Virus Genes

, Volume 41, Issue 3, pp 309–318

Interaction of orthopoxviruses with the cellular ubiquitin-ligase system

Authors

    • Institute of Cytology and GeneticsSiberian Branch of the Russian Academy of Sciences
Article

DOI: 10.1007/s11262-010-0519-y

Cite this article as:
Shchelkunov, S.N. Virus Genes (2010) 41: 309. doi:10.1007/s11262-010-0519-y

Abstract

Protein modification by ubiquitin or ubiquitin-like polypeptides is important for the fate and functions of the majority of proteins in the eukaryotic cell and can be involved in regulation of various biological processes, including protein metabolism (degradation), protein transport to several cellular compartments, rearrangement of cytoskeleton, and transcription of cytoprotective genes. The accumulated experimental data suggest that the ankyrin-F-box-like and BTB-kelch-like proteins of orthopoxviruses, represented by the largest viral multigene families, interact with the cellular Cullin-1- and Cullin-3-containing ubiquitin-protein ligases, respectively. In addition, orthopoxviruses code for their own RING-domain-containing ubiquitin ligase. In this review, this author discusses the differences between variola (smallpox), monkeypox, cowpox, vaccinia, and ectromelia (mousepox) viruses in the organization of ankyrin-F-box and BTB-kelch protein families and their likely functions.

Keywords

OrthopoxvirusSmallpox virusHost range genesAnkyrin-F-boxBTB-kelchUbiquitin-protein ligase

Ubiquitin ligase cascade

It has been recently discovered that protein degradation is among the most important elements of numerous cell processes [1, 2]. In the majority of cases, proteins in eukaryotic cells are degraded via an ubiquitin-directed pathway. Ubiquitin (Ub) comprises 76 amino acid (AA) residues and is among the most evolutionarily conserved polypeptides; Ub is covalently attached to target proteins by a coordinated action of three enzyme classes [3].

The ubiquitin-activating enzyme (E1) cleaves ATP to form a thioester bond between the Ub C-end and the cysteine in the active site of this enzyme. Thus, activated Ub is then transferred to the ubiquitin-conjugating enzyme (E2), also forming a thioester bond. E2–Ub interacts with ubiquitin-protein ligase (E3), which concurrently binds the target protein, frequently named the substrate. E3 causes transfer of the Ub from E2–Ub complex to the substrate by formation of the covalent isopeptide bond between the Ub C-end and the lysine residues in target protein (substrate). Attachment of a single Ub can change the function or localization of the protein in the cell. A tandem attachment of Ub molecules, producing a polyubiquitin chain, can also modify the function or cell localization of target protein or causes involvement of such protein in degradation by the cellular 26S proteasome leading to protein cleavage into short peptides and Ub release [1].

Ubiquitin is the first member of the ever increasing family of ubiquitin-like (Ubl) proteins, which are also involved in modification of various proteins and their functions. Such modification processes are frequently of transient character because of existence of Ub/Ubl-deconjugating enzymes (Ub/Ubl-specific proteases) along with Ub/Ubl-conjugating enzymes. It has been discovered that Ubl attachment to target protein can frequently enhance the interaction of this modified protein with other proteins or, on the contrary, block its interaction with the target [3].

The eukaryotic organisms encode very few or even single E1 (ubiquitin-activating) enzyme. The diversity of E2 (ubiquitin-conjugating) enzymes is considerably larger. For example, over 50 such enzymes have been found in humans [4]. As for E3 (ubiquitin-protein ligases), five main groups of such enzymes are known so far [5]. In this review, this author considers only two groups of E3 Ub-protein ligases related to poxviruses.

The RING-domain-containing Ub ligases have the simplest organization—a single polypeptide containing a substrate binding site. E2–Ub binds to the RING domain to transfer Ub to the substrate protein, which is in complex with such E3 ligase [6].

