Tatyana M. Kozlovska lndulis Cielens lnta Vasiljeva Anna Strelnikova Andris Kazaks Andris Dislers Dzidra Dreilina Velta Ose IndulisGusars Paul Pumpens
RNA Phage ap Coat Protein as a Carrier for Foreign Epitopes
Biomedical Research and Study Centre, University of Latvia, Riga, Latvia
Genetic engineering Cloning Expression Chimeric proteins RNAphageQ~ Coat proteins
The Q~ gene C has been proposed as a new carrier for the exposure of foreign peptide sequences. Contrary to well-known 'display vectors' on the basis of coat proteins of RNA phage group I, group III phage Q~-based vectors suggested application of the 195-amino acid extension of coat protein (CP) within the so-called Al protein for insertion of the appropriate immunological epitopes. 'Mosaic' capsids presenting model hepatitis B virus preSI and HIV-I gp120 epitopes and formed by Q~ CP together with AI-derived proteins were obtained as a result of (1) suppression ofleaky UGA stop codon of the CP gene and (2) simultaneous expression of 'pure' CP and full-length AI-derived genes obtained after the changing of CP-terminating UGA to strong UAA stop codon or sense GGA codon, respectively.
A detailed knowledge of the RNA-phage spatial structure and molecular mechanism of morphogenesis makes them a useful tool in protein engineering of viral capsids. The three-dimensional structure of phage particles and recombinant coat protein (CP) capsids of the two most distant representatives of RNA-phage serological group I, and namely MS2 [1, 2] and fr , has been determined at high resolution by X-ray crystallography. Quasisymmetrical icosahedral protein shells of both viruses and their recombinant CP capsids are very similar and consist of 180 copies of CP subunits forming a lattice with the triangulation number T = 3. RNA-phage CP has a fold which is different from the fold of all other viral coat proteins so far known . It is made up of a five-stranded ~-sheet
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'core' and a hairpin and two helices on the outside of the particle . CP subunits are represented as three conformers A, Band C which are chemically identical and spatially similar. Only the B conformer has a unique conformation in the 'non structured' loop involved in 5-fold interactions . According to classical functional data [for review see 4] and recent structural implications [1-3], initial building blocks of RNA phage capsids are CP dimers. Recently, a similar X-ray structure was obtained for RNA phage GA [Bundule et aI., unpubl. data], a representative of the more distantly related serological group II, but belonging together with group I to the major group A, and for far remote RNA phage Q~ from the serological group III belonging to another major group B [5, Golmohammadi et aI., unpubl. data].
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P.Pumpens Biomedical Research and Study Centre University of Latvia Kirchenstein Str. I
GP - 133 aa
- 195 aa / GP extension I
(between CP extension aa residues 72 ~d 73) I insertion of preSl epitope into BglII site
Independent expression of CP and At genes
Expression of CP and At genes from differentplasmids
Expression of CP and At genes from the same plasmid
Fig. 1. General scheme of expression in E. coli of gene C of R.'\A phage Q~ and its application for exposure of foreign epitopes. Figures represent nt positions of gene C start and end points according to existing full Q~ genome sequence . 'lmmunomarker' sequence DP AFRA introduced as an example of epitope cloning represents a fragment from HBV preSl sequence recognized by monoclonal anti-preSl antibody :\L\1817.
KozloyskalCielensN asiljeva/Strelnikoval KazakslDislers/Dreilina/Ose/ Gusars/ Pumpens
The recombinant capsids produced by highly efficient expressing of the MS2  and fr  CP genes in Escherichia coli, the natural host of the viruses, were proposed as first RNA-phage carriers for presentation of foreign epitopes on the outer surface of MS2  and fr [9, 10] CP shells, respectively. A limitation of studied RNA-phage CP models is the low capacity of CP 'display vectors' for foreign insertions  as a result of relatively high rigidity of capsid self-assembly requirements. To overcome these difficulties, we proposed recombinant capsids of RNA phage Q~, the coat (C) gene of which is fundamentally different from simple CP genes of group I and II phages, as one of the prospective new candidates for the role of the particulate carrier.
paracrystallinic areas of capsids (fig. 2A, C). Immunological tests with polyclonal anti-Q~ phage antibodies and electron microscopy verified the correct self-assembly of Q~ CP monomers into capsids which were indistinguishable morphologically and immunologic ally from Q~ virions. Electron microscopy pf recombinant QI3 CP purified on Sepharose CL4B column showed icosahedral phage-particle-like 25-nm capsids (fig. 2E) reactive with anti-Q~ (fig. 2F) but not with anti-fr (or anti-MS2) antibodies. According to UV absorbance of Q~ capsids, they retained their ability to incorporate nucleic acid [data not shown].
