Alexei V. Totmenin

Conserved surface-exposed K/R-X-K/R motifs and net positive charge on poxvirus complement control proteins serve as putative heparin binding sites and contribute to inhibition of molecular interactions with human endothelial cells: a novel mechanism for evasion of host defense.

ABSTRACT Vaccinia virus complement control protein (VCP) has been shown to possess the ability to inhibit both classical and alternative complement pathway activation. The newly found ability of this protein to bind to heparin has been shown in previous studies to result in uptake by mast cells, possibly promoting tissue persistence. It has also been shown to reduce chemotactic migration of leukocytes by blocking chemokine binding. In addition, this study shows that VCP—through its ability to bind to glycosaminoglycans (heparin-like molecules) on the surface of human endothelial cells—is able to block antibody binding to surface major histocompatibility complex class I molecules. Since heparin binding is critical for many functions of this protein, we have attempted to characterize the molecular basis for this interaction. Segments of this protein, generated by genetic engineering of the DNA encoding VCP into the Pichia pastoris expression system, were used to localize the regions with heparin binding activity. These regions were then analyzed to more specifically define their properties for binding. It was found that the number of putative binding sites (K/R-X-K/R), the overall positive charge, and the percentage of positively charged amino acids within the protein were responsible for this interaction.

Human monkeypox and smallpox viruses: genomic comparison

Monkeypox virus (MPV) causes a human disease which resembles smallpox but with a lower person‐to‐person transmission rate. To determine the genetic relationship between the orthopoxviruses causing these two diseases, we sequenced the 197‐kb genome of MPV isolated from a patient during a large human monkeypox outbreak in Zaire in 1996. The nucleotide sequence within the central region of the MPV genome, which encodes essential enzymes and structural proteins, was 96.3% identical with that of variola (smallpox) virus (VAR). In contrast, there were considerable differences between MPV and VAR in the regions encoding virulence and host‐range factors near the ends of the genome. Our data indicate that MPV is not the direct ancestor of VAR and is unlikely to naturally acquire all properties of VAR.

Comparison of the genetic maps of variola and vaccinia viruses

The complete genetic map of the variola major virus strain India‐1967 is built basing on the sequence data. The suggested map is compared with the maps of the sequenced genomic regions of Copenhagen and Western Reserve strains of vaccinia virus and Harvey strain of variola major virus. The principle differences revealed in the genomic organization of these viruses are discussed.

Molecular mimicry of the inflammation modulatory proteins (IMPs) of poxviruses: evasion of the inflammatory response to preserve viral habitat.

Microorganisms encode numerous immunomodulators that resemble, in structure and function, molecules captured over the millennia from their hosts [G. J. Kotwal J. Leukoc. Biol. 62, 415–429]. The vaccinia virus complement control protein (VCP) was the first soluble microbial protein to have a postulated role in the immunomodulation and evasion of host defense [G. J. Kotwal and B. Moss Nature 355, 176–179]. Purified bioactive VCP has been shown to bind to C3 and C4, block the complement cascade at multiple sites [G. J. Kotwal et al. Science 250, 827–830; R. Mckenzie, G. J. Kotwal et al. J. Infect. Dis. 166, 1245–1250] and exhibit a greater potency than the human complement 4b binding protein, C4b‐BP [G. J. Kotwal, Am. Biotech. Lab. 9,76]. The importance of this protein to poxviruses was further demonstrated in rabbits and guinea pigs through the use of recombinant virus lacking an intact DNA coding for VCP [Isaacs, G. J. Kotwal, and B. Moss Proc. Natl. Acad. Sci. 89, 628–672]. Studies in mice have shown that the homolog of VCP in cowpox virus (CPV), referred to as the inflammation modulatory protein (IMP) can, in a mouse model, significantly diminish the specific footpad swelling response [C. G. Miller, S. N. Shchelkunov, and G. J. Kotwal Virol. 229, 126–133]. To determine the precise cellular changes at the site of infection, BALB/c mice were subcutaneously injected (in the backs) with CPV or a recombinant virus lacking IMP, CPV‐IMP. Differences in histology were observed by staining the adjoining skin tissue sections with hematoxylin & eosin or by removal of the connective tissue and staining with May‐Grunwald‐Geimsa. All mice that were injected with the CPV‐IMP experienced severe tissue destruction and formation of nodular lesions compared with the mice injected with CPV. Microscopic examination indicated significantly greater cellular infiltration and destruction of skeletal muscle cells in the sections of connective tissue and adjoining skin tissue, respectively, of the mice injected with the CPV‐IMP [G. J. Kotwal et al. Mol. Cell. Biochem. in press]. Thus IMP preserves the tissue at the site of infection (viral habitat). In this review, we present evidence for molecular mimicry and evolutionary relationship to other homologs of IMP and discuss their relationships with other IMPs such as the poxviral chemokine and cytokine receptor‐like proteins. J. Leukoc. Biol. 64: 68–71; 1998.

Species-Specific Differences in Organization of Orthopoxvirus Kelch-Like Proteins

Organization of orthopoxvirus proteins of the kelch superfamily and their genes were analyzed and compared. Complete genomic sequences of variola (VAR), monkeypox (MPV), vaccinia (VAC), and species-specific regions of cowpox (CPV) viruses were used in the work. Despite the multiplicity of kelch-like proteins in orthopoxviruses, their function is still vague. It has been discovered that the genes of orthopoxvirus kelch-like proteins are localized only to the terminal variable regions of the genome and display species-specific differences in the lengths of the proteins they potentially encode. All the genes belonging to kelch superfamily in the genome of VAR, which has the only host–the man, are mutationally destroyed. However, CPV, displaying the widest host range among orthopoxviruses, encode the most numerous set of kelch-like proteins. Weak homologies between kelch-like proteins of one virus were demonstrated as well as high homologies between isologues of different orthopoxvirus species. The comparison performed suggest that CPV virus is most ancient and may be considered as the ancestor of other orthopoxviruses pathogenic for humans.