The most numerous group contains cullin-RING ubiquitin ligases (CRLs), multisubunit complexes comprising cullin proteins [7], RING H2 finger proteins (designated Rbx1, Roc1, or Hrt1) [6], variable substrate-recognition subunit (SRS), and, for the majority of CRLs, additional adaptor proteins uniting SRS with other CRL proteins [2]. Proteins of the cullin family are hydrophobic proteins playing the role of a backbone for assembly of the CRL complex [2, 7]. The CRL containing cullin-1 (CUL1), named SCF complex, has been most intensively studied. This complex comprises four subunits—Skp1, CUL1, F-box-containing protein, and Rbx1 (Fig. 1a). The N-end of CUL1 protein binds to the Skp1 adaptor, which, in turn, interacts with F-box-containing protein. The C-terminal part of CUL1 binds to Rbx1 protein, whose function is in the interaction with E2–Ub. In turn, F-box-containing protein [8, 9] provides for the interaction with substrate protein, which is ubiquitinated by the complex (see Fig. 1a).
https://static-content.springer.com/image/art%3A10.1007%2Fs11262-010-0519-y/MediaObjects/11262_2010_519_Fig1_HTML.gif
Fig. 1

A schematic representation of two classes of orthopoxvirus E3 ubiquitin ligases: a SCF E3 ligase; b BTB-kelch/Cul3 E3 ligase

The F-box motif is a specific sequence of 45–50 AA residues providing for protein–protein interaction. It was designated according to the protein cyclin F, where it was discovered. The F-box-containing proteins are abundant in eukaryotic organisms. At least 38 proteins of this family have been found in humans. Along with F-box, the majority of these proteins contain other domains causing specific protein–protein interactions [8, 9].

The adaptor protein Skp1 (humans contain three genes encoding proteins of this subgroup) contains the so-called BTB domain at its N-end; this domain provides for Skp1 binding to CUL1. The C-terminal sequence of Skp1 specifically interacts with the F-box motifs of numerous proteins, which realize interaction with substrate proteins via their additional domains of various types and further ubiquitination of these substrate proteins [10].

The CUL3-containing CRL complexes contain Rbx1; however, they differ from the other studied CRL classes by the absence of adaptor proteins [11]. The BTB-domain-containing protein, accomplishing binding with substrate protein of the complex via another additional domain [10], directly interacts with the N-terminal CUL3 region (Fig. 1b). Initially, BTB domain was detected as a conserved motif in Drosophila proteins, the so-called Bric-a-brac, Tramtrack, and Broad complex transcription regulators [12]. It has been demonstrated that this domain is important for protein–protein interaction. The BTB protein family is abundant in eukaryotes. Many of these proteins also contain additional domains providing for other specific protein–protein recognition. For example, over 150 human, two-domain BTB proteins have been discovered [10].

Thus, the information accumulated so far demonstrates that a tremendous diversity of CRL complexes can be formed in mammalian cells. This agrees with the modern understanding that the modification of proteins by ubiquitin or ubiquitin-like polypeptides is important for the fate and functioning of the majority of proteins in eukaryotic cell and can be involved in regulation of various biological processes, including protein metabolism (degradation), protein transport to several cell compartments, rearrangement of cytoskeleton, transcription of cytoprotective genes, and DNA repair [1315].

Orthopoxviruses and ubiquitin-ligase system

Taking into account the importance of ubiquitin-ligase and ubiquitin–proteasome systems for the function of eukaryotic cells, the role of viruses in regulation of these processes has been intensively studied recently. Although the data on this topic are sparse, it has been already discovered that viruses of various families can influence the protein ubiquitination to overcome the cell defense mechanisms, including apoptosis, type I interferon response, and antigen presentation by the class I major histocompatibility complex [14, 16, 17]. The issue of our interest is orthopoxviruses, including smallpox (variola, VARV), monkeypox (MPXV), cowpox (CPXV), vaccinia (VACV), and ectromelia (ECTV) viruses. The developmental cycle of orthopoxviruses takes place in the cell cytoplasm. Orthopoxviruses replicate in the discrete cytoplasmic structures called virus factories or virosomes. These structures are encompassed by endoplasmic reticulum membranes, resembling cytoplasmic mininuclei [18].

Recent experiments with VACV have demonstrated that proteasome inhibitors interfere with formation of virus factories in the cytoplasm of permissive cells and, as a consequence, lead to a radical decrease in virus reproduction [19, 20]. These results suggest that a normal development of orthopoxvirus infection requires a functioning ubiquitin–proteasome system. Since it has been discovered that ubiquitin constitutes at least 3% of the total protein in VACV virions [21], this suggests that either the ubiquitin-ligase system is important for modification of virus proteins and assembly of virus particles or ubiquitin-modified proteins are packaged into virions for further involvement in the early infection stages in sensitive cells.

