ABSTRACT
The formation of a genomic RNA dimer appears to be a critical step in the life cycle of all retroviruses. To investigate the site and nucleotide interactions involved in this process, a 531 bp DNA fragment encompassing sequences up- and downstream of the splice donor in human T cell leukaemia virus type 1 (HTLV-1) was inserted into a plasmid vector under the control of the SP6 promoter. RNA transcripts generated in vitro from this template formed dimers which could be dissociated by heating at 60-80 degrees C for 3 min. The physical properties of the dimeric RNA were not consistent with either Watson-Crick base pairing or guanine tetrad formation as being solely responsible for the interaction. Deletion mutagenesis identified a 32 nt sequence required for dimerisation. Computer modelling was carried out in order to identify putative RNA secondary structures within this essential region. A stem-loop structure was identified, the stem of which was conserved among different sequenced isolates of HTLV-1. This sequence also contains a 15 nt palindrome. We sought by disruptive and compensatory mutagenesis to define the possible roles of these two structures in dimer linkage.
Subject(s)
Human T-lymphotropic virus 1/chemistry , Nucleic Acid Conformation , RNA, Messenger/chemistry , RNA, Viral/chemistry , Base Sequence , Computer Simulation , Human T-lymphotropic virus 1/genetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , RNA, Messenger/genetics , RNA, Viral/genetics , Sequence Deletion , Structure-Activity RelationshipABSTRACT
Shiga-like-toxin (SLT)-producing Escherichia coli strains, especially serotype O157:H7, are important causes of bloody diarrhea and are associated with the development of hemolytic-uremic syndrome (HUS). Enzyme-linked immunosorbent assays (ELISAs) were developed for the serologic detection of immunoglobulin M (IgM), IgG, and IgA to Shiga toxin (ST) and SLT-I, IgG to SLT-II, and IgM and IgG reactive against E. coli O157 lipopolysaccharide (LPS). Serum samples were collected from 27 HUS patients (25 pediatric and 2 adult) and tested in the ELISAs. Of 27 patients, 10 (37%) were positive for at least one class of antibody to ST/SLT-I. None of the patients were positive for IgG antibody to SLT-II. Twenty-one of the 27 patients (78%) were positive for antibody to E. coli O157 LPS; 19 of 27 (70%) were positive for IgM, and 20 of 27 (74%) were positive for IgG. None of 48 control serum samples were positive in any of the toxin assays, and only 1 of 48 (2%) and 2 of 48 (4%) were positive for IgM and IgG, respectively, to E. coli O157 LPS. Twelve of the 24 patients (50%) from whom stool specimens were collected were positive by culture for E. coli O157. Overall, serology and culture produced confirmation of infection by SLT-producing organisms in 23 of 27 (85%) HUS patients. A combination of ELISA for antibodies to E. coli O157 LPS and culture provided evidence for 22 of 27 (82%) of these patients. The results indicate that while ELISAs for ST/SLT-I and SLT-II antibodies were of limited diagnostic value, the ELISAs for IgM and IgG to E. coli O157 LPS provided valuable and sensitive adjuncts to culture.
