1607-6729/02/0304- $27.00 © 2002

MAIK “Nauka /Interperiodica”0096

Doklady Biochemistry and Biophysics, Vol. 383, 2002, pp. 96–100. Translated from Doklady Akademii Nauk, Vol. 383, No. 2, 2002, pp. 271–275.Original Russian Text Copyright © 2002 by Shchelkunov, Totmenin, Kolosova, Sandakhchiev.

The

kelch

gene of

Drosophila

encodes the proteinwhich is a structural component of so-called annularchannels, which ensure the transport of cytoplasmiccomponents from the nurse cells to

Drosophila melan-ogaster

oocytes during ontogeny [1]. The amino acidsequence analysis showed that the kelch protein con-sisting of 688 amino acid residue contains six tandemrepeats comprised of approximately 50 amino acid res-idues each, which are located at the C-terminus of thisprotein. This repeat has been termed the kelch motif[2], and the group of these repeats is called the kelchdomain.

It was discovered that the kelch protein contains aso-called BTB domain at the N-terminus, which ischaracteristic of some other

Drosophila

proteins and iscomprised of approximately 115 amino acid residues[3]. Further studies showed that BTB domains are ableto dimerize and to interact with other BTB domains toform heterodimers [4].

It was shown that the kelch domain is required forbinding the kelch protein to the actin filaments in thecell. Dimerization of the BTB domains of two actin-associated kelch proteins results in cross-interactionbetween these filaments, which leads to formation ofintercellular annular channels of

Drosophila

oocytes [5].The search for the kelch repeats and BTB domains

in the databases showed that both motifs are ancientand have widely spread in different types of organismduring evolution [4, 6]. A superfamily of proteins con-taining the kelch repeats has been formed. The proteinsof this family are involved in different cellular func-tions, such as changes in the cytoskeleton and plasmamembrane, regulation of gene expression, mRNAsplicing, etc. The extent of homology between individ-ual kelch repeats in not high (25–50% for six motifs ofthe same kelch protein of

Drosophila

). This value maydecrease to 11% when comparing individual motifs ofdifferent proteins [2, 6]. An important characteristic ofthe kelch motif is a glycine duplex and a specificarrangement of aromatic amino acid residues [1, 2].

The number of the kelch motifs in different proteinsusually varies from four to seven. In addition, the loca-tion of the kelch domain in proteins may differ. Basedon the kelch motif location, kelch-like proteins aredivided into five groups [6]. Based on the structuralhomology, some poxviral proteins were classified withthe same group as the

Drosophila

kelch protein [1, 2, 6].

The purpose of this study was to analyze the organi-zation of orthopoxviral genes and the correspondingproteins belonging to the kelch family. Comparison ofgenomic nucleotide sequences of monkeypox, cowpox,variola, and vaccinia viruses allowed us to identify allorthopoxviral genes of the family studied and to revealsignificant species-specific differences in virus organi-zation. The explanations of the possible function of theunique multigenic kelch family and its evolution in theviral genome are suggested.

We analyzed the genomic nucleotide sequencesdetermined earlier for the monkeypox virus strain Zair-96-I-16 (MPV-ZAI, access number AF 346404 in theGenBank), variola virus strain India (VAR-IND) [7],cowpox virus strain GRI-90 (CPV-GRI) [8], as well asthe published data on the vaccinia virus strain Copen-hagen (VAC-COP) [9] using the BioEdit software [10].

First, we discovered that the genes encoding theorthopoxviral kelch-like proteins are located only in theterminal variable regions of the orthopoxviral genome(Fig. 1) and are characterized by tissue-specific differ-ences in the size of potentially encoded proteins (table).The cowpox virus encodes six proteins belonging to theanalyzed superfamily (with a size of approximately 500amino acid residues each) with 22–26% homology oftheir amino acid sequences. Vaccinia virus encodesonly three full-length kelch proteins (C2L, F3L, andA55R), which share much homology with the corre-sponding proteins of cowpox virus (99.4, 97.9, and98.6% homology, respectively). Monkeypox virusencodes only one kelch-like protein, C9L (97.3%homology with the CPV-GRI G3L). In variola virus, allpotential open reading frames (ORFs) are disrupted asa result of multiple mutations, and only short ORFs thatare nonfunctioinal fragments of the genes of the precur-sor virus have been found (Fig. 1, table).