It has been found that the modification of VACV protein A40R by SUMO-1, an ubiquitin-like protein (polypeptide with a size of 101 AA residues), is necessary for specific localization of this protein in the virus factories and normal virus replication. Moreover, it has been demonstrated that the SUMO modification prevents formation of A40R aggregates [22, 23].

The first orthopoxvirus ubiquitin ligase belonging to the class of single-subunit RING-domain-containing E3 ligases was discovered in ECTV. As was initially demonstrated, the RING-domain-containing protein p28 of ECTV is a virulence factor and inhibits the cell apoptosis induced by tumor necrosis factor [24]. This viral protein in the cells is localized to the cytoplasmic virus factories and is necessary for virus replication in macrophages [25]. Taking into account the accumulating information about the class of RING-domain-containing ubiquitin ligases, the properties of ECTV and VARV p28 protein were studied; it has been demonstrated that this protein itself performs the functions of ubiquitin ligase [26]. The gene encoding this protein (D4R in VARV-IND [27]) is transcribed from the virus early/intermediate promoter. This gene is highly conserved in VARV, MPXV, CPXV, and ECTV but is inactivated in the studied VACV strains [26, 28]. The molecular target (viral or cellular protein) for the orthopoxvirus ubiquitin ligase p28 has not been yet identified.

An orthopoxvirus proteins with ankyrin repeats as the factors determining virus host range

The discovery of F-box domain and study of its function in the formation of SCF ubiquitin ligases (CUL1 ubiquitin–ligase complexes) initiated a computer search for F-box-containing proteins in the available databases of protein sequences. It has been shown that many poxvirus ankyrin-like (ANK) proteins contain F-box sequences at their C-ends [29]. Such combination of domains is characteristic of poxviruses only. In the cellular proteins, F-box domain is usually localized to the N-terminal part. In addition, the combination of ANK and F-box domains has not been found in cellular proteins. Another specific feature of the poxvirus ANK proteins is that they most frequently contain at their C-end two or even one α-helical region of the three helices characteristic of a full-sized F-box [3032]. The analysis performed by this author allowed detection of F-box sequences in the C-terminal regions in 13 of the 14 CPXV ANK proteins (Fig. 2). No sequences homologous to F-box were detected only in the shortest ANK protein M1L (K1L for VACV-COP; Table 1).
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Fig. 2

Amino acid alignment of the C-terminal F-box sequences of the ANK proteins of CPXV strain GRI-90. Lines indicate location of three α-helices of the typical cellular F-box [32]. The residues corresponding to F-box consensus sequence (CON) are marked with gray blocks. An asterisk indicates the end of the protein. The names of the proteins with experimentally confirmed interaction with the cellular SCF complex are marked with hash

Table 1

Orthopoxviral ankyrin-like proteins

VAR-IND

VAR-GAR

MPV-ZAI

CPV-GRI

VAC-COP

VAC-MVA

ORF

Size, aa

ORF

Size, aa

ORF

Size, aa

ORF

Size, aa

ORF

Size, aa

ORF

Size, aa

J3L*

587

D3L*

586

C19L*

259

D4L*

672

C17L*

386

4L*

233

C15L*

91

D8L

661

D14L

764

D1L*

437

C1L

437

C3L

833

D6L

452

B8L

355

D7L

660

C9L

668

D7L

153

B12L

132

D9L

630

C11L

614

C9L

634

16L

297

O1L

446

Q1L

449

O1L

442

O1L

474

M1L

472

C1L

66

P1L

66

C1L

284

M1L

284

K1L

284

22L

98

B6R

558

H6R

558

B5R

561

B3R

558

B4R

558

171R

177

B19R

574

D8R

574

B16R

574

B18R

574

186R

574

B21R

787

D10R

787

B17R

793

B18R

795

B20R

127

K1R

581

B21R*

91

N4R*

437

H2R*

672

B23R*

386

190R*

233

G1R

585

G1R

585

J1R*

587

H3R*

586

B25R*

259

Asterisks denote a ORFs that are duplicated in left and right inverted terminal repeat regions of the viral genome

