Importance of the gene for maintenance of high virus loads and for the development of AIDS. repeat (US). In all cases, we have used the infectious, pathogenic SIVmac239 clone as the starting parental strain and have introduced large deletions in order to prevent reversion at the targeted locus (12). For deletions involving and coding sequence (13, 18, 19), it will be treated as a discrete entity for the purpose of this report. Previous publications have described the properties of (missing gene was amplified by PCR from 10 ng of pSIVSphSph5 plasmid template DNA with oligonucleotides 5SIVvifEcoRI and 3SIVvifBamHI. Amplified SIV DNA was molecularly cloned into bacterial plasmid pCRII, using a TA cloning kit (Promega) according to the manufacturers directions. The TA vector intermediate was purified, verified by DNA sequencing across the gene, and digested with genes between the virus, virus stocks were prepared by transfection of cloned DNA into CEMx174 cells by using DEAE-dextran (12). For SIVantigen, determined with a commercial antigen capture kit (Coulter, Hialeah, Fla.). Inocula containing 100 ng of p27 of SIV3, SIV3x, or SIV4 were used for each animal in experiment A (Table ?(Table1).1). Viruses containing 34, 396, and 325 ng of p27 were used for experiments B, D, and F, respectively, in Table ?Table1.1. We noticed no effect of inoculum dose on virus load. SIVcontaining 180 ng of p27, and animal 180-93 received heat-inactivated SIVcontaining 180 ng of p27. Peripheral blood samples were taken at intervals and used to assay the numbers of infectious cells in peripheral blood mononuclear cells (PBMC) (4, 35) and to determine anti-SIV antibody responses by enzyme-linked immunosorbent assay (ELISA) Mirabegron (6, 7, 11, 17, 35); PBMC were stored for DNA and PCR (4, 35), and plasma was stored for RNA quantitation. TABLE 1 Summary of attenuated mutants of?SIVmac239 virus stocks were prepared by transfection of cloned DNAs into CEMx174 cells as described previously (12). For SIVmac239antigen concentrations of the virus stocks were determined by using a commercial antigen capture kit (Coulter). Polyclonal Vpr-specific and Vif-specific antisera were raised in rabbits by using –galactosidase-Vpr and -Vif fusion proteins, respectively. Monoclonal antibody to SIV p27was harvested from the FA2 hybridoma cell line (33). Virus preparations containing 200 ng of p27were treated with Laemmli sample buffer, electrophoresed through an SDSC15% polyacrylamide gel, and electroblotted onto Immobilon-P membranes (Millipore, Bedford, Mass.). Membranes were first blocked with 8% skim milk in PBSC0.05% Tween 20 (PBST) for 1 h and then incubated with a 1:500 dilution of the corresponding antiserum in the same blocking solution for 1 h at room temperature. Primary antibodies were removed by washing the membranes three times with PBST at room temperature. Dilutions Mirabegron of the secondary antibodies and detection were performed according to the protocol for the Amersham ECL system. RESULTS Stocks of SIVmac2393, -3x, and -4 were produced by transfection of Rabbit Polyclonal to NBPF1/9/10/12/14/15/16/20 cloned DNA into CEMx174 cells and harvesting virus from the cell-free supernatant at or near the peak of virus production. The precise size and limit of deletions in these strains can be found in reference 12. Virus stocks were stored Mirabegron in the vapor phase of liquid nitrogen at approximately ?160C. All three of these multiply deleted viruses replicated well in CEMx174 cells (12). In one experiment (experiment A [Table 1]), four rhesus monkeys were inoculated intravenously with normalized amounts of each strain, 3, 3x, and 4, containing 100 ng of p27 antigen. Other monkeys were infected with these same strains at different times (experiments B to D [Table 1]). We observed no unusual variations in the behavior of these viruses with infections initiated at different times. Cell-associated virus loads were measured by limiting dilution culture of PBMC (Fig. ?(Fig.1).1). Peak virus loads in this assay around week 2 decreased stepwise in the order 33×4. This result is consistent with previous virus load measurements in animals infected with in which deletion of had a greater effect than deletion of and deletion of and had a Mirabegron much greater effect than deletion of either element alone (11). Peak viral loads occurred around week 2 postinoculation. With parental SIVmac239, an average of 305 PBMC were required Mirabegron to recover virus at peak, compared to 4,115 PBMC for 3, 106 PBMC for 3x, and 106 PBMC for 4. Thus, peak loads in this assay for.