Prevalence of acute enteric viral pathogens in acquired immunodeficiency syndrome patients with diarrhea.

Diarrhea due to enteric pathogens is an important complication of advanced human immunodeficiency virus infection. Whereas numerous bacterial and parasitic agents have been implicated, the role of pathogenic enteric viruses is less clear. Stools from 153 human immunodeficiency virus seropositive men were tested by electrophoresis, enzyme-linked immunosorbent assay, and immune electron microscopy for the presence of rotaviruses (group A and non-group A), adenoviruses, and Norwalk agent. Virus was detected in 9% of the patients with acquired immunodeficiency syndrome, 3% of the patients with acquired immunodeficiency syndrome-related complex, and none of the seropositive men without these diagnoses. Virus detection was not more likely in stool from patients with diarrhea.

Pertussis toxin S1 mutant with reduced enzyme activity and a conserved protective epitope.

Pertussis toxin (PTX) is a major virulence factor in whooping cough and can elicit protective antibodies. Amino acid residues 8 to 15 of PTX subunit S1 are important for the adenosine diphosphate-ribosyltransferase activity associated with the pathobiological effects of PTX. Furthermore, this region contains at least a portion of an epitope that elicits both toxin-neutralizing and protective antibody responses in mice. The gene encoding the S1 subunit was subjected to site-specific mutagenesis in this critical region. A mutant containing a single amino acid substitution (Arg9----Lys) had reduced enzymatic activity (approximately 0.02% of control) while retaining the protective epitope. This analog S1 molecule may provide the basis for a genetically detoxified PTX with potential for use as a component of an acellular vaccine against whooping cough.

Identification of a region in the S1 subunit of pertussis toxin that is required for enzymatic activity and that contributes to the formation of a neutralizing antigenic determinant.

The S1 subunit of pertussis toxin possesses two regions (homology boxes), each spanning 8 residues, that are nearly identical in sequence to similarly located regions in the enzymatically active A fragments of two other ADP-ribosylating toxins: cholera toxin and Escherichia coli heat-labile toxin. This observation suggests a functional role for one or both of these regions in enzymatic activity. We have examined the role of one of these regions, located near the amino terminus of the S1 subunit, by using a high-level recombinant expression system and progressive truncation of the gene sequence encoding the amino terminus of the molecule. A series of six truncated, recombinant proteins were produced at high levels in E. coli and examined for their enzymatic and antigenic properties. The three molecules that lacked most or all of the homology box delimited by amino acid residues 8 and 15 lacked detectable enzymatic activity. All of the three molecules in which the box was retained exhibited detectable activity. Only those recombinant molecules that possessed the homology box reacted with a neutralizing and passively protective monoclonal anti-S1 antibody. These findings identify the region of homology located near the amino terminus of S1 as an apparent enzymatic subsite and a potentially important antigenic determinant.

Infectious rotavirus enters cells by direct cell membrane penetration, not by endocytosis.

Rotaviruses are icosahedral viruses with a segmented, double-stranded RNA genome. They are the major cause of severe infantile infectious diarrhea. Rotavirus growth in tissue culture is markedly enhanced by pretreatment of virus with trypsin. Trypsin activation is associated with cleavage of the viral hemagglutinin (viral protein 3 [VP3]; 88 kilodaltons) into two fragments (60 and 28 kilodaltons). The mechanism by which proteolytic cleavage leads to enhanced growth is unknown. Cleavage of VP3 does not alter viral binding to cell monolayers. In previous electron microscopic studies of infected cell cultures, it has been demonstrated that rotavirus particles enter cells by both endocytosis and direct cell membrane penetration. To determine whether trypsin treatment affected rotavirus internalization, we studied the kinetics of entry of infectious rhesus rotavirus (RRV) into MA104 cells. Trypsin-activated RRV was internalized with a half-time of 3 to 5 min, while nonactivated virus disappeared from the cell surface with a half-time of 30 to 50 min. In contrast to trypsin-activated RRV, loss of nonactivated RRV from the cell surface did not result in the appearance of infection, as measured by plaque formation. Endocytosis inhibitors (sodium azide, dinitrophenol) and lysosomotropic agents (ammonium chloride, chloroquine) had a limited effect on the entry of infectious virus into cells. Purified trypsin-activated RRV added to cell monolayers at pH 7.4 medicated 51Cr, [14C]choline, and [3H]inositol released from prelabeled MA104 cells. This release could be specifically blocked by neutralizing antibodies to VP3. These results suggest that MA104 cell infection follows the rapid entry of trypsin-activated RRV by direct cell membrane penetration. Cell membrane penetration of infectious RRV is initiated by trypsin cleavage of VP3. Neutralizing antibodies can inhibit this direct membrane penetration.