ORFs with full length are italicized. The ORFs for the proteins with experimentally confirmed interaction with the cellular SCF complex are indicated by bold italic letters. Bold letters show the ORFs lacking the F-box sequence

An ANK repeat, a motif with a length of around 33 AA residues, was detected in ankyrin, a cytoskeleton protein containing 24 copies of this repeat [33]. ANK proteins have been discovered in all the three superkingdoms (Bacteria, Archaea, and Eukarya) and are the most abundant in eukaryotes [34]. Most frequently, proteins contain from two to six ANK repeats. Some proteins contain only ANK repeats, while the other also carry additional domains. ANK domains determine protein–protein interactions. The proteins of this family and the complexes they form are necessary for many cellular processes, such as determination of cell fate, endocytosis, transcription regulation, cell cycle control, and can be involved in the determination of host range (hr) of the viruses [35].

Metabolism of animal viruses depending on their specific features is directed to utilize the structures of the cell cytoplasmic or nuclear protein skeleton. In particular, it is assumed that the virus-specific cytopathic effect is determined not by cell damage meaningless from the virus standpoint but rather by a specific rearrangement of cytoskeleton elements that would create the conditions for virus reproduction [36, 37]. The cytoskeleton proteins are encoded by a large set of various genes with a tissue-specific expression. This determines the difference between the compositions of protein “backbone” in different cell types, which influences the functions of these cells [38]. These differences can influence the parameter of virus replication in the host organism, such as tissue tropism. In addition, the protein composition of cytoskeleton in various mammalian species (or cell cultures) can influence the overall sensitivity to certain viruses and determine the hr.

It is known that individual orthopoxvirus species considerably differ in the range of animal species where they can reproduce. For example, CPXV has a very wide range of sensitive hosts [39], and VACV also can reproduce in various animal species, although in a local manner at the infection site. On the contrary, VARV and ECTV have a very narrow hr: VARV under natural conditions infects only humans and ECTV only some mouse strains [40]. This diversity of orthopoxviruses with respect to the range of sensitive hosts gives the hope to detect the virus genes that control these properties. Active attempts to find the genes determining hr are undertaken. Unfortunately, these genes have not been identified for the in vivo system (according to the change in the range of sensitive animals). However, several genes determining the ability of viruses to reproduce in particular mammalian cell lines have been found for in vitro systems [41, 42].

Unlike CPXV, VACV is unable to reproduce in the Chinese hamster ovary (CHO) cell line. It has been shown that the gene named CHOhr (C9L in CPXV strain GRI-90 [28], Table 1) and encoding a 77-kDa protein is responsible for this ability of CPXV [43]. Integration of this CPXV gene into the VACV genome renders VACV able to reproduce in CHO cells. In addition, the gene K1L was identified in VACV genome [41, 44]; a damage of this gene makes the virus unable to reproduce in the rabbit kidney cell culture RK13. Note that the CPXV CHOhr gene is able to compensate for the function of VACV gene K1L but not vice versa, i.e., it is dominant relative to another hr gene [41]. The proteins CHOhr and K1L do not display a pronounced similarity in their AA sequences; however, their important specific feature is that they belong to the family of ANK proteins [33, 35, 45]. One of the functions of these proteins is to provide for the interaction between cellular integral membrane proteins and elements of cytoskeleton.

Taking into account the importance of ANK proteins in determining the virus properties, in particular, the hr, we performed a computer analysis of the AA sequences of open reading frames (ORFs) of VACV strains Copenhagen (VACV-COP) and MVA (VACV-MVA), CPXV strain GRI-90 (CPXV-GRI), MPXV strain Zaire-96 (MPXV-ZAI), and VARV strains India-1967 (VARV-IND) and Garcia-1966 (VARV-GAR). This analysis has demonstrated [28] that orthopoxviruses code for a large set of ANK proteins (Table 1). This is the largest family of orthopoxvirus proteins; moreover, each species has its specific set of the corresponding genes [46]. Of the natural orthopoxviruses, CPXV, displaying the widest hr, has 14 unique ANK genes in its genome (two of them are duplicated in the terminal genomic regions); MPXV, eight such genes; and VARV, a stringently anthroponosic virus, five ANK genes (Fig. 3, Table 1), VACV-COP encodes five ANK proteins (three match the VARV proteins), while the highly attenuated variant VACV-MVA, obtained by multiple passages in chick embryo fibroblasts and having a very narrow range of sensitive cell cultures, retained only one ANK gene (Table 1) [47, 48]. Note that the ANK proteins of orthopoxviruses display a low homology even in the regions of ANK repeats (about 25%), which can suggest difference in their properties. Different sets of ANK proteins and the level of their synthesis are likely to provide for fine regulation and determination of the tissue tropism of virus reproduction in the organism of infected animal.
https://static-content.springer.com/image/art%3A10.1007%2Fs11262-010-0519-y/MediaObjects/11262_2010_519_Fig3_HTML.gif
Fig. 3