Structure of A 1 Protein and 'Display Vectors' on the Basis of 013 Gene C Structure, Cloning and Expression of Gene C Encoding 013 CP and A 1 Proteins
E. coli cells harboring the appropriate expression plasmids supported high-efficient synthesis of 14-kD protein comigrating upon PAGE with control Q~ CP isolated from Q~ phage particles. One of the constructed plasmids, pQ~lO, assured up to 20% of the total protein synthesis of Q~ CP within E. coli strain K802 cells . Electron microscopy of cell cross sections revealed accumulation of
Taking into account an above-mentioned advantage of the Q~ model in comparison with previously used group I RNA phages, we considered 195-amino acid extension of CP within Al protein as an ideal target for the insertion of foreign sequences (fig. 1). Whilst nothing is known about the spatial organization and localization of CP extension, we found that it contained elements typical for protruding spike-like structures exposed on the surface of particles. For example, mathematical prediction showed clear-cut colinearity ofCP extension and preS sequence of the hepatitis B virus (fig. 3). Unexpectedly, with an eye to the I % suppression level of UGA stop codon within wild-type E. coli cells , recombinant Q~ capsids grown in E. coli K802 revealed only traces, if nothing, of 329-amino-acid Al protein. In order to realize the idea of CP lA 1 mixed, or 'mosaic', capsids harboring foreign epitopes inserted into the CP extension part of AI, we have chosen two possible strategies depicted on figure 1 and providing (1) expression of native gene C in conditions of enhanced UGA suppression, and (2) separate expression of pure CP gene and full-length Al gene after mutational changing of the UGA stop codon to UAA stop or GGA sense codons, respectively. In the latter case expression of CP and full-length Al genes is possible from (1) a single plasmid harboring the appropriate genes within a cassette under the control of strong promoter, and (2) two different plasmids conveying two antibiotic resistances to cells. As a model foreign epitope short enough and easy to test with well-characterized antibodies we chose the 20DPAFRA-25 sequence from HBV, subtype ayw, preSl sequence where DP AF is sufficient to be recognized by the monoclonal virus-neutralizing anti-preSl antibody
Q~ Coat Protein as a Carrier
Unlike group I and II phages, gene C of group III (including Q~) and IV phages codes not only for CP but also for the C-terminally prolonged CP variant called Al protein  which arises by read-through of UGA-termination codon at the end ofCP  (fig. 1). Although biological function of the Al protein and read-through mechanism remains to date unclear, several molecules of Al are found in Q~ virions and seem to be essential for the formation of infectious phage particles . 'Full-length gene C of Q~ phage was amplified from native Q~ RNA using a reverse transcription-PCR technique and the appropriate 1,062-bp PCR fragment encoding the 133-amino-acid Q~ CP and an additional 195-amino-acid C-terminal sequence, separated from CP by leaky UGA stop codon, was expressed under the control of the strong E. coli trp promoter (Ptrp) within a pGEM-derived plasmid . Sequencing showed the full identity of the cloned fragment to available QI3 sequence .
Synthesis and Self-Assembly CPCapsids
Fig. 2. Electron microscopy of recombinant protein-producingE. coli cellsand purified recombinant Q~ CP particles. Cross sections of E. coli K802 cells producing Q~ CP(A) and Q~ CP and full-length Al simultaneously (B);rectangles mark regions which are magnified on C and D respectively. Negative contrast of recombinant Q~ CP capsids purified on Sepharose CL-4B column (E) and immunogold staining of recombinant Q~ CP capsids with polyclonal anti-Q~ antibodies (F). Bar represents 50 nm.
