The Choice of Translation Initiation Site of the Rep Proteins from Goose Parvovirus P9-Generated mRNA Is Governed by Splicing and the Nature of the Excised Intron

The goose parvovirus (GPV) Rep 1 and Rep 2 proteins are encoded by P9-generated mRNAs that are either unspliced or spliced within the rep gene region, respectively. These mRNAs are present in an approximately equal ratio. The translation of Rep 1 was initiated from the first AUG in unspliced P9-generated mRNA; however, this AUG was bypassed in spliced P9-generated RNA and Rep 2 translation initiated predominately at the next initiating AUG downstream. We show that the choice of the site of initiation of translation of GPV Rep-encoding mRNAs is governed both by the splicing process itself and by the nature of the excised intron.Goose parvovirus (GPV) has identical hairpin termini, is most similar in both nucleotide sequence and protein homology to adeno-associated virus 2 (AAV2), and has been classified as a member of the Dependovirus genus (10-12); however, unlike the AAVs, GPV can replicate efficiently without the aid of a helper virus (12). The RNA expression profile of GPV is a surprising hybrid of features of the Parvovirus and Dependovirus genera of the Parvovirinae (7). Similar to the Dependovirus AAV5, RNAs transcribed from the GPV upstream P9 promoter, which encode the viral Rep protein(s), are polyadenylated at high efficiency at a polyadenylation [(pA)p] site located within the small intron in the center of the genome (7). No promoter analogous to the Dependovirus P19 promoter has been detected; however, similar to minute virus of mice (MVM) and other members of the Parvovirus genus, approximately half of the pre-mRNAs generated from the P9 promoter are additionally spliced within the putative GPV Rep coding region between a donor site located at nucleotide (nt) 814 and an acceptor site at nt 1198 (7). The GPV RNA profile has been shown to be the same in both human 293T and goose CGBQ cells (7). Thus, the mechanism that GPV uses for the expression of its nonstructural gene is more like that used by members of the autonomous Parvovirus group.In this report, we describe the coding strategy for the nonstructural proteins of GPV. We demonstrate that the large Rep 1 protein is encoded uninterruptedly in open reading frame 1 (ORF 1) from the unspliced P9-generated mRNA using an initiating AUG codon at nt 537. The smaller Rep 2 protein is encoded by the spliced P9-generated mRNA; it initiates in ORF 2 at an AUG at nt 650 and continues in ORF 1 after the splice. Strikingly, the first upstream AUG at nt 537 is not utilized in spliced P9-generated mRNA. We show that the choice of initiation site is governed by the splicing process itself and by the nature of the excised intron.     

Identification of the trans-acting Rep proteins of adeno-associated virus by antibodies to a synthetic oligopeptide.

Prior genetic analysis provided evidence for trans-acting regulatory proteins (Rep) coded by the left-hand open reading frame (orf-1) of adeno-associated virus (AAV). We have used immunoblotting analysis to identify four protein products of orf-1. Antibodies elicited against an oligopeptide encoded by orf-1 were reacted with extracts of cells that were infected with AAV or transfected with AAV recombinant vectors in the presence or absence of helper adenovirus. The antibody recognized four polypeptides with apparent molecular weights of 78,000, 68,000, 52,000, and 40,000. The 78,000-dalton (78K) (Rep78) and 68K (Rep68) proteins appear to be encoded by the unspliced 4.2-kilobase (kb) and spliced 3.9-kb mRNAs, respectively, transcribed from the p5 promoter. The 52K (Rep52) and 40K (Rep40) proteins appear to be the products of the unspliced 3.6-kb and the spliced 3.3-kb mRNAs, respectively, transcribed from the p19 promoter. Rigorous identification of Rep68 as an AAV-coded protein is compromised by a cross-reacting cellular protein of similar size. All four proteins were expressed in the human cell lines 293, HeLa, HT29, and A549 infected with AAV together with adenovirus. Rep78 and Rep52 were detected at lower levels in cells infected with AAV at high multiplicity in the absence of adenovirus. Human 293 cells transfected with a recombinant AAV vector (pAV2) also expressed Rep proteins in the presence or absence of adenovirus. Mutations introduced into the Rep region of pAV2 further identified the Rep proteins. The amount of each Rep protein varied between nuclear and cytoplasmic extracts, but all four proteins accumulated during the lytic cycle of the viral infection. Other studies have indicated that the Rep proteins have independent trans-acting functions in viral DNA replication and negative and positive regulation of gene expression. Correlation of each trans-acting function with individual Rep proteins will be facilitated with the antibodies described herein.     