Graphical alignment of a left and b right terminal species-specific genomic regions of CPXV-GRI, MPXV-ZAI, VACV-COP, VARV-IND, and VARV-GAR [28]. Arrows indicate location, direction, and size of ankyrin-like ORFs. Names of ORFs are given above the arrows. Asterisks mark the genes whose host range function was demonstrated experimentally. Fine lines indicate deletions in DNAs of one virus relative to the others

Further studies have demonstrated that the product of the gene CHOhr in CHO cells prevents the translation blocking of virus intermediate mRNAs, thereby providing expression of the virus late genes [49]. The VACV K1L gene in RK13 cells induces synthesis of the cell factor VITF-2, necessary for transcription of the virus intermediate genes [50]. In the cells permissive for VACV K1L, VITF-2 is synthesized in the absence of any infection. In addition, it has been demonstrated for VACV K1L that translation of the virus early mRNAs is blocked in RK13 cells. Introduction of gene CHOhr (CP77) into the genome of VACV K1L renders VACV K1LCP77+ able to reproduce in RK13 cell line, although forming plaques on cell monolayer of smaller size as compared with the wild-type virus. Moreover, this virus variant initially (during the first 6 h) behaves in RK13 cells as VACV K1L, i.e., blocking of early protein synthesis is observed; however, the synthesis of virus early proteins is then induced, and the virus undergoes the complete developmental cycle [51].

It is known that viral infections activate the cell antiviral signaling and inflammatory responses. The nuclear factor kappa-B (NF-κB), which regulates transcription of the genes involved in development of the immune response, inflammation, apoptosis, and cell proliferation [52], plays an important role in these responses. Activation of NF-κB is controlled by ANK proteins of the IκB family, which interact with this factor. In an inactive form, NF-κB (the dimer p65/p50) is bound to the inhibitory protein IκBα, which via six ANK repeats interacts with p65 subunit. In response to molecular signals of infection, IκB kinase (IKK) is phosphorylated by cellular protein kinase to phosphorylate IκBα at the serine residues at positions 32 and 36. The phosphorylated IκBα is polyubiquinated by the SCF complex at lysine 48 and degraded by the 26S proteasome complex, thereby removing the NF-κB inhibition; this factor moves to the cell nucleus and stimulates gene transcription via the interaction with specific DNA sequences [30].

Different VACV strains inhibit activation of the cellular transcription factor NF-κB, thereby providing inhibition of inflammatory response development, which is among the first reactions of nonspecific protection from infectious agents. It was demonstrated that the highly attenuated VACV strain MVA failed to inhibit NF-κB activation [53]. Recombination-based introduction of the K1L gene from VACV strain WR to the MVA genome restored the ability of the virus to inhibit the activation of cellular factor NF-κB [47]. It is assumed that the ANK-repeat-containing virus protein K1L can inhibit degradation of the cellular IκBα via competing with it for phosphorylation by the enzyme IKK and subsequent ubiquitination and degradation. Another ANK-containing protein, CPXV CHOhr (not synthesized by VACV) is likely to act in an analogous manner [30] and, consequently, is able to rescue the mutation VACV K1L (see above).

Recently, it has been experimentally demonstrated for the ANK-F-box proteins C9L (CHOhr) of CPXV [30], G1R of VARV (D3L/H3R in CPXV) [54], 186R of VACV-MVA (B16R in CPXV) [55], as well as EVM002, EVM005, and EVM154 of ECTV (D3L/H3L, D8L, and B3L in CPXV) [56] (see Table 1) that they interact with the cellular SCF complex and, presumably, provide for specific interaction with substrate proteins of cellular or viral origins, which are then ubiquitinated by this complex. An important problem is to detect these substrate proteins for each of the numerous orthopoxvirus proteins of the considered family. This will enhance understanding of the functions of these virus proteins.

