwell Hall, 720 20th Street South, UAB Station, Birmingham, Alabama 35294, USA ~-Department of Biophysics, University of Oxford, The Rex Richards Building, South Parks Road, Oxford OX1 3QU, UK; e-mail: [email protected] § e-mail: [email protected],uk Current Opinion in Structural Biology 1996, 6:157-161 © Current Biology Ltd ISSN 0959-440X
Abbreviations BTM bluetongue virus CLP core-bikeparticles FHV flock house virus FMDV foot and mouth disease virus HSV herpes simplex virus MPMV Mason-Pfizer monkey virus TMV tobaccomosaic virus VLP virus-like particle VP virus protein
Introduction
Despite the increasing recognition of the role of specific proteins (chaperones) in the folding and assembly of macromolecular structures [1], it is, nonetheless, generally true that in many cases, complex protein structures will assemble spontaneously provided that the level of interacting proteins is sufficiently high, that all components are present in the correct molar ratios and that, in some cases, a scaffolding (e.g. a membrane or cytoskeletal component) is provided for the process of assembly. With the advent of expression systems that produce high levels of protein, therefore, there has been an increasing number of reports of the assembly of complex protein structures directly following the expression of the components concerned. 'Assembly' may range from the relatively simple oligomerization of higher order structures from a single gene product to the assembly of enzyme complexes or virus-like particles in which several proteins participate. Coupled with improved methods for the visualization of complex structures (e.g. cryoelectron microscopy), the opportunity to study and understand macromolecular assemblies has never been greater. In this review, we
describe two systems that are widely used for protein expression and some examples of the macromolecular assemblies that have been observed following their use. The different expression systems Escherichia coil and recombinant baculoviruses are widely
used for the production of recombinant proteins. Both systems are renowned for the high yield of protein that is possible and, for both systems, vectors have been developed that allow the expression of one or more open reading frames. In what respects do these systems differ? T h e E. coil expression system represents one of the first described systems for the production of a recombinant protein and currently encompasses a plethora of different vectors and methods. In general, vectors are designed to express the desired protein transiently following the addition of an inducer (chemical or heat). T h e sudden burst of protein synthesis that ensues often provides high yields of recombinant protein, but can sometimes lead to the production of inclusion bodies in which the protein is present in a largely denatured and aggregated form. In such cases, the likelihood of the generation of a higher-order assembly of structure is small, although it is still possible (see, for example, [2*]). In many cases, however, the soluble protein is expressed at high levels and oligomerization, or co-assembly with a second (or more) protein(s), can occur. E. coli has no mechanism that enables it to carry out the addition of carbohydrate to proteins and although some secondary modifications can bc engineered to occur (e.g. myristoylation [3]), the level of secondary protein modification that can be achieved in this system is limited. Thus, if the assembly of protein complexes following expression requires a modification that is specific to eukaryotic cells, it may be impossible to achieve without in vitro modification prior to attempted in vitro assembly. In these cases, whilst E. coil expression is undoubtedly enabling, complex formation per se does not occur within the bacterial cell. Expression in recombinant baculoviruses, on the other hand, allows proteins to be modified in the same way as in the majority of higher eukaryotic cells and the yield of protein is often as high or higher than that achievable in E. coli. Proteins are expressed late in the viral life cycle over a two or three day period and, consequently, the frequency of inclusion body formation is low and most expressed proteins are soluble and available to engage in protein-protein interactions. Vectors have been developed to allow the expression of one, two, three or four proteins simultaneously 14"] and, as co-infection is relatively efficient, there is the possibility of expressing up to eight proteins in the same cell [5]. Recently it was
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also shown that the level of misfolded proteins could be minimized by co-expressing proteins with chaperone-like activity (see, for example, [6"1). Accordingly, there are more examples of heterologous macromolecular assembly in the baculovirus expression system than in the E. coil expression system, particularly when the genes expressed are of eukaryotic origin.
