Adeno-associated virus Rep protein inhibits human immunodeficiency virus type 1 production in human cells.

The adeno-associated virus (AAV) rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40) required for AAV DNA replication and AAV gene regulation. In addition, the Rep proteins may have pleiotropic regulatory effects in heterologous systems, and in particular Rep78 may mediate a negative regulatory effect. We analyzed the effects of the AAV rep gene on human immunodeficiency virus type 1 (HIV-1) gene expression. The rep gene proteins of AAV type 2 (AAV2) inhibited the trans-activating ability of HIV-1. Constructs containing the AAV2 rep gene (pHIVrep) or a CAT gene (pBennCAT) expressed from the 5' HIV-1 long terminal repeat were inducible for Rep78 and Rep68 or CAT expression, respectively, when cotransfected with a plasmid containing the HIV-1 tat gene (pARtat). When equivalent amounts of pHIVrep and pBennCAT were cotransfected with increasing amounts of pARtat, expression of CAT activity was decreased. The pHIVrep construct was more inhibitory than plasmids expressing rep from the wild-type AAV2 p5 transcription promoter. rep expression from pHIVrep almost completely inhibited the replication of an HIV-1 proviral clone as measured by reverse transcriptase activity and p24 protein levels. Inhibition of HIV-1 production by Rep protein was also seen at the transcriptional level in that all HIV-1 transcripts were decreased when pHIVrep was present. The inhibitory effects of pHIVrep appear to be mediated primarily by Rep78 and perhaps Rep68. These results suggest that a trans-acting protein from a heterologous virus might be used to inhibit HIV-1 growth.

Femoral head osteonecrosis. Detection by magnetic resonance imaging versus single-photon emission computed tomography.

Magnetic resonance imaging (MRI) and single-photon emission computed tomography (SPECT) examinations were compared for detection of femoral head osteonecrosis. Of 29 hips with clinical and roentgenographic evidence of osteonecrosis (18 histologically confirmed), 15 were Stage II, three transitional, six Stage III, and five Stage IV. MRI identified osteonecrosis in all 29 cases (100% sensitivity), and there were no false-positives (100% specificity). Of 24 osteonecrotic hips with technically adequate examinations, SPECT identified 14 (sensitivity 58%), and there were four false-positives (78% specificity). If Stages III and IV were eliminated, SPECT correctly identified ten of 15 (67% sensitivity).

Cholesterol assays in the Seralyzer instrument.

We evaluated the Seralyzer instrument for the assay of serum cholesterol and compared it to the Kodak Ektachem method. The Seralyzer showed good accuracy in the analysis of cholesterol in Abell-Kendall-verified serum pools, and the bias from the expected value was small in all cases but one. The Seralyzer exhibited CVs of less than 5% in all cases and good comparison with the Ektachem method. The Seralyzer is easy to use; however, some training in the proper pipetting technique is necessary. The Seralyzer meets medical needs criteria of total error greater than 5% from the true cholesterol value.

Effect of a viral rep gene on transformation of cells by an adeno-associated virus vector.

Adeno-associated virus (AAV) vectors readily express the gene for geneticin-resistance under control of the AAV p40 promoter when chromosomally integrated at low copy number in mammalian cells. We show that a truncated AAV rep gene, transcribed from the p5 and p19 promoters, mediates a negative effect on expression of geneticin-resistance in human 293 cells and a positive effect in HeLa cells. Also, we describe a novel phenotype for a mutant expressing the p19 rep gene alone which has a negative effect in 293 cells but no positive effect in HeLa cells.

Adeno-associated virus vector for high-frequency integration, expression, and rescue of genes in mammalian cells.

We describe the construction of an adeno-associated virus (AAV) vector in which the coding sequence of the procaryotic gene neo is expressed under the control of the major AAV promoter p40. This AAV-neo vector allowed stable expression of neo as a dominant selective marker in mammalian cells by selection of cells which were resistant to the antibiotic geneticin (G418). When the vector was introduced into human (293 or HeLa) cell lines by a DNA transfection procedure, stable geneticin-resistant colonies were obtained. When the vector was first packaged into AAV particles and then introduced into cells via particle infection, geneticin-resistant cells were obtained at higher frequencies than those obtained by DNA transfection. In geneticin-resistant cells the AAV-neo vector was integrated at low copy number and could be rescued by subsequent infection with wild-type AAV and the helper adenovirus or, in some cases, by infection with adenovirus alone. The rescued AAV-neo vector could then be recovered as amplified unintegrated DNA from a Hirt lysate. These results demonstrate that AAV can be used as a transducing viral vector for stable integration and expression of a foreign gene in mammalian cells. The high frequency of integration and the ability to rescue the integrated vector suggest that this vector system may be useful for selecting genes from cDNA libraries. This vector may also be useful for introduction of genes into cells which are refractory to transfection in procedures such as those involving the use of CaPO4 or DEAE-dextran.