Distance-dependent processing of adeno-associated virus type 5 RNA is controlled by 5' exon definition

Adeno-associated virus type 5 (AAV5) is unique among human AAV serotypes in that it uses a polyadenylation site [(pA)p] within the single small intron in the center of the genome. We previously reported that inhibition of polyadenylation at (pA)p, necessary for read-through of P41-generated capsid gene pre-mRNAs which are subsequently spliced, requires binding of U1 snRNP to the upstream donor. Inhibition was reduced as the distance between the cap site and the donor was increased (increasing the size of the 5' exon). Here, we have demonstrated that U1-70K is a key component of U1 snRNP that mediates inhibition of polyadenylation at (pA)p. Furthermore, introduction of a U-rich stretch, predicted to target TIA-1 and thus increase the affinity of U1 snRNP binding to the intervening donor site, significantly augmented inhibition of (pA)p, while depletion of TIA-1 by siRNA increased (pA)p read-through. Finally, artificially tethering the cap binding complex (CBC) components CBP80 and CBP20 upstream of the intron donor increased inhibition of polyadenylation at (pA)p. Our results suggest that interaction with the CBC strengthens U1 snRNP binding to the downstream intron donor in a manner inversely proportional to the size of the 5' exon, thus governing the competition between intron splicing and polyadenylation at (pA)p. This competition must be optimized to program both the levels of polyadenylation of P7- and P19-generated RNA at (pA)p required to produce proper levels of the essential Rep proteins and the splicing of P41-generated RNAs to produce the proper ratio of capsid proteins during AAV5 infection.     

Asymmetric replication in vitro from a human sequence element is dependent on adeno-associated virus Rep protein.

The DNA of human parvovirus adeno-associated virus type 2 (AAV) integrates preferentially into a defined region of human chromosome 19. Southern blots of genomic DNA from latently infected cell lines revealed that the provirus was not simply inserted into the cellular DNA. Both the proviral and adjoining cellular DNA organization indicated that integration occurred by a complex, coordinated process involving limited DNA replication and rearrangements. However, the mechanism for targeted integration has remained obscure. The two larger nonstructural proteins (Rep68 and Rep78) of AAV bind to a sequence element that is present in both the integration locus (P1) and the AAV inverted terminal repeat. This binding may be important for targeted integration. To investigate the mechanism of targeted integration, we tested the cloned integration site subfragment in a cell-free replication assay in the presence or absence of recombinant Rep proteins. Extensive, asymmetric replication of linear or open-circular template DNA was dependent on the presence of P1 sequence and Rep protein. The activities of Rep on the cloned P1 element are analogous to activities on the AAV inverted terminal repeat. Replication apparently initiates from a 3'-OH generated by the sequence-specific nicking activity of Rep. This results in a covalent attachment between Rep and the 5'-thymidine of the nick. The complexity of proviral structures can be explained by the participation of limited DNA replication facilitated by Rep during integration.     

Biologically active Rep proteins of adeno-associated virus type 2 produced as fusion proteins in Escherichia coli.

Four Rep proteins are encoded by the human parvovirus adeno-associated virus type 2 (AAV). The two largest proteins, Rep68 and Rep78, have been shown in vitro to perform several activities related to AAV DNA replication. The Rep78 and Rep68 proteins are likely to be involved in the targeted integration of the AAV DNA into human chromosome 19, and the full characterization of these proteins is important for exploiting this phenomenon for the use of AAV as a vector for gene therapy. To obtain sufficient quantities for facilitating the characterization of the biochemical properties of the Rep proteins, the AAV rep open reading frame was cloned and expressed in Escherichia coli as a fusion protein with maltose-binding protein (MBP). Recombinant MBP-Rep68 and MBP-Rep78 proteins displayed the following activities reported for wild-type Rep proteins when assayed in vitro: (i) binding to the AAV inverted terminal repeat (ITR), (ii) helicase activity, (iii) site-specific (terminal resolution site) endonuclease activity, (iv) binding to a sequence within the integration locus for AAV DNA on human chromosome 19, and (v) stimulation of radiolabeling of DNA containing the AAV ITR in a cell extract. These five activities have been described for wild-type Rep produced from mammalian cell extracts. Furthermore, we recharacterized the sequence requirements for Rep binding to the ITR and found that only the A and A' regions are necessary, not the hairpin form of the ITR.