Takayuki Miyazawa

Localization of the viral antigen of feline immunodeficiency virus in the lymph nodes of cats at the early stage of infection

SummaryImmunohistochemical examinations of localization of feline immunodeficiency virus (FIV) Gag protein were performed on lymph nodes of cats experimentally inoculated with three different strains of FIV (infectious molecular clone of TM 1, Petaluma, and KYO-1 strains), using rabbit anti-FIV Gag serum. The FIV Gag antigens were observed in many follicular dendritic cells (FDCs) and sparsely in small lymphocytes of paracortical area in the lymph nodes of cats inoculated with Petaluma and KYO-1 strains. However, the antigens were present only in small lymphocytes, and not in FDCs of a cat inoculated with infectious molecular clone of the TM 1 strain. The cell type differences in expression of the viral antigen in vivo might reflect on the cell tropisms of the FIV strains in vitro. By double immunohistochemical staining with rabbit anti-FIV Gag serum and monoclonal antibodies which recognize feline CD 4, feline CD 8 or feline pan-T molecules, the FIV Gag-positive lymphocytes were characterized as feline CD 4-positive T cells. Since the distributions of FIV Gag antigens were mainly in the FDCs, the FDCs may play an important role as a major reservoir and may be a primary target of FIV at early stages of infection.

Induction of apoptosis in a T lymphoblastoid cell line infected with feline immunodeficiency virus

SummaryThe mechanism of cell death induced by feline immunodeficiency virus (FIV) infection was investigated in an interleukin 2(IL-2)-dependent T-lymphoblastoid cell line (MYA-1). DNA extracted from FIV-infected MYA-1 cells showed a ladder of nucleosomal DNA, indicating that the cytopathic effect (CPE) observed in these cells was due to apoptosis. Infection of MYA-1 cells with FIV was associated with suppression of the proliferative response of the cells to exogenous IL-2 prior to DNA fragmentation. These findings suggest that FIV-induced CPE in these T-lymhoblastoid cells is associated with apoptosis possibly due to a defect in the IL-2 signal transduction pathway.

The AP-1 binding site in the feline immunodeficiency virus long terminal repeat is not required for virus replication in feline T lymphocytes

Sequences of 31 bp containing putative AP-1 and AP-4 binding sequences in the U3 region of the feline immunodeficiency virus (FIV) long terminal repeat (LTR) were deleted and the basal promoter activity of the LTR was measured by the chloramphenicol acetyltransferase (CAT) assay. The activity of the FIV LTR was reduced in Felis catus whole foetus 4 (fcwf-4) cells and Crandell feline kidney cells by this deletion. Cotransfection of murine c-Fos or c-Jun expression plasmids with the FIV LTR-CAT reporter plasmid into fcwf-4 cells revealed that FIV LTR could be activated by c-Fos but not c-Jun in the cells. The mutated LTR was introduced into an infectious molecular clone of FIV and the replication rate and the cytopathogenic activity of the mutant were compared with those of the wild-type in two feline CD4-positive T lymphoblastoid cell lines. It was found that the rate and activity of the mutant were almost the same as those of the wild-type. From these data, we conclude that the 31 bp fragment is important for achieving maximal expression of the FIV genome, but not required for the replication of FIV in feline T lymphocytes.

Feline immunodeficiency virus gene expression : analysis of the RNA splicing pattern and the monocistronic rev mRNA

The transcription pattern of the feline immunodeficiency virus (FIV) genome in a feline CD4+ cell line was examined. In addition to the genomic RNA (9.2 kb), at least five FIV-specific transcripts [5.2, 4.4 (doublet), 1.7 and 1.4 kb] were detected by using subgenomic restriction enzyme fragments of an FIV molecular clone or FIV-specific oligonucleotides as probes. Among these transcripts, the 9.2, 5.2 and 4.4 (doublet) kb mRNAs were not expressed in the cytoplasm of cells transfected with a rev- mutant. To determine the location of splice junctions in the FIV genome, we used PCR to amplify and clone cDNAs corresponding to the viral mRNAs from infected cells. The region between pol and env was found to contain at least two splice donor and three splice acceptor sites. Two splice acceptor sites were detected in the 3 region of env. By hybridization analysis and sequencing of cDNA clones, it was revealed that the medium sized mRNAs are derived from a single splice event, with different splice acceptor sites, and that the two smaller transcripts are doubly or triply spliced mRNAs. Our results demonstrate a complex pattern of alternative splicing of FIV mRNAs. Furthermore, we identified monocistronic rev mRNA species that employ a unique splice acceptor site.

Comparison of biological properties of feline immunodeficiency virus isolates using recombinant chimeric viruses

The biological properties of homogeneous populations of feline immunodeficiency viruses derived from infectious, molecular clones of the TM1, TM2 and Petaluma strains were compared. Differences in infectivity for Crandell feline kidney (CRFK) cells, and in syncytium formation and replication kinetics in a feline T lymphoblastoid cell line (MYA-1 cells) were observed. To investigate the basis of these differences between the TM2 and Petaluma strains, we first compared the basal promoter activity of the long terminal repeat which is a highly divergent region, but no significant difference in activities was found in CRFK cells. We then constructed two recombinant chimeric clones which carry gag, pol, vif, and ORF A from the heterologous virus. From analyses using the chimeric clones, it was revealed that efficient virus growth in CRFK cells and MYA-1 cells was regulated by the gag, pol, vif and ORF A regions, whereas viral determinants of infectivity for CRFK cells, and syncytium formation and cytopathogenicity in MYA-1 cells, were located in the env region.

Inhibition of feline immunodeficiency virus gene expression and replication by alphaherpesvirus ICP4 homologues

We investigated the effects of pseudorabies virus (PRV), herpes simplex virus type 1 (HSV-1), and equine herpesvirus type 1 (EHV-1) ICP4 homologues on feline immunodeficiency virus (FIV) long terminal repeat (LTR)-directed gene expression. This was done by using the transient expression chloramphenicol acetyltransferase (CAT) assay in Crandell feline kidney (CRFK) and Felis catus whole fetus 4 cells transfected with a chimeric FIV LTR-CAT reporter construct in combination with effector plasmids expressing the PRV, HSV-1 or EHV-1 ICP4 homologue. The experiments demonstrated that the ICP4 homologues could significantly inhibit FIV LTR-directed gene expression. Moreover, the ICP4 homologues also exhibited a marked inhibitory effect on FIV replication in CRFK cells cotransfected with an infectious molecular clone of FIV.