BTB-kelch proteins of orthopoxviruses

When studying the kelch gene of Drosophila, it was found that it encoded the protein with a size of 688 AA residues containing in its C-terminal region six copies of tandemly arranged elements with a length of about 50 AA residues [57]. The detected repeat was named as kelch motif [58] and the block of these repeats, a kelch domain. It was also found that kelch protein contains a BTB domain in its N-terminal region [12]. It has been experimentally demonstrated that kelch domain is necessary for kelch protein to bind to the actin of cell filaments and the dimerization of two BTB domains of two actin-bound kelch proteins results in a cross interaction of these filaments, leading to formation of Drosophila ovarian intercellular ring channels [59].

The search for kelch repeats and BTB domains over the databases of AA sequences suggested that both motifs were ancient and had widely spread among organisms of various types during the evolution [60, 61]. The superfamily of proteins containing kelch repeats was formed. This protein family is involved in various aspects of cell functioning, such as rearrangement of cytoskeleton and cell plasma membrane, regulation of gene expression, and mRNA splicing. As has been shown, the identity between individual kelch motifs is not high, about 20–25% for six motifs of one Drosophila kelch protein; this value can even decrease to 11% when comparing individual motifs from different proteins [58, 60]. An important specific feature of kelch motif is the presence of duplicated glycine residue and specifically arranged aromatic AAs [57, 58]. The number of kelch motifs in various proteins usually varies from four to seven. In addition, the location of kelch domain can be different, which divides the kelch-like proteins into five groups [60].

Among all the viruses, only the representatives of the family Poxviridae contain the genes of kelch-like proteins in their genomes. According to structural similarity, they are ascribed to the same group as Drosophila kelch protein (BTB-kelch) [6264]. These proteins contain the N-terminal BTB domain and C-terminal kelch domain. Computer analysis of orthopoxvirus genomes has demonstrated that CPXV codes for six BTB-kelch family proteins with a size of about 500 AA residues each and mutual identity in AA sequence in the range of 22–26%. VACV genome codes for only three full-sized kelch-like proteins, which are highly homologous to the corresponding CPXV proteins; as for the highly attenuated strain VACV-MVA, unable to replicate in the majority of mammalian cell lines, it retained only one gene of this family [48]. The same gene is the only gene in MPXV genome encoding a BTB-kelch protein. As for VARV genome, all the genes of this family are destroyed due to multiple mutations; consequently, only short potential ORFs, which are nonfunctional fragments of the genes of a precursor virus, are detectable in this virus [65] (Fig. 4; Table 2). ECTV codes for the four genes of the considered family—EVM18, EVM27, EVM150, and EVM167, which correspond to the CPXV-GRI genes C18L, G3L, A57R, and B19R [66, 67].
https://static-content.springer.com/image/art%3A10.1007%2Fs11262-010-0519-y/MediaObjects/11262_2010_519_Fig4_HTML.gif
Fig. 4

Graphical alignment of a left and b right terminal species-specific genomic regions of CPXV-GRI, MPXV-ZAI, VACV-COP, VARV-IND, and VARV-GAR [28]. Arrows indicate location, direction, and size of BTB-kelch-like ORFs. Names of ORFs are given above the arrows. Fine lines indicate deletions in DNAs of one virus relative to the others

Table 2

Orthopoxviral kelch-like proteins

VAR-IND

VAR-GAR

MPV-ZAI

CPV-GRI

VAC-COP

VAC-MVA

ORF

Size, aa

ORF

Size, aa

ORF

Size, aa

ORF

Size, aa

ORF

Size, aa

ORF

Size, aa

D11L

521

D13L

201

B19L

154

D15L

105

C18L

512

C2L

512

D16L

77

D13.5L

79

B20L

65

D17L

98

D18L

107

C7L

179

E3L

179

C9L

487

G3L

485

F3L

480

31L

476

J7R

71

K7R

71

A57R

564

A55R

564

J8R

172

K8R

70

B1R

70

B9R

501

B10R

166

178R

158

B22R

70

D11R

70

B18R

70

B19R

557

B23R

83

D12R

127

B24R

88

D13R

88

ORFs with full length are italicized. The ORFs for the proteins with experimentally confirmed interaction with the cellular CUL3-containing CRL complex are indicated by bold italic letters