Genetic analysis of adeno-associated virus: properties of deletion mutants constructed in vitro and evidence for an adeno-associated virus replication function.

Transfection of a pBR322-based, recombinant plasmid, pAV2, containing the entire adeno-associated virus (AAV) type 2 genome into human 293 cells in the presence of helper adenovirus resulted in rescue and replication of AAV to yield infectious particles. We constructed mutants of pAV2 containing deletions within the AAV sequence. We describe here the phenotypes of these AAV deletion mutants. The results can be summarized as follows. Mutants (cap-) with deletions between map positions 53 and 85 did not synthesize capsid antigen or progeny single-stranded DNA but accumulated normal levels of duplex replicating form DNA. Mutants (rep-) with deletions between map positions 17 and 36 failed to rescue or replicate any AAV DNA. The rep- mutants could be complemented for replicating form DNA synthesis by a cap- mutant. This clearly demonstrates an AAV-coded replication function which is different from the capsid antigen. Other mutants (inf-) with deletions in the region between map positions 40 and 52 synthesized abundant amounts of replicating form DNA and capsid antigen but gave a low yield of infectious particles. This suggests that there may be an additional region of AAV, perhaps within the intron, which is required for efficient particle assembly. This work shows that AAV is genetically complex and expresses at least three clearly different functions.

Genetic alterations in potassium transport in L cells.

Starting with mutagenized cultures of the mouse fibroblastic cell line LM(TK-), we have selected mutant clones by their ability to grow at 0.2 micrometer K+, a concentration unable to support the growth of the parent cell. The mutants fall into two classes on the basis of their potassium transport properties. Both classes maintain a high intracellular K+ concentration when growing in low-potassium medium, and both are unaltered in the ouabain-sensitive Na/K pump. One class shows an increased activity of a ouabain-resistant, furosemide-sensitive K+ transport system; the other class shows a decreased activity of a specific component of K+ efflux.

Overexpression of an unstably inherited gene in cultured mouse cells.

The specific activity of hypoxanthine-guanine phosphoribosyltransferase (IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) is increased up to 58-fold in unstable gene transferents produced by the transfer of cell-free chromosomal material from one mouse L cell line to another; the specific activity of this enzyme returns to normal levels when the transferred gene becomes stabilized. This phenomenon, which is not observed in comparable heterospecific transfers, may be an effect of gene dosage (multiple copies of the transferred genetic fragment in the unstable gene transferents), or it may represent an escape of the unstably inherited gene from the normal regulatory mechanisms of the recipient cell.

Chromosome-mediated gene transfer between closely realted strains of cultured mouse cells.

Gene transfer between two closely related mouse cell lines has been carried out, using as the vector a cell-free preparation of metaphase chromosomes and nuclei. Distinction between gene transferents and revertants of the recipient mutant phenotype was achieved by the use of a donor strain carrying a mutationally altered (8-azaguanine-resistant) hypoxanthine-guanine phosphoribosyltransferase (HPRTase; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8). The transferred HPRTase gene is initially unstable; in nonselective medium, it is lost at a rate of about 0.1 per cell per generation. Stabilization occurs as a rare event, with a frequency on the order of 1 X 10(-5) per cell per generation. The unstable state can be maintained for at least 200 generations through serial passages of the transferent in selective medium. Under the conditions of cultivation used in these experiments, the unstable HPRTase-positive cells are eventually replaced by the stable HPRTase-positive cells in the population.

Comparison of deoxyribonucleic acid uptake and marker integration in bacilli and protoplasts of Bacillus subtilis.

trp(+)his(-) donor deoxyribonucleic acid (DNA) was added to highly competent trp(-)his(+) recipient bacilli and to protoplasts prepared from these bacilli, and the cell-DNA complexes were incubated for 30 min. The complexes were then washed and lysed, and their DNA was analyzed on a trp(-)his(-) strain for the donor marker trp(+), the resident marker his(+), and for the recombinant trp(+)his(+) combination. The extracts of the bacillary complexes contained a normal percentage of donor markers (0.1-0.2%), and the number of trp(+)his(+) doubles (20% of all trp(+) transformants) indicated that the donor DNA had become integrated into the resident genomes. The protoplast complexes contained 10 to 1,000 times fewer donor markers and almost no recombinants. This indicated that, in protoplasts, marker uptake was minimal and recombination was absent. Uptake was also measured with (3)H-labeled DNA. On the average, protoplasts took up one-fiftieth as much DNA as bacilli. It was concluded that, probably, protoplasts took up no DNA at all, that there were no DNA affinity sites on the surface of the protoplasts, and that the residual marker and radioactivity uptake was due to imperfections in the experimental system. The data and conclusions differed sharply from earlier ones of Hirokawa and Ikeda despite the fact that the techniques of these authors were followed in repeat experiments.