Assembly of complex structures in E. coli Despite the limitations of E. co/i expression, a number of examples of complex structure formation have been reported. In common with the baculovirus system outlined below, the expression of virally encoded proteins has given notable results. T h e replication strategy of many viruses has evolved to maximize both the number of progeny virions and the most economical usage of coding capacity. As a consequence, self assembly is a common property of a number of virus capsids and has been observed in an increasing number of viruses since early observations on tobacco mosaic virus (TMV) assembly [7]. T h e expression of genes from bacterial viruses has, in a number of cases, given rise to subviral structures, allowing detailed progress to be made in understanding the assemblages and assembly pathways being made. A few of the well studied cases include phi 29, MS2 and the lambda-like phages. In an analysis of phage HK97, for example, the different intermediates of assembly have been identified through the use of temperature-sensitive mutations that block the assembly process and allow the build up of part-assembled structures [8]. From this analysis, genes deduced to be responsible for the assembly of the phage head structure were subsequently expressed in isolation to confirm that they do, indeed, assemble into head structures [9"]. A similar analysis has been carried out on the Bacillus subtilis phage phi29, where expression of three phage-encoded proteins in E. coil are necessary for the production of active phage proheads [10 °] whilst the expression of only one or two proteins led to only partial or aberrant prohead formation. One of the proteins required is a scaffold protein that is necessary to enable the assembly of the other two, but is not itself a component of the prohead. These data illustrate well how E. coil can supply a suitable background for the assembly of quite complex structures of non-E, coli origin, provided that all the necessary components, including non-structural components, are present in sufficient yield. An analysis of the E.coli phage MS2 coat protein has delineated a role in assembly and also in the specific incorporation of the RNA genome to such a degree that the coat protein-RNA interaction is understood at atomic level [11,12]. Moreover, in a practical application of assembly studies, the MS2 coat protein gene has been engineered to express multiple foreign peptides on the surface of an RNA-free particle in positions that are known to elicit a strong antibody response [13]. Such applications demonstrate the value of dissecting complex
structure formation by recombinant means and suggest the use of viral structures for a variety of biotechnological applications in the future. Virus assembly following expression in E. coil has not been limited to phage systems. The capsids of Mason-Pfizer monkey virus (MPMV; a type D retrovirus) assemble within inclusion bodies after expression in E. co/i, although capsids of the related HIV (a type C retrovirus) do not [2°]. Interestingly, in normal infections, assembly of type D retroviruses occurs in the cytoplasm, whereas that of type C retroviruses occurs at the plasma membrane concomitant with virus budding. It is possible, therefore, that the specific requirement for co-budding at the membrane is the cause of the failure of type C capsids to assemble in E. coll. The TMV coat protein has been shown to assemble in E. coli to form pseudovirions [14] as has the 70S empty capsid of foot and mouth disease virus (FMDV) [15]. For FMDV, many proteins are necessary for the assembly of the virus particle and all are encoded in a large precursor, polyprotein Pl. Rather than express each protein directly, Lewis et al. [15] made only two expression plasmids, one encoding the Pl precursor and the other the 3C protease responsible for Pl cleavage. Upon co-expression, Pl was correctly processed and 70S particles were assembled. As with the phi 29 scaffold protein, therefore (above), it was necessary to provide an enabling protein function that was not itself directly involved in the final structure. The assembly of complex structures is not restricted to those of viral origin. T h e oligomerization domain of the HIV transmembrane protein gp41 assembles in E, co/i [16"] as do a number of multicomponent enzymes derived from prokaryotic [17] or mammalian origin [18].