The absence of the genes from this family in various VARV isolates and the possibility of their deleting in VACV without any loss in its viability in cell culture [68] indicate that these genes are not vitally important for orthopoxviruses. Presumably, these genes are important for manifestation of species-specific properties of orthopoxviruses in vivo. It has been assumed that these genes can play a role in adaptation, i.e., they can determine the hr (tissue tropism) and/or the possibility of virus persistence in animal body [66]. In particular, CPXV, low pathogenic for humans and displaying the widest range of sensitive animals in nature, codes for the largest set of BTB-kelch proteins. In VARV, highly pathogenic for its only host, human, in organism of which this virus cannot persist, all the genes of BTB-kelch subfamily are mutationally destroyed.

The available data suggest that various BTB-kelch proteins interact with CUL3 (Fig. 1b) rather than with the other cullins, i.e., BTB-kelch proteins are substrate-specific adaptors for CUL3 ubiquitin–ligase complex and regulate modification and/or degradation of various proteins [11]. Note that any BTB-kelch proteins must not obligatory be subunits of CUL3-containing E3 ligases but can be components of the protein complexes that determine modifications of cell morphology, modulation of cytoskeleton, elongation of pseudopodia, and so on [60, 69].

When studying the properties of orthopoxvirus BTB-kelch proteins, it has been found that the ECTV proteins EVM150 and EVM167 are involved in formation of active CUL3-containing ubiquitin ligases [70]. Two other proteins, EVM18 and EVM27, also interact with CUL3 [5]. Since the mutual homology of these viral proteins is low, it is likely that their functions are different and they interact with different targets. It has been experimentally demonstrated that a directed deletion of individual genes encoding EVM18, EVM27, or EVM167 radically decreases the ECTV virulence for white mice, while the damage of EVM150 gene has no effect on the virulence [71].

It was found for the sheeppox virus (genus Capripoxvirus, family Poxviridae), whose genome contains three BTB-kelch genes, that deletion of single gene 019, belonging to this family, led to a considerable decrease in the virulence of this pathogen for sheep [72].

So far, the CPXV mutants with directed deletions of individual genes from BTB-kelch family were obtained as well as with simultaneous deletions of two, three, or four genes of the six [71, 73, 74]. It has been found that the damage of genes belonging to the family in question has practically no effect on the in vitro virus reproduction in 11 studied continuous mammalian cell cultures except for the canine kidney cell line MDCK. In this line, the CPXV with three inactivated BTB-kelch genes gave considerably fewer progeny, while the CPXV variant with four deleted BTB-kelch genes completely lost the ability to reproduce. Successive removal of kelch-like genes led to a decrease in the size of pocks developed on chick embryo CAMs and in the virus yield as well as a decreased virulence for BALB/c mice. This suggests a dose-dependent effect of BTB-kelch genes on the in vivo properties of CPXV [74].

Electron microscopy examination detected the electron dense inclusions composed of the aggregates of thin fibers, amorphous substance, and tubular structures in the CAM cells infected with the CPXV deletion mutants for three BTB-kelch genes, which are absent in the cells infected with wild-type virus [73]. Presumably, this results from Ub/Ubl modification of yet unidentified virus and/or cellular proteins, which normally requires the virus BTB-kelch proteins encoded by the deleted genes. As is mentioned above, it has been experimentally determined that, for example, a SUMO modification prevents formation of aggregates of VACV protein A40R [22, 23]. Deletion of four CPXV BTB-kelch genes led to a decrease in the cytopathic effect on cell culture and statistically significant reduction in formation of the virus-induced cytoplasmic pseudopodia [71].

Deletion of individual kelch-like genes in the VACV genome provided for demonstrating that the damage of genes C2L or A55R (see Table 2) led to similar effects, appearing as changes in the morphology of virus plaques on cell culture monolayer, decrease in virus-induced cytoplasmic pseudopodia, decrease in Ca2+-independent adhesion of VACV-infected cells, and induction of larger lesions in the model of intradermal infection of mouse ear pinnae as compared with the wild-type virus [75, 76]. Damage of the VACV kelch-like gene F3L did not cause so pronounced effects [77]. Interestingly, this particular single BTB-kelch gene remained in the highly attenuated VACV strain MVA and MPXV (see Table 2).

