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.

We describe the construction of an adeno-associated virus (AAV) vector

The adeno-associated virus (AAV) rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep4O) 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 transacting protein from a heterologous virus might be used to inhibit HIV-1 growth.

The lytic action of lysozyme upon Bacillus subtilis walls was studied by following the disappearance of bacillary-colony-forming units and the appearance of L-colony-forming-units. The rapidity of cell wall removal by lysozyme fluctuated markedly during growth in a chemically defined medium, presumably because subtle changes in the cell wall were constantly occurring. When lysozyme-sensitive bacilli were grown with chloramphenicol 10 pg./ml. for 3 hr they showed a notable increase in lysozyme resistance; at the same time, their walls almost doubled in thickness. As lysozyme attack proceeded in a given culture, the bacilli passed first through a rod-shaped osmotically sensitive stage, and then a spherical stage characterized by incomplete removal of cell wall before finally reaching the naked protoplast stage. The spherical forms with adherent wall residues formed L colonies on a medium containing the reversion inhibitor D-methionine and bacillary colonies on the same medium without D-methionine. Under the latter conditions, the cell wall residue served as a starting point for rebuilding of complete wall, much as residual wall permits reversion of Gram-negative spheroplasts to the bacillary state. In the presence of Dmethionine, the feedback sequence required for wall formation was severed, resulting in heritable propagation of the protoplast state.

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 mM 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.Mammalian cells maintain a high internal potassium concentration and a low internal sodium concentration relative to their environment; the resulting cation gradients play a central role in cell physiology, being responsible for such diverse functions as volume regulation (1), nutrient transport (2), and membrane excitability (3). Mechanistically, the potassium and sodium gradients reflect the steady-state concentrations at which all passive ion movements (leaks) are balanced exactly by all active ion movements (pumps) (1). Among the active pathways, the ouabain-sensitive Na+,K+-ATPase is the best characterized (4), but additional ouabain-insensitive active cation movements have been detected (for example, see refs. 5-7). There are also several modes of passive K+ and Na+ movement that are determined by the specific membrane permeabilities to the ions and are thought to occur through ion-selective channels (3). It is the ability of a class of these channels to alter permeability in response to membrane potential or receptor-bound neurohormone that confers excitability upon a membrane (8). These complex and interrelated pathways are difficult to sort out unambiguously either by kinetic measurements or with the use of inhibitors.In view of the successful application of genetics to the analysis of complex transport processes in microorganisms (9), we have undertaken a genetic study of the mechanisms for cation movement in mammalian cells. One class of mutants, resistant to the cardiac glycoside ouabain, has already been isolated in a number of laboratories (for example, see refs. 10 and 11). In this paper we introduce two new classes of mutants, both selected for their ability to grow at a potassium concentration below that minimally required for the growth of parent cells. MATERIALS AND METHODSCell Lines and Culture Conditions. The parental cell line used for the isolation of mutants was LM(TK-), a thymidine kinase-deficient derivative of the mouse fibroblastic L cell line (12). All cell lines were maintained at 370C in a humidified 5% CO2 atmosphere in flasks containing a medium (13) with horse serum added to a final concentration of 10% (vol/vol