Two types of deletions in orthopoxvirus genomes

The genome nucleotide sequences of two strains of variola major virus and one strain of vaccinia virus were compared. One hundred and sixty-eight short (less than 100 bp in length) and eight long (more than 900 bp in length) deletions, four deletion/insertion regions, and four regions of multiple mutational differences between variola and vaccinia virus DNAs were revealed. Short deletions generally occur at directly repeated sequences of 3–21 bp. Long deletions showed no evidence of repeated sequences at their points of junction. We suggest the presence of a consensus sequence characteristic of these junctions and propose that there is a virus-encoded enzyme that produces this nonhomologous recombination/deletion in the cytoplasm of the infected cell.

Analysis of the nucleotide sequence of 48 kbp of the variola major virus strain India-1967 located on the right terminus of the conservative genome region

Computer analysis of a variola major virus (VAR) genomic fragment bounded by the open reading frames (ORFs) D1R and A33L, which is 47,961 bp long, revealed 46 potential ORFs. The VAR proteins were compared to the analogous proteins of vaccinia virus strain Copenhagen. The subunits of DNA-dependent RNA polymerase, as well as the transcription factors, mRNA-capping enzymes, and proteins necessary for the virion morphogenesis proved to be highly conservative within orthopoxviruses. The most pronounced differences between the VAR genome fragment under study and the corresponding vaccinia virus fragment were revealed in the vicinity of the gene encoding the A-type inclusion bodies protein. Possible functions of the analysed viral proteins are discussed.

Multiple genetic differences between the monkeypox and variola viruses.

The elimination of variola, an extremely dangerous human epidemic disease, was the first and still remains the only example of a successful fight of the international community against an infectious disease under the aegis of World Health Organization [1]. The success of this campaign was to a large extent due to the fact that variola virus, which belongs to the genus Orthopoxvirus of the family Poxviridae and causes variola, has a very narrow host spectrum (only humans). Longterm medical practice has demonstrated the effectiveness of protection against variola based on the use of live vaccine designed on the basis of closely related vaccinia virus. Mass vaccination of people and thorough epidemiological control allowed variola elimination all over the world by 1977. Thereupon, vaccination against variola and other infections induced by closely related orthopoxviruses was stopped everywhere [1].

Species-specific differences in the organization of genes encoding kelch-like proteins of orthopoxviruses pathogenic for humans.

The kelch gene of Drosophila encodes the protein which is a structural component of so-called annular channels, which ensure the transport of cytoplasmic components from the nurse cells to Drosophila melanogaster oocytes during ontogeny [1]. The amino acid sequence analysis showed that the kelch protein consisting of 688 amino acid residue contains six tandem repeats comprised of approximately 50 amino acid residues each, which are located at the C-terminus of this protein. This repeat has been termed the kelch motif [2], and the group of these repeats is called the kelch domain. It was discovered that the kelch protein contains a so-called BTB domain at the N-terminus, which is characteristic of some other Drosophila proteins and is comprised of approximately 115 amino acid residues [3]. Further studies showed that BTB domains are able to dimerize and to interact with other BTB domains to form heterodimers [4]. It was shown that the kelch domain is required for binding the kelch protein to the actin filaments in the cell. Dimerization of the BTB domains of two actinassociated kelch proteins results in cross-interaction between these filaments, which leads to formation of intercellular annular channels of Drosophila oocytes [5]. The search for the kelch repeats and BTB domains in the databases showed that both motifs are ancient and have widely spread in different types of organism during evolution [4, 6]. A superfamily of proteins containing the kelch repeats has been formed. The proteins of this family are involved in different cellular functions, such as changes in the cytoskeleton and plasma membrane, regulation of gene expression, mRNA splicing, etc. The extent of homology between individual kelch repeats in not high (25–50% for six motifs of the same kelch protein of Drosophila ). This value may decrease to 11% when comparing individual motifs of different proteins [2, 6]. An important characteristic of the kelch motif is a glycine duplex and a specific arrangement of aromatic amino acid residues [1, 2]. The number of the kelch motifs in different proteins usually varies from four to seven. In addition, the location of the kelch domain in proteins may differ. Based on the kelch motif location, kelch-like proteins are divided into five groups [6]. Based on the structural homology, some poxviral proteins were classified with the same group as the Drosophila kelch protein [1, 2, 6].

Species-specific differences in the organization of the complement-binding protein of orthopoxviruses.

The human complement system consists of more than twenty serum proteins and at least ten membraneassociated proteins. The mechanism of antiviral action of the complement system involves virus neutralization and opsonization, lysis of infected cells, and enhancement of nonspecific inflammatory and specific immune response [1]. The vaccinia virus protein secreted by the cells infected with this virus and controlling the reactions of complement activation (vaccinia virus complement control protein, VCP) was the first identified microbial protein to exhibit a complement-binding activity. This protein consists of four short (approximately 60 amino acids each) degenerate repeats or short consensus repeats (SCRs), which are characteristic of the family of proteins regulating complement activation (RCA) [2]. VCP shares the highest homology with the amino acid sequence of the first four out of the eight SCRs of α -chain of the human complement C4b-binding protein [3]. It is believed that the VCP gene has originated as a result of incorporation of the coding sequence of the host RCA protein or its fragment into the viral genome and subsequent adaptation (alteration) of this gene to the functions required by the virus. The results of X-ray analysis showed that the SCR sequences of VCP form discrete compact domains that are sequentially tightly bound to each other [4].