Assembly of complex structures in recombinant baculovirus infected cells In common with the findings in recombinant E. co/i (above), there are many examples in the literature of the assembly of viruses and subviral particles following expression using recombinant baculoviruses. The assembly of a single viral capsid protein, p55 Gag, into a virus-like particle (VLP) was observed for HIV as early as 1989 [19,20]. Several other retroviral Gag proteins have also been shown to assemble into VLPs following baculovirus expression (e.g. simian immunodeficiency virus [21], feline immunodeficiency virus [221, bovine immunodeficiency virus [23], MPMV [241 and feline leukaemia vires [25]). For HIV in particular, these observations have spawned a detailed analysis of the sequence requirements for VLP formation and the essential domains within the Gag protein have been delineated in each of the subdomains of the molecule: the N-terminal matrix domain [26°,27°], the central capsid domain [28] and the C-terminal nucleocapsid domain [29]. T h e abundant quantities of Gag VLP that are synthesized from this system coupled with the uniform dimensions of the VLP produced have facilitated an examination of higher order Gag structure
Assembly of macromolecular complexes in bacterial and baculovirus expression systems Roy and Jones
by high-resolution electron microscopy and a tentative model of the Gag shell has been suggested [30]. T h e expression of other single viral proteins has also resulted in the assembly of VLPs. For Norwalk virus, the use of baculoviruses to express the single 58 kDa capsid protein resulted in a source of VLPs that proved uniform and suitable for cryoelectron microscopy and permitted an image reconstruction of the vition shell to a resolution of 22/~, [31"]. Similarly, the expression of a single rotavirus gene encoding the virus protein (VP)2 led to the production of a single-shell particle with icosahedral symmetry discernible by electron microscopy analysis [32]. T h e expression of the VP1 protein of polyomavirus led to the production of single-shell structures resembling the authentic virus [33] and expression of the flock house virus (FHV) coat protein resulted in the synthesis of FHV particles that were indistinguishable from the authentic virions [34]. Interestingly, single point mutations in the FHV coat protein led to altered morphologies including half shells and shells with holes, emphasizing the fact that the three-dimensional representation of the VLP is encoded within the single protein expressed and can be separated from the ability to assemble per se. A mutant virion form of FHV was subsequently crystallized and shown to diffract to 3.3 ,~, suggesting that the uniform particles produced by this system are suitable for crystallographic analysis [35]. T h e r e have been numerous examples of the assembly of papillomavirus L1 protein into VLPs (see, for example [36-38]) and, in some cases, these reports also document the incorporation of the second capsid protein VP2. T h e first report of the expression of two proteins that interacted to form a distinct VLP was that describing bluetongue virus (BTV) [39], in which the expression of structural proteins VP3 and VP7 was shown to result in the synthesis of BTV core-like particles (CLP) that were similar to authentic BTV cores. T h e C L P were shown by cryoelectron microscopy to be faithful mimics of authentic BTV cores [40] and other work established that a double-shell structure could be formed by the additional synthesis of the outer capsid proteins VP5 and VP2 [41]. Cryoelectron microscope studies of these particles has shown them to be near exact mimics of the complete BTV virion [42]. In separate studies the co-expression of the an internal BTV protein, VP1, with either the C L P or VLP led to incorporation of VP1 into the virus-like structures [43]. Thus, although VP1 was not necessary for the synthesis of either C L P or VLP, it could be incorporated into the assembling particle if present, suggesting that it will ultimately be possible to reconstruct a variety of BTV assembly intermediates by co-expression of a select number of BTV proteins. Similar to the exploitation of MS2 phage outlined earlier, the BTV CLPs have been engineered to express new epitopes on their surface with a view to novel vaccine
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applications [44]. CLPs themselves are exceptionally potent vaccines [45], so there is a precedent to suggest that chimeric CLPs will be equally effective. Similar, though less extensive, studies on the assembly of multicomponent viral components have been reported for the hepatitis B virus core and surface antigens [46], for papillomavirus VP1 and VP2 capsid proteins [38] and for rabies virus N and M1 proteins [47]. T h a t more complex viral assemblies are also possible has been demonstrated by the assembly of thc herpes simplex virus (HSV) capsid in insect cells following the expression of six HSV proteins. An outer icosahcdral shell composed of four proteins and an inner scaffolding layer composed of a further two proteins were assembled [48]. Subsequently, the role of the two scaffold proteins encoded by HSV genes UL26 and UL26.5 was further investigated and the proteins were observed to assemble in their own right to produce intermediate capsids. T h e products of both genes are normally processed during assembly, although the role of the processing event was unclear. Capsid assembly from discrete mutated proteins, however, clearly demonstrated that assembly precedes processing [49"]. These studies, and others like them, bode well for the use of these systems to unravel assembly pathways as well as to achieve an understanding of the assembled structure itself. The assembly of poliovirus capsids following the expression of the entire polyprotein in a recombinant baculovirus has been dcscribed [50], suggesting the possibility that this type of system can be extended to viruses where, despite a cloned genome being available, no productive tissue-culture system for the derivation of large numbers of virions for structural studies exists (e.g. hepatitis C virus). As with E. coli expression, although the instances of known complex assembly concern predominantly structures of viral origin, other multicomponent systems have been shown to assemble. When a class I MHC heavy chain was expressed in conjunction with 132-microglobulin, about 10% of the total class 1 chains were in the form of a heterodimer whose assembly was driven by peptide binding to the complex [51] and similar reports have documented the assembly of antibody heavy and light chains following their co-expression [6"]. T h e assembly of the rodent Na+/K+-ATPase has been reported following the expression of ctl and 131 subunits in insect cells to produce a detergent-resistant functional complex [52] and similarly, the subunits of the bovine GABAA receptor assemble to form ion channels with distinctive physiological behavior I53].