Species-Specific Differences in the Organizationof Genes Encoding KELCH-like Proteins

of Orthopoxviruses Pathogenic for Humans

S. N. Shchelkunov, A. V. Totmenin, I. V. Kolosova, and

Academician

L. S. Sandakhchiev

Received November 1, 2001

State Research Center of Virology and Biotechnology“Vector,” Kol’tsovo, Novosibirsk oblast, 633159 Russia

BIOCHEMISTRY, BIOPHYSICS,AND MOLECULAR BIOLOGY

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SPECIES-SPECIFIC DIFFERENCES 97

Despite the abundance of the orthopoxviral kelch-like proteins, their function remains obscure. Theabsence of genes of this family in different isolates ofvariola virus [11] and the possibility of their deletionfrom vaccinia virus without loss of its viability in thecell culture [12] indicate that these genes are not vitalfor orthopoxviruses. Apparently, these genes areimportant for expression of species-specific properties

of orthopoxviruses

in vivo.

Earlier, we assumed thatthese genes may be assigned to “buffer” viral genes[13], which attenuate the adverse effect of virus infec-tion on the organism. These genes may also play anadaptive role, i.e., determine the spectrum of hosts orthe possibility of persistence of the virus in animals.For example, cowpox virus, which is characterized bythe broadest spectrum of susceptible animals and low

Fig. 1.

A scheme of ORF location in the kelch-like proteins of cowpox virus (CVP-GRI), monkeypox virus (MPV-ZAI), variolavirus (VAR-IND), and vaccinia virus (VAC-COP) in the (a) right and (b) left terminal variable regions of the orthopoxviral genome.The arrows indicate the direction of these ORFs (their denotations are shown above the arrows).

Comparative analysis of the ORFs of orthopoxviral kelch-like proteins

BOK-GRI BOB-COP BHO-IND BOO-ZAI

ORF length, amino acid resides ORF length, amino

acid resides ORF length, amino acid resides ORF length, amino

acid resides

D11L 521 – – – – – –

C18L 512 C2L 512 D13L 201 D16L 105

– – D17L 77

D13.5L 79 D18L 98

– – D19L 107

C3L 485 F3L 480 C7L 179 C9L 487

A54R 564 A55R 564 J7R 71 – –

J8R 172 B1R 70

B9R 501 B10R 166 – – – –

B19R 557 – – B22R 70 B18R 70

B23R 83 – –

B24R 88 – –

CPV-

MPV-

VAR-

VAC-

CPV-

MPV-

VAR-

VAC-

A54R B9R B19R

B18RB1R

B10RA55R

J7R J8R B22R-B24R

C18L G3L

D16L-D19L C9L

C7L

C2L

D13L D13.5L

F3L

(‡)

(b)

GRI

ZAI

IND

COP

D11L

GRI

ZAI

IND

COP

98

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et al

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pathogenicity for humans, encodes the largest numberof kelch proteins. In variola virus, which is highlypathogenic for the only host, human (in whose organ-ism its persistence is absent), all genes of the kelchfamily are disrupted by mutations.

All the six kelch-like CPV-GRI proteins contain theN-terminal BTB domain (Fig. 2) and the C-terminalkelch domain (Fig. 3). However, only the ORF of theA54R protein contains five classic kelch motifs. In theB9R protein, the C terminus lacks the duplicated gly-cine; in the G3L and C18L proteins, the fourth motifcontains a single glycine residue instead of the glycineduplex, and, in the fifth motif of the C18L protein, theglycine duplex is absent. In the B19R protein, the kelchmotifs are disrupted in the fourth and fifth repeats, andin the D11L protein, the first and third repeats containdisrupted kelch motifs (Fig. 3).

