Marchal (285) and Downie (126) bodies, respectively. Because of the ambiguity associated with describing structures solely on the basis of their reaction with various histochemical stains, Kato et al. (253) carefully examined the virusspecific cytoplasmic structures of a large number of poxvirus species and, using a criterion based on morphologic observations, histochemical staining, and immunochemical reactivity, suggested that the acidophilic and basophilic inclusion bodies be referred to as A-and B-type inclusion bodies, respectively. Under the auspices of a Poxvirus Subcommittee created at the Sixth International Congress for Microbiology in 1953, Fenner and Burnet (161) wrote a review which summarized the characteristics of the poxvirus group. This article remains the basis for the subsequent classification of poxviruses. The poxvirus family is divided into two subfamilies: Entomopoxvirinae (poxviruses of the insects) and Chordopoxvirinae (poxviruses of the vertebrates); the latter group of viruses is the focus of this review (Table 1). The vertebrate poxviruses share a group-specific NP antigen (511) and are able, in a reciprocal fashion, to participate in nongenetic reactivation (160, 168). This is a process whereby an infectious poxvirus is able to reactivate a second, heatinactivated poxvirus, as indicated by the production of progeny virus. Poxviruses were further classified into genera by comparing cross-protection in animal studies, crossneutralization of infectivity in tissue culture, and crosshybridization of genomic DNA from virions. This latter criterion is now the method of choice for preliminary classification of poxvirus isolates. In general, members of the same genera have similar morphology and biological properties. Virion Morphology Poxviruses are the largest of all animal viruses and can be visualized by light microscopy, although the details of the
Ultraviolet irradiation of the [3Hjcolchicine-tubulin complex leads to direct photolabeling of tubulin with low but practicable efficiency. The bulk (70% to >90%) of the labeling occurs on (8-tubulin and appears early after irradiation, whereas a-tubulin is labeled later. The labeling ratio of .8-tubulin to a-tubulin (13/a ratio) is reduced by prolonged incubation, prolonged irradiation, urea, high ionic strength, the use of aged tubulin, dilution of tubulin, or large concentrations of colchicine or podophyllotoxin. Glycerol increases the (3/a ratio. Limited data with (3Hlpodophyllotoxin show that it covalently bound with a similar fl/a distribution.Vinblastine, on the other hand, exhibits preferential attachment to a-tubulin. The possibilities that colchicine binds at the interface between a-tubulin and j3-tubulin, that the drug spans this interface, and that both subunits may contribute to the binding site are suggested.Despite the fact that colchicine has been used as an antimicrotubule agent for many years, there is no unanimity regarding the location of the high-affinity binding site for the drug in the tubulin dimer, which is formed by the noncovalent association ofthe similar but not identical q and (3 monomers.Several studies have assigned the site to the a-subunit, but uncertainties exist regarding the specificity of the' reactions used. Thus, N-bromoacetyldesacetylcolchicine showed nonspecific alkylation (1), photoaffinity labels used long spacer arms (2, 3), and studies with limited proteolysis could have been affected by rearrangements during proteolysis in the damaged protein (4). Colchicine binding to a site on ,f-tubulin has been proposed on the basis of indirect experiments dealing with the reactivity ofcysteine residues in ,3-tubulin (5) and by findings that most tubulin mutations that confer colchicine resistance occur in 83-tubulin genes (6-8).The excitation maximum of colchicine occurs at a higher wavelength than that of the tryptophan residues of tubulin; hence, direct photolabeling of tubulin with colchicine, without irradiating the protein, appeared to be feasible. However, stoichiometric covalent binding would not be expected for such a reaction because the efficiency of direct photolabeling tends to be <25% (9) because of the short colchicine fluorescence lifetime (of 1.14 ns) (10) with little intersystem crossing to the triplet state or long lifetimes (11), and because of the powerfully competing photoisomerization reaction to form lumicolchicines from excited-state colchicine, which causes dissociation of the ligand (12-14). Nevertheless, such a reaction might be less subject to the specificity problems noted above and thus increases the probability that colchicine will cross-link to the "correct" site. The following study explores the conditions for the direct photolabeling reaction, the localization of the covalently bound colchicine, and the factors influencing the distribution of the drug on tubulin. A portion of this material has been presented (15). MATERIALS AND METHODST...
Poxviruses are a highly successful family of pathogens, with variola virus, the causative agent of smallpox, being the most notable member. Poxviruses are unique among animal viruses in several respects. First, owing to the cytoplasmic site of virus replication, the virus encodes many enzymes required either for macromolecular precursor pool regulation or for biosynthetic processes. Second, these viruses have a very complex morphogenesis, which involves the de novo synthesis of virus-specific membranes and inclusion bodies. Third, and perhaps most surprising of all, the genomes of these viruses encode many proteins which interact with host processes at both the cellular and systemic levels. For example, a viral homolog of epidermal growth factor is active in vaccinia virus infections of cultured cells, rabbits, and mice. At least five virus proteins with homology to the serine protease inhibitor family have been identified and one, a 38-kDa protein encoded by cowpox virus, is thought to block a host pathway for generating a chemotactic substance. Finally, a protein which has homology with complement components interferes with the activation of the classical complement pathway. Poxviruses infect their hosts by all possible routes: through the skin by mechanical means (e.g., molluscum contagiosum infections of humans), via the respiratory tract (e.g., variola virus infections of humans), or by the oral route (e.g., ectromelia virus infection of the mouse). Poxvirus infections, in general, are acute, with no strong evidence for latent, persistent, or chronic infections. They can be localized or systemic. Ectromelia virus infection of the laboratory mouse can be systemic but inapparent with no mortality and little morbidity, or highly lethal with death in 10 days. On the other hand, molluscum contagiosum virus replicates only in the stratum spinosum of the human epidermis, with little or no involvement of the dermis, and does not spread systemically from the site of infection. The host response to infection is progressive and multifactorial. Early in the infection process, interferons, the alternative pathway of complement activation, inflammatory cells, and natural killer cells may contribute to slowing the spread of the infection. The cell-mediated response involving learned cytotoxic T lymphocytes and delayed-type hypersensitivity components appears to be the most important in recovery from infection. A significant role for specific antiviral antibody and antibody-dependent cell-mediated cytotoxicity has yet to be demonstrated in recovery from a primary infection, but these responses are thought to be important in preventing reinfection.
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