MA 18/7 . This 'immunomarker' sequence allowed us to screen potential insertion sites on the gene. Next, a longer epitope, namely the HIV-l gp120 V3 loop [18, 19], sub type MN, of 39-amino-acid residues was used. Figure 4 presents constructed variants of C gene-containing insertions within the native as well as UGA-terminationdepleted gene.
Expression of Q~ Gene C in UGA Suppressor Cells
As early as 1974, Hofstetter et al.  found out that the content of Al within Q~ phage particles could be elevated in conditions of UGA suppression. For this purpose, we introduced the pISM3001 plasmid harboring cloned gene of opal suppressor tRNA which recognized UGA codon as Trp codon  into E. coli K802 cells bearing pQ~ 10 and its derivatives with inserted foreign epitopes (fig. 4). As a result, we obtained an enhanced synthesis (about 50% of CP synthesis) of Al or Al[DPAFRA], which could be incorporated into capsids.
Kozlovska/CielensN asiljeva/Strelnikova/ Kazaks/Dislers/Dreilina/Ose/Gusars/ Pumpens
Fig. 3. Sensitive homology between amino acid sequences of gene C of RNA phage Q~ and preS region of HBV (subtype ayw). Vertical dotted line marks the end of proper CP and start ofCP extension. Arrows indicate regions of colinear homology of CP extension and preS sequence participating in their full-length relatedness. N-terrninal homologous sequences are deciphered at the bottom. Reference points of homology are boxed.
Expression of CP and A 1 Genes
With the aim of expressing 'pure' CP gene without any traces of the read-through Al protein we changed the leaky UGA terminator of the CP gene into the strong UAA stop codon (fig. I). 'Pure' CP was able to self-assemble and resulting capsids showed no changes in their shape and stability in comparison to capsids produced by native gene C. The expression of full-length AI, Al [DPAFRA] or AI[V3 loop] genes led to the synthesis of insoluble products unable to self-assemble and localized in inclusion bodies within the cell (fig. 4).
Q~ Coat Protein as a Carrier
In the case of simultaneous expression of 'pure' CP and full-length AI-derived genes from a single plasmid or from two different plasmids ensuring ampicillin (AIderived genes) and kanamycin, but not tetracycline (CP gene) resistance and vice versa, we detected integration of prolonged proteins into capsid particles formed mainly by helper CP (fig. 2B, D, 4).
Capsid-forming ability Structure of recombinant gene 72 ~
with helper CP helper gene I without CP gene
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Fig. 4. Chimeric derivatives of gene C-encoded proteins and their ability to self-assembly. Capsid-forming was detected by double radial immunodiffusion and electron microscopy. ND = Not determined.
The DNA fragment corresponding to the Q~ RNA region 1,299-2,360 in terms of the existing Q~ RNA sequence  and containing the gene C has been cloned and expressed in E. coli. It was suggested that 'display vectors' on the basis of the Q~ gene C used both proper CP and CP extension within Al protein for insertion offoreign peptide sequences. Firstly, a model allowing suppression of UGA and enhanced synthesis of Al was created. Secondly, 'pure' CP genes terminated by the strong TAA stop codon and full-length Al gene with TGA converted to GGA and its foreign epitope-containing derivatives were expressed simultaneously within the same E. coli cell. Formation of 'mosaic' capsids built by CP and Al proteins was detected using epitope-specific monoclonal antibodies. We expected (1) high cloning capacity of the CP extension part of the Al protein, (2) exposure of the
inserted peptides on the outer surface of the capsid, and (3) retention of the dimer-forming and capsid-forming ability of chimeras. Vectors were created which retained only a short amino acid stretch of Al adjacent to CP and provided a full exchange of Al against foreign sequences.
Acknowledgments The authors wish to thank Juris Ozols for skilful technical assistance and Dr. Eva Stankevica for synthesis of oligonucleotide primers. They also thank Dr. F.e. Minion for suppressor strains and plasmid pISM300l and Prof. Elmars Grens for his continued interest in the project and for many helpful discussions. This work was supported by grant No. 93.557 from the Latvian Council of Science.
Kozlovska/Cielens/V asiljeva/Strelnikova/ Kazaks/Dislers/Dreilina/Ose/Gusars/ Pumpens
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Q0 Coat Protein as a Carrier
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