Each kelch repeat is comprised of four antiparallel

β

-chains (Fig. 3), which form a sheet or a so-calledblade of the

β

propeller formed by all repeats [14]. This

β

-propeller structure is, apparently, important for spe-cific protein–protein interactions. Note that each repeatin the region of

β

chains 2 and 3 (Fig. 3) has different

amino acid sequence and length. It is believed that theloops formed by these sequences ensure the specificityof protein–protein interaction of each individual

β

pro-peller [6].

Based on the results of comparative analysis per-formed in this study and our previously published dataon the organization of the CPV-GRI genome [8], it canbe concluded that the cowpox virus is the most ancientand can be regarded as an ancestor of other orthopoxvi-ruses pathogenic for humans. There are two mainexplanations of the low homology between the kelchproteins of the same virus and the high homologybetween the isologues of different orthopoxvirus spe-cies. The first explanation is based on the fact that DNAcopies of different kelch proteins of the same or differ-ent hosts were inserted into the genome of the precursorpoxvirus as the spectrum of hosts broadened. As aresult, the modern variant of cowpox virus occurred,from which other orthopoxvirus species originated.According to the second assumption, the codingsequence of one of the cellular kelch proteins was

Fig. 2.

Comparative analysis of the amino acid sequences of the N-terminal BTB domain of the kelch protein of

Drosophila

and thekelch-like proteins A54R, G3L, B19R, C18L, and D11L of the cowpox virus stain GRI-90. Black and gray vertical rectangles indi-cate the conserved and similar (with respect to physicochemical characteristics) amino acid residues, respectively, which weredetected in more than half the compared sequences. The number in parentheses on the left denotes the number of amino acid resi-dues of the kelch protein that are located upstream of this sequence. The numbers on the right of the analyzed sequences denote theposition of the last (right) amino acid residue in the line from the N terminus of the corresponding protein.

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inserted in the genome of the ancient poxvirus. There-after, this gene was multiplied, and each copy under-went long-term independent evolution within the sameviral genome. If this is true, the precursor orthopoxvi-rus has a very ancient origin, but its splitting to modernspecies occurred evolutionarily recently.

ACKNOWLEDGMENTS

We are grateful to Dr. J. Esposito for kindly pro-viding us with MPV-ZAI DNA preparations andP.F. Safronov and V.V. Gutorov, who kindly providedus with the data on MPV-ZAI genome sequence.

Fig. 3.

Comparative analysis of the amino acid sequences of (1–5) the kelch motifs of the A54R, B9R, G3L, C18L, B19R, and D11Lproteins of the cowpox virus strain GRI-90 and (1–6) the analogous motifs of the kelch protein of

Drosophila melanogaster.

Eachkelch motif contains four

β

chains denoted above with the arrows and numbered from 1 to 4. The dashes denote the deletions ofamino acid residues relative to other sequences compared. The numbers in parentheses denote the number of additional amino acidresidues contained in the given motif. Black vertical rectangles indicate glycine duplexes (GG) and other conserved amino acid res-idues. Gray vertical rectangles show the amino acid residues with similar physicochemical properties that were detected in morethan half the sequences compared. The numbers on the left and on the right denote the positions of the first and last amino acidresidue, respectively, in the kelch sequence of each protein.

A54R–1A54R–2A54R–3A54R–4A54R–5B9R–1B9R–2B9R–3B9R–4B9R–5G3L–1G3L–2G3L–3G3L–4G3L–5C18L–1C18L–2C18L–3C18L–4C18L–5B19R–1B19R–2B19R–3B19R–4B19R–5D11L–1D11L–2D11L–3D11L–4D11L–5kelch–1kelch–2kelch–3kelch–4kelch–5kelch–6

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This study was supported by the Russian Founda-tion for Basic Research (projects nos. 97-04-49716 and00-04-49558) and International Science and Technol-ogy Center (grant no. 884-2p).

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