The p53 tumor suppressor gene product is negatively regulated by the product of its downstream target, mdm2. The deletion of mdm2 in the mouse results in embryonic lethality at 5.5 days post coitum (d.p.c.) which can be overcome by simultaneous loss of the p53 tumor suppressor, substantiating the importance of the negative regulatory function of MDM2 on p53 function in vivo. Hence, the loss of MDM2 allowed the unregulated p53 protein to continuously exert its growth-suppressing activity, which either led to a complete G1 arrest or induced the p53-dependent apoptotic pathway, resulting in the death of the mdm27/7 embryos. To determine which of these possibilities is occurring, mouse embryo ®broblasts (MEFs) from p53 null and p53/mdm2 double null embryos were transfected with a retroviral vector carrying a temperature-sensitive p53 (tsp53) cDNA. Shifting of single-cell clonal populations to the permissive temperature caused the p537/7mdm27/7 ®bro-blasts expressing tsp53 to undergo apoptosis in a dosedependent manner. This phenotype was not observed in the tsp53 expressing p537/7 clones nor the parental cell lines. Thus, our data indicate that the simple loss of mdm2 can induce the p53-dependent apoptotic pathway in vivo.

We have derived and characterized a highly pathogenic molecular isolate of feline immunodeficiency virus subtype C (FIV-C) CABCpady00C. Clone FIV-C36 was obtained by lambda cloning from cats that developed severe immunodeficiency disease when infected with CABCpady00C (Abbotsford, British Columbia, Canada). Clone FIV-C36 Env is 96% identical to the noninfectious FIV-C isolate sequence deposited in GenBank (FIV-Cgb; GenBank accession number AF474246) (A. Harmache et al.) but is much more divergent in Env when compared to the subgroup A clones Petaluma (34TF10) and FIV-PPR (76 and 78% divergence, respectively). Clone FIV-C36 was able to infect freshly isolated feline peripheral blood mononuclear cells and primary T-cell lines but failed to productively infect CrFK cells, as is typical of FIV field isolates. Two-week-old specific-pathogen-free cats infected with FIV-C36 tissue culture supernatant became PCR positive and developed severe acute immunodeficiency disease similar to that caused by the uncloned CABCpady00C parent. At 4 to 5 weeks postinfection (PI), 3 of 4 animals developed CD4+-T-cell depletion, fever, weight loss, diarrhea, and opportunistic infections, including ulcerative stomatitis and tonsillitis associated with abundant bacterial growth, pneumonia, and pyelonephritis, requiring euthanasia. Histopathology confirmed severe thymic and systemic lymphoid depletion. Interestingly, the dam also became infected with a high viral load at 5 weeks PI of the kittens and developed a similar disease syndrome, requiring euthanasia at 11 weeks PI of the kittens. This constitutes the first report of a replication-competent, infectious, and pathogenic molecular clone of FIV-C. Clone FIV-C36 will facilitate dissection of the pathogenic determinants of FIV

Feline immunodeficiency virus (FIV) causes progressive immunodeficiency in domestic cats

Feline immunodeficiency virus (FIV) is an important viral pathogen worldwide in the domestic cat, which is the smallest animal model for the study of natural lentivirus infection. Thus, understanding the molecular mechanisms by which FIV carries out its life cycle and causes an acquired immune deficiency syndrome (AIDS) in the cat is of high priority. FIV has an overall genome size similar to HIV, the causative agent of AIDS in man, and shares with the human virus genomic features that may serve as common targets for development of broad-based intervention strategies. Specific targets include enzymes encoded by the two lentiviruses, such as protease (PR), reverse transcriptase (RT), RNAse H, and integrase (IN). In addition, both FIV and HIV encode Vif and Rev elements essential for virus replication and also share the use of the chemokine receptor CXCR4 for entry into the host cell. The following review is a brief overview of the current state of characterization of the feline/ FIV model and development of its use for generation and testing of anti-viral agents.