Conclusions T h e use of recombinant systems to study multicomponent assembly is a growing area and the expression systems we have outlined here play a key role. Whilst there are clearly limits to the type of interaction that can occur within bacteria, the examples we have described provide ample evidence to suggest a continuing role for E. co/i expression in the analysis of complex structures. An area wherc E. co/i has much to offer is in the combined
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use of multiple expression with bacterial genetics. T h e examples we have highlighted largely concern phage genetics but we see no reason why, given the assembly of a complex with a recognized phenotype, in E. coil, genetic studies should not allow the complete dissection of other assembly processes. Such studies are also possible in recombinant baculoviruses but the advantages of eukaryotic expression are tempered by the slower rate of analysis. Furthermore, baculovirus development should see the construction of 'moving windows' of assembly, representing different stages in a complex assembly process, by the expression of select combinations of proteins. That complex structures can assemble is well demonstrated but the order of many assembly pathways remains unclear. In both systems, there is also the exciting possibility of chimeric complex formation to provide multicomponent structures with new and novel properties of biotransformation or immunogenicity.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: ,, of special interest • e of outstanding interest 1.
Wynn RM, Davie JR, Cox RP, Chuang DT: Molecular chaperones: heat-shock proteins, foldases, and matchmakers. J Lab C/in Med 1994, 124:31-36.
2. •
Klikova M, Rhee SS, Hunter E, Rural T: Efficient in vivo and in vitro assembly of retroviral capsids from Gag precursor proteins expressed in bacteria. J Virol 1995, 69:1093-1098. This paper gives details of the first demonstration of retrovirus Gag assembly in a prokaryote. The particles formed within inclusion bodies but could be isolated by differential centrifugation. They lacked membrane and so were particularly suitable for cryoelectron microscopy. 3.
Duronio RJ, Jackson-Machelski E, Heuckeroth RO, Olins PO, Devine CS, Yonemoto W, Slice LW, Taylor SS, Gordon Jh Protein N-myristoylaflon in Escherichie co//: reconstitution of a eukaryotic protein modification in bacteria. Proc Nat/Acacl Sci USA 1990, 87:1506-1510.
4. •
Belyaev AS, Hails RS, Roy P: High-level expression of five foreign genes by a single recombinant beculovirus. Gene 1995, 156:229-233. This paper describes the construction of a novel baculovirus expression vector with the ability to express four different genes at the same time. When this vector was used to create a recombinant virus which already contained one foreign gene, a virus expressing five proteins in the same cell was isolated. Dual infections would allow 10 proteins to be expressed together, although this was not directly demonstrated in this paper. 5.
Roy P: Orbivirus structure and assembly. Virology 1996, 215:1-11.
Hsu TA, Eiden JJ, Bourgarel P, Meo T, Betenbaugh MJ: Effects of co-expressing chaperone BiP on functional antibody production in the baculovirus system. Protein Expr Purif 1994, 5:595-603. The correct folding of proteins, especially after expression at high level, represents a grey area in the application of expression systems. In this report, chaperonins are co-expressed with antibody heavy chains and shown to be beneficial for the amount of functional protein made. The study suggests that folding pathways may be engineered to maximize the recovery of fully functional product.
