Analysis of the cell and erythrocyte binding activities of the dimple and canyon regions of the canine parvovirus capsid.

Canine parvovirus (CPV) binds to a number of cell and erythrocyte receptors, some of which are involved in cell infection, while others are used for other viral functions. Little is known about the regions of the virus capsid which bind to the cell receptors. CPV binds sialic acid through a region within or adjacent to the dimple on the surface of the capsid (Barbis, D. P., Chang, S-F., and Parrish, C. R., 1992, Virology 191, 301-308). In order to map the sialic acid binding site in more detail and to examine other regions of the capsid for cell receptor binding, a variety of mutant capsids were analyzed which had changes in two depressions within the surface of the capsid--the "canyon" and "dimple." In most cases recombinant VP1 and VP2 proteins were stably expressed together in canine A72 cells from a plasmid expression vector. The purified empty capsids were tested for their ability to bind sialic acid and thereby hemagglutinate (HA) erythrocytes and for binding to permissive host cells. In addition, the ability of neutralizing monoclonal antibodies to block cell attachment was also examined. Mutations of amino acids on a wall of the dimple eliminated or severely decreased HA. Changing various residues within the canyon had no effect on binding to either sialic acids or other receptors on feline lymphoblastoid cells, suggesting that the canyon is not the site of cell receptor attachment. Neutralizing monoclonal antibodies against both major antigenic determinants had variable effects on cell binding, but no consistent inhibition of binding was observed by antibodies directed against either of those two major antigenic determinants of the capsid.

Primary structure of p19 species of avian sarcoma and leukemia viruses.

The internal structural proteins of avian sarcoma and leukemia viruses are derived from a precursor polypeptide that is the product of the viral gag gene. The N-terminal domain of the precursor gives rise to p19, a protein that interacts with the lipid envelope of the virus and that may also interact with viral RNA. The C terminus of p19 from the Prague C strain of Rous sarcoma virus was previously assigned to a tyrosine residue 175 amino acids from the N terminus. We have used metabolic labeling and carboxypeptidase digestion to show that the C terminus of p19 is actually tyrosine 155. This implies the existence of a sixth gag protein 22 amino acids in length and located between p19 and p10 on the gag precursor. The p19 species of some recombinant avian sarcoma viruses and of the defective endogenous virus derived from the ev-1 locus migrate on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as if they were about 4,000 daltons smaller than p19. We have elucidated the structure of these forms, called p19 beta, by analysis of the proteins and determination of the DNA sequence of the p19 region of the gag gene from ev-1 and ev-2. Esterification of carboxyl groups completely suppressed the differences in migration of p19 and p19 beta. Peptide mapping showed the altered mobility to be determined by sequences in the C-terminal cyanogen bromide fragment of the proteins. We conclude from the DNA sequence that a single glutamate-lysine alteration is responsible for the altered electrophoretic mobility.

Heparin induced thrombocytopenia: a prospective study.

Thrombocytopenia occurred in 11.6% of 43 patients prospectively randomized to receive either bovine lung or porcine mucosal heparin. Four patients received bovine heparin and developed mild to moderate thrombocytopenia after eight to twelve days which resolved when therapy was stopped in one to six days. One patient received porcine heparin and developed mild thrombocytopenia after four days. The thrombocytopenia resolved in one day in spite of continued heparin therapy. Serial blood samples from the five thrombocytopenic patients were studied for their in vitro effect upon normal platelets. These samples induced a high release of serotonin from normal platelets or caused normal platelets to aggregate in the presence of additional heparin. Release or aggregation coincided with or occurred shortly after nadir platelet counts were reached and returned toward normal following cessation of heparin. Our data constitute the first prospective study of heparin induced thrombocytopenia in which serial blood samples were studied before, during and following the onset of thrombocytopenia. They strongly support the presence of a heparin dependent co-factor, presumably an antibody, as the cause for thrombocytopenia.