Dissertation for Scientifist Degree of Dr.habil.biol. (in Molecular Biology)
Tatjana Kozlovska.
Cloning and expression of viral genomes in prokaryotic and
eukaryotic systems.
University of Latvia, Riga, 1997
Supplement (16 papers).
SUMMARY
We chose for our study some typical representatives of two families of small viruses of
eukaryotic and prokaryotic origin, namely, of Hepadnaviridae and Leviviridae families.
Human hepatitis B virus (HBV) and RNA bacteriophages fr and Qβ from serological
groups I and III, respectively, were involved in the present cloning and expression
investigations. These viruses, being very different and related to very different worlds of
organisms, harbored, at the same time, structural genes encoding envelope and/or
capsid shells and therefore prospective for employement in protein engineering
experiments, in order to create multitasking display systems. Special interest to
recombinant HBV proteins was dictated also by their medical significance as possible
diagnostic and vaccine tools. In general, novel generation of particulate models for
presenting of foreign polypeptide sequences were elaborated on the basis of cloned and
expressed hepadnaviral and leviviridal structural genes.
First of all, full-length HBV genome of Latvian origin, subtype ayw, so-called HBV320,
was cloned. and its genes were subjected to expression in Escherichia coli cells, with
special attention to the expression of short form of gene S (SHBs), which was expressed
directly from its ATG and as a fusion protein with N-terminal fragments of bacterial
antibiotic-resistance proteins. Full-length region preS and gene X of HBV genome were
also expressed in E.COLI. Moreover, HBV320 served as a source of HBV genes for
expression in bacteria, yeast and mammalian systems world-widely.
The gene C of RNA phage fr was expressed in E.coli cells and evaluated on the
dependence on the used promoters and the presence of original S'-upstream
sequences. In contrast to earlier data, the level of production of fr coats was found
practically independent of the 5'-upstream sequences. Deletion, insertion and
substitution mutagenesis allowed us to localize regions of the fr coat protein, which are
capable to include foreign amino acid insertions without an appreciable effect on the
capsid self-assembly. N- and C-terminal insertions (positions 2 and 129), as well as the
insertion into codon 51 in the RNA-binding region were found dispensable for
self-assembly, to some extent, in opposite to α-helical region 96-112, that was
characterized as absolutely unappropriate for introduction of foreign sequences.
Insertion of oligonucleotide linkers, coding short amino acid sequences and bearing also
convenient restriction sites, into different sites of the fr gene C, first of all, into its N- and
C-terminal regions, lead to creation of a set of first display vectors on the basis of the fr
coat.
Full-length gene C of RNA phage Qβ was cloned in E.coli by reverse transcription polymerase chain reaction (RT-PCR) technique and expressed in both short (Qβ CP)
and extended (Qβ protein A1) forms. Cloned gene C of Qβ genome was employed to
construct new class of display vectors, which appeared to have the greatest promise for
exposure of foreign sequences by enabling the formation of "mosaic" structures. As the
first model epitopes, fragments of HBV pre31 (as a short epitope consisting of 4-6 amino
acid residues) and HIV-1 gp120 (V3 loop, as a 39 amino acid sequence) proteins have
been empoyed.
In order to extend our studies to elucidation of basic principles of organization and
self-assembly of full virion particles, we tried to express structural genes S and C of
cloned HBV genome in homologous mammalian system under the control of strong
eukaryotic CMV promoter within pcDNA3 plasmid. Further, we chose the Semliki Forest
virus (SFV) model as the most promising system for modeling of intimate details of HBV
self-assembly. All structural HBV genes were expressed in SFV vectors pSFV1 and
pSFVC, allowing direct and SFV core-fused production of target proteins, respectively.
Maximal production of HBV proteins was achieved using pSFVC vectors where target
products were obtained after processing of SFV core-HBV protein fusions.
The data documented in the present thesis are of considerable importance for further
studies in protein engineering of particulate carriers, construction of DNA and RNA
vaccines, gene therapy tools, as well as in molecular mechanisms of complicated viral
processes, such as self-assembly, budding, attachment to cell receptors and entry into
the target cell.
The present work is described in 16 scientific papers and 12 patents, it was
presented in about 100 international and local conferences, 8 scientific papers in
peer-reviewed journals were published before defending of Cand.biol. Award of
Latvian Academy of Sciences, 1992.
Papers (after defending of Cand.biol. in 1985)
I.
Pumpen, P.P., Kozlovskaya. T.M. Borisova, G.P., Bichko, V.V., Pushko, P.P.,
Dishler, A.V., Kalis, J.V., Gren, E.J., Tsibinogin, V.V. & Kukaine, R.A. (1985).
Synthesis of antigens of hepatitis B virus in the bacterial cell. Biotechnologiya, 2,
II.
II. Kozlovskaia. T.M. & Pumpen, P.P. (1986). Structure and expression of
human hepatitis B virus surface antigen. Mot. Biol. (Mosk) 20, 884-901.
III.