A thorough analysis of the requirements for assembly of the prohead of phage HK97. In contrast to the data in [10"], prohead formation occurs with a single gene product in the absence of a scaffold protein. Co-expression of the viral protease led to maturation of the prohead and provides a model for understanding the interaction of the protease with the prohead substrata. 10. Lee CS, Guo P: Sequential interactions of structural proteins in • phage phi 29 procapsid assembly. J Viro11995, 69:5024-5032. An elegant study demonstrating the assembly of the prohead of the Bacillus subtilis phage phi29 in E. colt. The work concludes that correct prohead assembly is inherent as long as all necessary components are present and that neither the gene order of the expression system used nor the molar ratios of protein is important as long as they reach the threshold level for assembly to occur.
1 1.
Valeg&rdK, Murray JB, Stockley PG, Stonehouse NJ, Liljas L: Crystal structure of an RNA bacteriophage coat protein-operator complex. Nature 1994, 371:623-626.
12.
Stockley PG, Stonehouse NJ, Valeg&rd K: Molecular mechanism of RNA phage morphogenesis'/nt J Biochem 1994, 26:1249-1 260.
13.
Mastico RA, Talbot S J, Stockley PG: Multiple presentation of foreign peptides on the surface of an RNA-free spherical bacteriophage capsid. J Gen Viro/1993, 74:541-548.
14.
Hwang DJ, Roberts IM, Wilson TM: Expression of tobacco mosaic virus coat protein and assembly of pseudovirus particles in Escherichia coil Prec Nat/Acad Sci USA 1994, 91:906'7-9071.
15.
Lewis SA, Morgan DO, Grubman MJ: Expression, processing, and assembly of foot-and-mouth disease virus capsld structures in heterologous systems: induction of a neutralizing antibody response in guinea pigs. J Viro11991,65:6572-6580.
16. •
Bernstein HB, Tucker SP, Kar SR, McPherson SA, McPherson DT, Dubay JW, Lebowitz J, Compans RW, Hunter E: OIIgomerlzation of the hydrophobic heptad repeat of gp41. J Viro11995, 69:2745-2750. Eukaryotic glycoproteins are seldom well expressed in E. coil but the use of fragments here demonstrates that inherent properties of self assembly can be visualized in E. coIL The rapid analysis of mutants that is possible in E. colt allows an analysis of the residues involved in the oligomerization. 17.
Lessard IA, Perham RN: Expression in Escherichie colt of genes encoding the E1 alpha and E1 beta subunits of the pyruvate dehydrogenase complex of Bacillus stearothermophilus and assembly of a functional E1 component (alpha 2 beta 2) in vitro. J Biol Chem 1994, 269:10378-10383.
18.
Davie JR, Wynn RM, Cox RP, Chuang DT: Expression and assembly of a functional E1 component (alpha 2 beta 2) of mammalian branched-chain alpha-ketoacid dehydrogenase complex in Escherichie colt. J Biol Chem 1992, 267:16601-16606.
19.
Overton HA, Fujii Y, Price I, Jones IM: The protease and Gag gene products of the human immunodefictency virus: authentic cleavage and post-translational modification in an insect cell expression system. Virology 1989, 170:107-116.
20.
Gheysen D, Jacobs E, De-Foresta F, Thiriart C, Francotte M, Thines D, De-Wilde M: Assembly and release of HIV-1 precursor Pr55gag virus-like particles from recombinant baculovirus-infected insect cells, Cell 1989, 59:103-112.
21.
Delchambre M, Gheysen D, Thines D, Thiriart C, Jacobs E, Verdin E, Horth M, Burny A, Bex F: The Gag precursor of simian immunodeficiency virus assembles into virus like particles. EMBO J 1989, 8:2653-2660.
22.
Morikawa S, Booth TF, Bishop DH: Analyses of the requirements for the synthesis of virus-like particles by feline immunodeficiency virus Gag using baculovirus vectors. Virology 1991,183:288-297.
23.
RasmussenL, Battles JK, Ennis WH, Nagashima K, Gonda MA: Characterization of virus-like particles produced by a recombinant baculovirus containing the Gag gene of the bovine immunodeficiency-like virus. Virology 1990, 178:435-451.
24.
