Hemorrhage in lesions caused by cowpox virus is induced by a viral protein that is related to plasma protein inhibitors of serine proteases.

Abstract

Several recombinant cowpox viruses were constructed and used to identify a viral gene that controls the production of hemorrhage in lesions caused by the Brighton Red strain of cowpox virus (CPV-BR). This gene is located in the KpnD fragment of CPV-BR DNA, between 31 and 32 kilobases from the end of the genome. This position corresponds well with that predicted from analyses of the DNA structures of spontaneously generated deletion mutants. The gene responsible for hemorrhage encodes a 38-kDa protein that is one of the most abundant early gene products. The 11-basepair sequence GAAAATATATT present 84 base pairs upstream of its coding region is also present upstream of three other early genes of vaccinia virus; therefore, this sequence may be involved in the regulation of transcription. There is extensive similarity between the predicted amino acid sequence of the 38-kDa protein and the amino acid sequences of several plasma proteins that are inhibitors of various serine proteases involved in blood coagulation pathways. This suggests that the viral protein may possess a similar biological activity, which may enable it to effect hemorrhage by inhibiting one or more of the serine proteases involved in the host's normal processes of blood coagulation and wound containment.The inoculation of cowpox virus (CPV) into the skin of various mammals (guinea pigs, rabbits, humans) results in lesions that exhibit edema, hypertrophy ofthe epidermis, and hemorrhage. Similar effects are produced in CPV lesions (pocks) in the chorioallantoic membrane of developing chicken embryos; there is extensive proliferation of ectodermal and mesodermal cells, edema, and localized hemorrhage that gives the pocks a deep red color (1).CPV variants that do not produce hemorrhage have been isolated from the white pocks that usually comprise up to 1% of the pocks present on chorioallantoic membranes infected with wild-type CPV (2, 3). The existence of these white-pock variants demonstrates that a viral function controls the production of the hemorrhage.Studies of the structures of the DNAs of these white-pock variants have shown that their genomes are generated from the genome of the wild-type virus by large deletions and duplications of sequences (4, 5). Similar results have been obtained from studies of the DNAs of white-pock variants of rabbitpox virus and monkeypox virus (6-8). The rearrangements of DNA that generate the white-pock variants of CPV typically involve the replacement of up to 39 kilobases (kb) of one end of the genome with an inverted copy of up to 50 kb of DNA from the other end (4, 5). The mechanisms by which these rearrangements proceed are unknown; but it is clear that there are no identifiable homologous sequence elements at the sites of the duplication/deletion end points (5). This result suggests that it is the location of essential genes rather than DNA sequence content that limits where rearrangements may occur. The position ofgenes that encode markers, such as production of hemorrhage, will place a further cons...

Non-essential Genes in the Vaccinia Virus HindIII K Fragment: a Gene Related to Serine Protease Inhibitors and a Gene Related to the 37K Vaccinia Virus Major Envelope Antigen

“…match of antithrombin III to another poxvirus gene is shown, namely to the cowpox virus gene that causes the red pock phenotype (Pickup et al, 1986). ORF K3 shows a similar high level of identity (28~ identity plus 29~ conservative changes) with a previously sequenced vaccinia virus gene which has been shown to encode a major 37K envelope antigen (Hirt et al, 1986).…”

“…Hence both of these genes map in that central region of the orthopoxvirus genome which appears to be highly similar between vaccinia and cowpox viruses, by restriction mapping (Mackett & Archard, 1979) and probably at a sequence level (Pickup et al, 1986). The positioning and degree of identity of these two genes suggests therefore that they are not direct equivalents.…”

“…Thus this is probably the 32.5K host range gene (Gillard et al, 1986) most of which is in HindlII M. The eight possible genes in (Pickup et al, 1986) and in fowlpox virus (Tomley et al, 1988). The cowpox gene, whose nucleotide sequence predicts a protein of size 38K, has been shown to be involved in the haemorrhaginous lesions, or red pocks, caused by the virus (Pickup et al, 1986). Lack of the gene causes white pock variants, presumably because inhibition of proteins involved in blood clotting no longer occurs.…”

A vaccinia serine protease inhibitor which prevents virus-induced cell fusion

“…In other poxviruses, serpins are known to be involved in the pathology and virulence of infection. In cowpox virus, a 38K protein (equivalent to the vaccinia virus B13R) is, at least partly, responsible for the haemorrhagic phenotype of pocks produced following infection of the chorioallantoic membranes of fertile hens' eggs (Pickup et al, 1986). It also prevents the migration of white cells in vitro (Chua et al, 1990) and to infected lesions in vivo (Palumbo et al, 1989).…”

“…malignant rabbit fibroma virus (MRV) (Upton et al, 1990), fowlpox virus (Tomley et al, 1988) and cowpox virus (Pickup et al, 1986), respectively. Three such ORFs have been identified in vaccinia virus, a member of the orthopox genus and the prototypical poxvirus.…”