Undoubtedly, further studies into the functions of kelch-like proteins encoded by poxviruses are necessary.

Conclusions

Poxviruses are eukaryotic DNA viruses with the developmental cycle taking place in the cellular cytoplasm [28]. These viruses encode a large set of proteins providing for extranuclear synthesis of virus mRNAs, replication of virus DNA, and assembly of complex virions and are involved in the regulation of multifactorial interactions of the virus with both individual cells and infected host organism. The unique properties of poxviruses attract close attention of researchers. The viruses belonging to the genus Orthopoxvirus are best studied among other viruses of the family Poxviridae, because this genus contains VARV, MPXV, CPXV, and VACV, pathogenic for humans.

It is believed that viruses during co-evolution with the host organism had incorporated into their genomes the coding sequences of various cellular genes and modified them for adapting to provide for their viability and preservation in the biosphere [64, 66]. Acquiring the knowledge about how viruses overcome numerous protective systems in the animal body, which are represented by molecular factors and cells of the immune system, we will not only get a deeper understanding, but also discover new patterns in organization and functioning of these most important mammalian organism responses directed against infectious agents. The virus that maintains the balance between its pathogenic effect on the host organism and the possibility of its effective development in the animal organism for a relatively long period is the best adapted from the evolutionary standpoint. Such virus is able to transmit efficiently from animal to animal under a low population density. Among orthopoxviruses, CPXV most pronouncedly displays such properties.

The CPXV genome has the largest size as compared with the other orthopoxviruses and contains the complete set of all the genes characteristic of other viruses from the genus Orthopoxvirus [66]. The fact that CPXV nonetheless does not display an increased virulence suggests that orthopoxviruses have a certain regulatory system. To describe this regulatory system, earlier I introduced the concept of buffer genes, whose role is to neutralize the negative effects developing in the body during infection [64]. Presumably, CPXV possesses the widest set of these genes as compared with VARV, MPXV, and VACV. We believe that the multigenic families of orthopoxvirus ANK and kelch-like proteins, considered above, can be of great importance in determining the interactions between the virus and infected host.

CPXV, which has the widest hr among all the orthopoxviruses, has also the largest set of genes encoding the proteins with multiple ankyrin repeats. At least two of these ANK genes belong to the hr genes. A comparatively low level of homology between the hr genes and other genes of the ANK family proteins although does not guarantee the identity of their biological functions but nonetheless suggest the involvement of the proteins belonging to this family in determination of the virus hr. Recent studies have demonstrated that many of orthopoxvirus ankyrin-F-box-like proteins interact with Cullin-1-containing ubiquitin-protein ligase. The ability of orthopoxvirus BTB-kelch-like proteins to interact with Cullin-3-containing ubiquitin-protein ligase to a considerable degree relates this family to the family of ANK proteins.

Most likely, the proteins of these two families are involved in organization of the multifactorial intricate system of interactions of virus proteins with one another and cellular components. We believe that such interactions can determine a wide range of animal tissues and species sensitive to CPXV as well as for the tolerant mode of relationships between this virus and the host. Destruction of the majority of the genes/proteins belonging to these two families, characteristic of VARV, is the most likely reason underlying a drastic narrowing of the VARV hr and its transition to an “aggressor” mode. Note that VARV retained five genes encoding ankyrin-F-box proteins, whereas the genes for BTB-kelch proteins are completely destroyed. The highly attenuated VACV strain MVA, unable to reproduce in the majority of mammalian cell cultures, retained only one gene for each ankyrin-F-box and BTB-kelch proteins (Tables 1, 2).

Undoubtedly, further studies into the properties of orthopoxvirus kelch-like and ANK proteins are necessary to acquire a deep understanding of the interactions between these viruses and both the infected cell and the overall host organism.

Acknowledgments

The study was supported by the Russian Foundation for Basic Research (Grant No. 09-04-00055a).

Copyright information

© Springer Science+Business Media, LLC 2010