Sommen~eltMA, Roberts CR, Hunter E: Expression of simian type D retroviral (Mason-Pfizer monkey virus) capsids in insect cells using recombinant baculovirus" Virology 1993, 192:298-306.
25.
ThomsenDR, Meyer AL, Post LE: Expression of feline leukaemia virus gp85 and Gag proteins and assembly into virus-like particles using the baculovirus expression vector system. J Gen Viro/1992, 73:1819-1824.
6.
•
7
FraenkeI-ConratH, Williams RC: Reconstitution of active tobacco mosaic virus from its inactive protein and nucleic acid components. Proc Nat/Acad Sci USA 1955, 41:690-698.
8.
Popa MP, McKelvey TA, Hempel J, Hendrix RW: Bacteriophage HK97 structure: wholesale covalent cross-linking between the major head shell subunits. J Viro/1991, 65:3227-3237.
9. •
Duda RL, Martincic K, Hendrix RW: Genetic basis of bacteriophage HK97 prohead assembly. J Mol Bio11995, 247:636-647.
Assembly of macromolecular complexes in bacterial and baculovirus expression systems Roy and Jones
26. •
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Chazal N, Gay B, Carriere C, Tournier J, Boulanger P: Human immunodeficiency virus type 1 MA deletion mutants expressed in baculovirus-infected cells: cis and trans effects on the Gag precursor assembly pathway. J Vim/1995, 69:365-375. This paper describes the use of the baculovirus system to map the sites of assembly of the HIV Gag protein into virus-like particles. It also includes an analysis of trans-dominant negative phenotypes which prevent assembly of the wild-type molecule by formation of hetero-oligomeric VLE
39.
FrenchTJ, Roy P: Synthesis of bluetongue virus (BTV) corelike particles by a recombinant baculovirus expressing the two major structural core proteins of BTV. J Viro11990, 64:1530-1536.
40.
27. •
PrasadBV, Yamaguchi S, Roy P: Three-dimensional structure of single-shelled bluetongue virus. J Virol 1992, 66:2135-2142.
41.
FrenchTJ, Marshall JJ, Roy P: Assembly of double-shelled, viruslike particles of bluetongue virus by the simultaneous expression of four structural proteins. J Viro/1990, 64:5695-5700.
42.
Hewat EA, Booth TF, Roy P: Structure of correctly selfassembled bluetongue virus-like particles. J Struct Bio11994, 112:183-191.
43.
Loudon PT, Roy P: Assembly of five bluetongue virus proteins expressed by recombinant baculoviruses: inclusion of the largest protein VP1 in the core and virus-like proteins. Virology 1991, 180:798-802.
44.
Belyaev AS, Roy P: Presentation of hepatitis B virus preS2 epitope on bluetongue virus core-like particles. Virology 1992, 190:840-844.
45.
Roy P, Urakawa T, Van-Dijk AA, Erasmus BJ: Recombinant virus vaccine for bluetongue disease in sheep. J Virol 1990, 64:1998-2003.
46.
TakeharaK, Ireland D, Bishop DH: Co-expression of the hepatitis B surface and core antigens using baculovirus multiple expression vectors. J Gen Viro11988, 69:2763-2777
47
PrehaudC, Nel K, Bishop DH: Baculovirus-expressed rabies virus M1 protein is not phosphorylated: it forms multiple complexes with expressed rabies N protein. Virology 1992, 189:766-770.
48.
ThomsenDR, Roof LL, Homa FL: Assembly of herpes simplex virus (HSV) intermediate capsids in insect cells infected with recombinant baculoviruses expressing HSV capsid proteins. J Viro/1994, 68:2442-2457.
Morikawa Y, Kishi T, Zhang W-H, Nermut MV, Hockley DJ, Jones IM: A molecular determinant of HIV particle assembly located in the matrix antigen pl Z J Viro/1995, 69:4519-4523. This paper constitutes the first analysis of HIV Gag VLPs that includes an interpretation of mutational data in the knowledge of protein structure and shows the clustering of mutations that affect assembly into a single (z helix. 28.
