ABSTRACT
Caprine arthritis-encephalitis virus (CAEV) is a lentivirus which is closely related by nucleotide sequence and biological properties to visna virus. Sequence analysis of the CAEV genome revealed the presence of a small open reading frame (ORF) which shares amino acid identity with the visna virus tat gene. Using an infectious molecular clone of CAEV the role of the tat ORF in viral replication was examined. Mutations were made in the tat ORF that introduced two in frame stop codons six amino acids downstream of the tat AUG; in addition, a deletion mutant was made that removed most of the tat ORF. Both of these mutants had greatly reduced virus titers (> 1000-fold less than the wild type infectious clone). Co-transfection of a tat expressing plasmid with these viruses containing the tat ORF mutations resulted in higher levels of virus production demonstrating that the effects of both mutants are tat specific. These mutants provide data that the CAEV tat gene is necessary for efficient virus replication. Analysis of the RNA in these transfected cells showed that complementation of the tat gene was in trans and not the result of recombination. Analysis of the gag and rev proteins in the transfected cells demonstrated that these proteins were not detectable in cells transfected with the tat mutants but could be readily detected when the mutations were complemented in trans with a tat expression vector. To test for tat mediated trans-activation a plasmid expressing the CAEV tat ORF was co-transfected with plasmids containing either the CAEV or visna virus LTR driving transcription of the bacterial chloramphenicol acetyltransferase gene (CAT). These experiments indicate that one function of the CAEV tat protein is to trans-activate gene expression from the viral promoter. RNase protection analysis of CAT mRNA from co-transfected cells demonstrated that CAEV Tat trans-activates gene expression by increasing steady-state levels of mRNA.
Subject(s)
Arthritis-Encephalitis Virus, Caprine/genetics , Arthritis-Encephalitis Virus, Caprine/physiology , Gene Expression Regulation, Viral , Genes, tat , Repetitive Sequences, Nucleic Acid , Virus Replication , Amino Acid Sequence , Animals , Cloning, Molecular , Gene Deletion , Genes, gag , Genome, Viral , Goats/immunology , Molecular Sequence Data , Mutagenesis, Site-Directed , Open Reading Frames , Point Mutation , Polymerase Chain Reaction , RNA, Viral/biosynthesis , RNA, Viral/metabolism , Restriction Mapping , TATA Box , TransfectionABSTRACT
Visna virus is a pathogenic lentivirus of sheep that is distantly related to the primate lentiviruses, including the human immunodeficiency virus type 1 (HIV-1). Replication of HIV-1 in cell culture requires the expression of a virus-encoded protein, Tat, which is a potent trans-activator of viral gene expression. Visna virus encodes an analogous Tat protein that greatly increases gene expression directed by the visna viral LTR. This report uses a stable vero cell line that constitutively expresses visna virus Tat to investigate the molecular mechanism of action of Tat on viral gene expression. Transient expression assays, using the visna virus LTR to drive transcription of the bacterial gene for chloramphenicol acetyltransferase (CAT), demonstrate that Tat trans-activates gene expression by increasing steady-state mRNA levels. The increase in steady-state mRNA levels is sufficient to account for the increase in protein observed and is due, in part, to an increase in the rate of transcription initiation. Tat mediates the accumulation of mRNA through AP-4 and AP-1 binding sites located in the U3 region of the LTR. Deletion of the upstream AP-1 and AP-4 binding sites results in a residual low level of trans-activation by Tat. Further experiments, using LTRs with R-U5 sequences deleted to +10, demonstrate AP-1 and AP-4 mediated responses to TAT at the RNA level, but no increase was observed in CAT protein.
Subject(s)
Gene Products, tat/physiology , Visna-maedi virus/genetics , Base Sequence , Binding Sites , Cloning, Molecular , DNA Mutational Analysis , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Viral , Molecular Sequence Data , Promoter Regions, Genetic , Protein Biosynthesis , Proto-Oncogene Proteins c-jun/metabolism , RNA, Messenger/genetics , Regulatory Sequences, Nucleic Acid , Repetitive Sequences, Nucleic Acid , Transcription Factors/metabolism , Transcription, Genetic , Transcriptional ActivationSubject(s)
Gene Expression Regulation, Viral , Visna-maedi virus/genetics , Base Sequence , Binding Sites , Cell Line , Humans , Macrophages/metabolism , Molecular Sequence Data , Promoter Regions, Genetic , Repetitive Sequences, Nucleic Acid , T-Lymphocytes/metabolism , Virus Activation , Visna-maedi virus/growth & developmentABSTRACT
Enzyme immunoassays for detection of Clostridium difficile toxins A and B were developed with use of a double-sandwich microtiter plate format. Each assay was specific for its respective toxin and was sensitive to 0.1 ng of toxin. Neither assay was reactive with 13 other species of clostridia. One hundred fifty fecal specimens submitted for tissue culture cytotoxicity assay were evaluated by enzyme-linked immunosorbent assay (ELISA). Of the 79 tissue culture-positive specimens, 72 (91%) were positive in the A assay, 63 (80%) were positive in the B assay, and 75 (95%) were positive in either assay. Specimens with tissue culture titers of greater than or equal to 10(3) were uniformly positive in both assays. The specificities of the toxin A and B ELISAs were 98.6% and 100%, respectively. An ELISA for both toxins could serve as a substitute for the tissue culture cytotoxicity assay.