In vivo tests were performed to assess the influence of the protease inhibitor TL-3 on feline immunodeficiency virus (FIV)-induced central nervous system (CNS) deficits. Twenty cats were divided into four groups of five animals each. Group 1 received no treatment, group 2 received TL-3 only, group 3 received FIV strain PPR (FIV-PPR) only, and group 4 received FIV-PPR and TL-3. Animals were monitored for immunological and virological status, along with measurements of brain stem auditory evoked potential (BAEP) changes. Groups 1 and 2 remained FIV negative, and groups 3 and 4 became virus positive and seroconverted by 3 to 5 weeks postinoculation. No adverse effects were noted with TL-3 only. The average peak viral load for the virus-only group 3 animals was 1.32 ؋ 10 6 RNA copies/ml, compared to 6.9 ؋ 10 4 copies/ml for TL-3-treated group 4 cats. Group 3 (virus-only) cats exhibited marked progressive delays in BAEPs starting at 2 weeks post virus exposure, which is typical of infection with FIV-PPR. In contrast, TL-3-treated cats of group 4 exhibited BAEPs similar to those of control and drug-only cats. At 97 days postinfection, treatments were switched; i.e., group 4 animals were taken off TL-3 and group 3 animals were treated with TL-3. BAEPs in group 3 animals returned to control levels, while BAEPs in group 4 animals remained at control levels. After 70 days on TL-3, group 3 was removed from the drug treatment regimen. Delays in BAEPs immediately increased to levels observed prior to TL-3 treatment. The findings show that early TL-3 treatment can effectively eliminate FIV-induced changes in the CNS. Furthermore, TL-3 can counteract FIV effects on the CNS of infected cats, although continued treatment is required to maintain unimpaired CNS function.

Expression of the feline immunodeficiency virus (FIV) accessory protein OrfA (or Orf2) is critical for efficient viral replication in lymphocytes, both in vitro and in vivo. OrfA has been reported to exhibit functions in common with the human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) accessory proteins Vpr and Tat, although the function of OrfA has not been fully explained. Here, we use microarray analysis to characterize how OrfA modulates the gene expression profile of T-lymphocytes. The primary IL-2-dependent T-cell line 104-C1 was transduced to express OrfA. Functional expression of OrfA was demonstrated by trans complementation of the OrfA-defective clone, FIV-34TF10. OrfA-expressing cells had a slightly reduced cell proliferation rate but did not exhibit any significant alteration in cell cycle distribution. Reverse-transcribed RNA from cells expressing green fluorescent protein (GFP) or GFP+OrfA were hybridized to Affymetrix HU133 Plus 2.0 microarray chips representing more than 47,000 genome-wide transcripts. By using two statistical approaches, 461 (Rank Products) and 277 (ANOVA) genes were identified as modulated by OrfA expression. The functional relevance of the differentially expressed genes was explored by Ingenuity Pathway Analysis. The analyses revealed alterations in genes critical for RNA post-transcriptional modifications and protein ubiquitination as the two most significant functional outcomes of OrfA expression. In these two groups, several subunits of the spliceosome, cellular splicing factors and family members of the proteasome-ubiquitination system were identified. These findings provide novel information on the versatile function of OrfA during FIV infection and indicate a fine-tuning mechanism of the cellular environment by OrfA to facilitate efficient FIV replication.

Background: The protease inhibitor, TL-3, demonstrated broad efficacy in vitro against FIV, HIV and SIV (simian immunodeficiency virus), and exhibited very strong protective effects on early neurologic alterations in the CNS of FIV-PPR infected cats. In this study, we analyzed TL-3 efficacy using a highly pathogenic FIV-C isolate, which causes a severe acute phase immunodeficiency syndrome, with high early mortality rates.

Poster #3732 * expected fold changes measured by orthogonal methods No CNV called in 24 normal samples (in duplicates) CNV specificity = 100% *excluding known polymorphism sites For Research Use Only. Not for use in diagnostic procedures.