Chazal N, Carriere C, Gay B, Boulanger P: Phenotypic characterization of insertion mutants of the human immunodeficiency virus type 1 Gag precursor expressed in recombinant baculovirus-infected cells. J Viro/1994,
type 33 into virus-like particles and tubular structures in insect cells. Virology 1994, 200:504-512.
68:111-122.
29.
30.
Carriere C, Gay B, Chazal N, Morin N, Boulanger P: Sequence requirements for encapsidation of deletion mutants and chimeras of human immunodeficiency virus type 1 Gag precursor into retrovirus-like particles. J Virol 1995, 69:2366-237?. NermutMV, Hockley DJ, Jowett JBM, Jones IM, Garreau M, Thomas D: Fullerene-like organisation of HIV Gag-protein shell in virus-like particles produced by recombinant baculoviruses. Virology 1994, 198:288-296.
31.
Prasad BV, Rothnagel R, Jian9 X, Estes MK: Three-dimensional structure of baculovirus-expressed Norwalk virus capsids. J Virol 1994, 68:5117-5125. The authors provide an excellent demonstration of the possibilities of combining baculovirus expression of virus capsid protein with the high-resolution observational power of cryoelectron microscopy. •
32.
Zeng CQ, Labbe M, Cohen J, Prasad BV, Chen D, Ramig RF, Estes MK: Characterization of rotavirus VP2 particles. Virology 1 9 9 4 , 201:55-65.
33.
Montross L, Watkins S, Moreland RB, Maroon H, Caspar DL, Garcea RL: Nuclear assembly of polyomavirus capsids in insect cells expressing the major capsid protein VP1. J Virol 1991,65:4991-4998.
34.
Schneemann A, Dasgupta R, Johnson JE, Ruecked RR: Use of recombinant baculoviruses in synthesis of morphologically distinct viruslike particles of flock house virus, a nodavirus. J Virol 1993, 67:2756-2763.
35.
36.
37.
38.
49.
ThomsenDR, Newcomb WW, Brown JC, Homa FL: Assembly of the herpes simplex virus capsid: requirement for the carboxylterminal twenty-five amino acids of the proteins encoded by the UL26 and UL26.5 genes. J Viro11995, 69:3690-3703. An excellent example of the use of the system described in [48] to dissect stages of the process, including the role of post-translational modifications. •
50.
Brautigam S, Snezhkov E, Bishop DH: Formation of polioviruslike particles by recombinant baculoviruses expressing the individual VPO, VP3, and VP1 proteins by comparison to particles derived from the expressed poliovirus polyprotein. Virology 1993, 192:512-524.
51.
Rose RC, Bonnez W, Reichman RC, Garcea RL: Expression of human papillomavirus type 11 L1 protein in insect cells: in vivo and in vitro assembly of viruslike particles. J Viro/1993, 67:1936-1944.
Godeau F, Casanova JL, Luescher IF, Fairchild KD, Delarbre C, Saucier C, Gachelin G, Kourilsky P: Binding of low concentration of peptide to H-2Kd produced in insect cells requires mouse beta 2-microglobulin co-expression. /nt/mmuno/1992, 4:265-275.
52.
Kirnbauer R, Taub J, Greenstone H, Roden R, Durst M, Gissmann L, Lowy DR, Schiller JT: Efficient self-assembly of human papillomavirus type 16 L1 and L1-L2 into virus-like particles. J Virol 1993, 67:6929-6936.
DeTomasoAW, Xie Z J, Liu G, Mercer RW: Expression, targeting, and assembly of functional Na,K-ATPase polypeptides in baculovirus-infected insect cells. J Bio/Chem 1993, 268:1470-1478.
53.
Atkinson AE, Bermudez I, Darlison MG, Barnard EA, Earley FG, Possee RD, Beadle DJ, King LA: Assembly of functional GABAA receptors in insect cells using baculovirus expression vectors. Neuroreport 1992, 3:597-600.
FisherAJ, McKinney BR, Schneemann A, Rueckert RR, Johnson JE: Crystallization of viruslike particles assembled from flock house virus coat protein expressed in a baculovirus system. J Viro11993, 67:2950-2953.
Volpers C, Schirmacher P, Streeck RE, Sapp M: Assembly of the major and the minor capsid protein of human papillomavirus