Efficient replication of adeno-associated virus type 2 vectors: a cis-acting element outside of the terminal repeats and a minimal size.

Recombinant adeno-associated virus type 2 (AAV2) can be produced in adenovirus-infected cells by cotransfecting a plasmid containing the recombinant AAV2 genome, which is generally comprised of the viral terminal repeats flanking a transgene, together with a second plasmid expressing the AAV2 rep and cap genes. However, recombinant viruses generally replicate inefficiently, often producing 100-fold fewer virus particles per cell than can be obtained after transfection with a plasmid containing a wild-type AAV2 genome. We demonstrate that this defect is due, at least in part, to the presence of a positive-acting cis element between nucleotides 194 and 1882 of AAV2. Recombinant AAV2 genomes lacking this region accumulated 14-fold less double-stranded, monomer-length replicative-form DNA than did wild-type AAV2. In addition, we demonstrate that a minimum genome size of 3.5 kb is required for efficient production of single-stranded viral DNA. Relatively small recombinant genomes (2,992 and 3,445 bp) accumulated three- to eightfold less single-stranded DNA per monomer-length replicative-form DNA molecule than wild-type AAV2. In contrast, recombinant AAV2 with larger genomes (3,555 to 4,712 bp) accumulated similar amounts of single-stranded DNA per monomer-length replicative-form DNA compared to wild-type AAV2. Analysis of two recombinant AAV2 genomes less than 3.5 kb in size indicated that they were deficient in the production of the extended form of monomer-length replicative-form DNA, which is thought to be the immediate precursor to single-stranded AAV2 DNA.

Post-transcriptional regulation of RNA polymerase II levels in Caenorhabditis elegans.

To investigate the regulation of RNA polymerase II levels in Caenorhabditis elegans, we have constructed nematode strains having one, two, or three copies of ama-1, the gene for the largest subunit of RNA polymerase II. Steady-state levels of RNA polymerase II polypeptides and solubilized enzyme activity are invariant with gene dosage, indicating regulatory compensation. However, steady-state levels of ama-1 mRNA are directly proportional to gene dosage. These results imply that RNA polymerase II levels in C. elegans are regulated post-transcriptionally.

The minor capsid protein VP1 of the autonomous parvovirus minute virus of mice is dispensable for encapsidation of progeny single-stranded DNA but is required for infectivity.

The two capsid proteins of minute virus of mice, VP1 and VP2, are generated from a single large open reading frame by alternate splicing of the capsid gene mRNA. Examination of the replication of a series of mutants that express only VP1, only VP2, or neither capsid protein demonstrates that VP2 is necessary for the accumulation and encapsidation of virus progeny single-stranded DNA. VP1 is dispensable for these functions but is required to produce an infectious virion. Virus that lacks VP1 binds to cells as efficiently as wild-type minute virus of mice but fails to initiate a productive infection. Because neither capsid protein is required for viral-DNA replication, these results suggest that virus lacking VP1 is blocked at a step during virus entry, subsequent to cell binding and prior to the initiation of DNA replication.

The trypsin-sensitive RVER domain in the capsid proteins of minute virus of mice is required for efficient cell binding and viral infection but not for proteolytic processing in vivo.

Analysis of a series of mutations in the trypsin-sensitive RVER region of the amino terminal domain in the capsid proteins (VP1 and VP2) of the autonomous parvovirus, minute virus of mice (MVM), demonstrates that this sequence is not essential for proteolytic processing of VP2 into VP3 in vivo, but specific amino acids within this domain are important for viral infection. Analysis of the most deficient of these mutants, VP(delta 2842-2863), a 7-aa deletion of aa 159-165 in VP1 and 17-23 in VP2, has identified at least two steps in MVM infection in which this domain is important. VP(delta 2842-2863) was 3-fold defective in binding to murine A9(2L) cells and, when an equivalent amount of virus was bound to cells, additionally 10-fold deficient compared to wild-type in initiating a productive infection. However, in those cells effectively infected, VP(delta 2843-2863) replicated similar to wild-type. These results suggest that these seven amino acids constitute a region important for both binding and a subsequent step prior to the start of DNA replication such as viral uptake or transport to the nucleus.

Nonsense mutations inhibit splicing of MVM RNA in cis when they interrupt the reading frame of either exon of the final spliced product.

mRNAs R1 and R2 of the autonomous parvovirus minute virus of mice (MVM), which encode the viral nonstructural proteins NS1 and NS2, respectively, are processed in an ordered splicing pathway in which R2 is generated from mature spliced R1. Introduction of translation termination signals into these genes alters the processing of these RNAs; there is a significant (up to fourfold) increase in the accumulated steady-state levels of R1 relative to R2, when compared with wild-type levels, although the total accumulated levels of R1 plus R2 remain the same. The increase in accumulated R1 relative to R2 in mutant infected or transfected murine cells is independent of RNA stability and transport and decreases, in a polar manner, with the distance of the inserted termination signal from the shared initiation codon for NS1 and NS2 at nucleotide 260. The increased ratio of R1 to R2 is a consequence of the artificially introduced translation termination signals acting in cis rather than in the absence of a functional viral gene product. These mutations have an effect when they interrupt previously open reading frames in either exon of the spliced product R2. Nonsense mutations that are located in the second exon of R2 inhibit splicing of R1 to R2 only when they interrupt an open reading frame (ORF) that has the potential, after normal splicing, to be joined in-frame with the initiating AUG. These results suggest that nonsense mutations inhibit splicing of R1 to R2 by influencing the mechanism by which exons are defined in murine cells.

Generation and characterization of a temperature-sensitive mutation in the NS-1 gene of the autonomous parvovirus minute virus of mice.

In-phase single-codon insertion mutations were constructed in the open reading frames of the NS-1 and NS-2 genes of the autonomous parvovirus minute virus of mice. A viral mutant containing an isoleucine insertion exclusively within NS-1 between residues 229 and 230 was isolated that produced approximately 3 orders of magnitude fewer plaques at 39 degrees C than at 32 degrees C. Preliminary characterization of the mutant demonstrated that the NS-1 gene product is independently required for both genome amplification and the regulation of the temporal expression between the two viral transcription units during lytic infection.