III. Kozlovskaia. T.M., Pumpen, P.P., Dreilinia, D.E., Tsimanis, A.lu,, Ose, V.P.,
Tsibinogin, V.V. & Gren, E.J. (1986). Formation of capsid-like structures as a
result of expression of the cloned gene of the envelope protein of the
RNA-containing bacteriophage fr. Dokl. Akad. Nauk SSSR, 287, 452-455.
IV.
IV. Pushko, P.M., Kozlovskaia, T.M.. Dreilina, D.E. & Pumpen, P.P. (1987).
Application of DNA dideoxysequencing for the direct selection of recombinant
clones. Preprint. Riga. Institute of Organic Synthesis of Latv. Acad. Sci. 10 pp.
V.
V.Borisova, G., Bundule, M., Grinstein, E., Dreilina, D., Dreimane, A., Kalis, J.,
Kozlovskaya. T, Loseva, V., Ose, V., Pumpen, P., Pushko, P., Snikere, D.,
Stankevica, E., Tsibinogin, V. & Gren, E.J. (1987) Recombinant capsid structures
for exposure of protein antigenic epitopes. Mol. Gen. (Life Sci. Adv.) 6, 169-174.
VI.
Kozlovskaya. T.M.. Sominskaya, I.V., Sergeyeva, S.M., Tsibinogin, V.V.,
Pumpen, P.P. & Gren, E.J. (1988). Genetically engineering's approaches to
direct investigation of Dane particles and antibodies against them for diagnostic
purpose. In "Novels in hepatology:methods, facts, conceptions" (A.F.BIuger, Ed),
(in Russian), pp.40-49. RMI Press, Riga
VII.
. Kozlovskaia. T.M.. Pushko, P.M., Stankevich, E.I., Dreimane, A.la., Sniker,
D.la., Grinstein, E.E., Dreilina, D.E., Vejina, A.E., Ose, V.P., Pumpen, P.P. &
Gren, E.J. (1988). Genetically engineered mutants of the envelope protein of the
RNA-containing bacteriophage fr. Mol. Biol. (Mosk) 22, 731-740
VIII.
Borisova, G.P., Bundule, M.A., Grinstein, E.E., Dishler, A.V., Dreilina, D.E.,
Dreimane, A.J.., Kalis, J.V., Kozlovskaya. T.M . Loseva, V.J, Ose, V.P., Pushko,
P.M., Pumpen, P.P., Snikere, D.J., Stankevica, E.I., Tsibinogin, V.V., Shklennik,
R.C. & Gren, E.J. (1989). Structure of viral antigens and expression of antigenic
determinants in bacteria. In: "Search for physiologically active compounds".
Collection of papers of 7th Ail-Union Symp., Riga, pp. 286-295.
IX.
Ulrich, R., Pushko, P., Kozlovskaya. T. Siakkou, H., Pumpen, P, Dreilina, D. &
Platzer, C. (1991) Precise localization of the epitope of major BLV envelope
protein. Acta Virol. 35, 302.
X.
Pumpen, P., Tzybinogin, V., Kozlovskaya. T.. Borisova, G., Bychko, V.,
Pushko, P., Dyshler, A., Kalis, Ya., Dreylinya, D., Loseva, V. & Gren, E. (1992).
Cloning and expression of hepatitis B viral genome. In: Sov. Med. Rev. E:
Virology Reviews, Harwood Academic Publishers GmbH, 5, pp. 43-83.
XI.
Sominskaya, I., Pushko, P., Dreilina, D., Kozlovskaya. T. & Pumpen, P. (1992).
Determination of the minimal length of preS1 epitope recognized by a monoclonal
antibody which inhibits attachment of hepatitis B virus to hepatocytes. Med.
Microbiol. Immunol. 181, 215-226.
XII.
Pushko, P., Kozlovskaya. T.. Sominskaya, I., Brede, A., Stankevica, E., Ose, V.,
Pumpens, P. & Grens, E. (1993) Analysis of RNA phage fr coat protein assembly
by insertion, deletion and substitution mutagenesis. Protein Eng. 6, 883-891.
XIII.
Kozlovska. T.M.. Cielens, I., Dreilina, D., Dislers, A., Baumanis, V., Ose, V. &
Pumpens, P. (1993). Recombinant RNA phage Qp capsid particles synthesized
and self-assembled in Escherichia coli. Gene, 137, 133-137.
XIV.
.Kozlovska. T.M.. Cielens, I., Vasiljeva, I., Strelnikova, A., Kazaks, A., Dislers, A.,
Dreilina, D., Ose, V., Gusars, I. & Pumpens, P. (1996). RNA phage Qp coat
protein as a carrier for foreign epitopes. Intervirology, 39, 9-15.
XV.
Kozlovska. T.M.. Cielens, I., Vasiljeva, I., Bundule, M., Strelnikova, A., Kazaks,
A.., Dislers, A.., Dreilina, D.., Ose, V., Gusars, I. & Pumpens, P. (1997). Display
vectors. II. Recombinant capsid of RNA bacteriophage Qp as a display moiety.
Proc. Latv.Acad.Sci. 51, 8-12.
XVI.
I.Kozlovska. T.M.. Ose, V. & Garoff, H. (1997). Synthesis of all hepatitis B
structural proteins in Semliki Forest Virus expression system. Prepared for
publication.