Degradation of de novo-generated adeno-associated virus type 5 (AAV5) Rep52 and capsid proteins is part of the limited target specificity displayed by adenovirus type 5 E4Orf6-E1B-55k as part of a cullin 5-containing E3 ligase complex. Both Rep and capsid proteins can be found in the ligase complex, and their presence is dependent on interaction between E4Orf6 and elongins B and C. Degradation of AAV5 proteins can be inhibited by a dominant-negative ubiquitin that prevents chain elongation or by small interfering RNA directed against cullin 5.

Adeno-associated virus (AAV) type 2 and 5 proteins Rep52 and Rep40 were polyubiquitinated during AAV-adenovirus type 5 (Ad5) coinfection and during transient transfection in either the presence or absence of Ad5 E4orf6 and E1b-55k. Polyubiquitination of small Rep proteins via lysine 48 (K48) linkages, normally associated with targeting of proteins for proteasomal degradation, was detected only in the presence of E4orf6. The small Rep proteins were ubiquitinated via lysine 63 (K63) following transfection in either the presence or absence of E4orf6 or following coinfection with Ad5. E4orf6/E1b-55k-dependent K48-specific polyubiquitination of small Rep proteins could be inhibited using small interfering RNA (siRNA) to cullin 5.Together, adenovirus type 5 (Ad5) early gene products E1a, E1b-55k, E2a, E4orf6, and virus-associated (VA) RNA can support efficient replication of adeno-associated virus (AAV) (4, 31). E4orf6 and E1b-55k are known to interact with cellular cullin 5 (cul5), elongins B and C, and the ring box protein Rbx1 to form an E3 ubiquitin ligase complex that specifically targets a small population of cellular proteins for degradation by the proteasome (1,7,21,22,24,27). This property has been implicated in a number of functions presumed to be required for both Ad and AAV replication (3, 8-10, 17, 23, 24, 34, 35).Previously, only p53, Mre11, DNA ligase IV, and integrin ␣3 had been shown to be substrates of the Ad5 E3 ubiquitin ligase complex (1,7,21,22,24,27); however, we have recently shown (16,17) that the small Rep proteins and capsid proteins of AAV5 are also degraded in the presence of Ad E4orf6 and E1b-55k in a proteasome-dependent manner. These proteins were restored to levels required during infection by the action of VA RNA (17). The targeting for degradation of AAV5 protein by the E4orf6/E1b-55k E3 ubiquitin ligase complex required functional BC-box motifs in E4orf6 and could be inhibited by depletion of the scaffolding protein cullin 5 using directed small interfering RNA (siRNA) (16). In addition, the degradation of AAV5 protein was partially prevented by overexpression of pUBR7, a plasmid that generates a dominantnegative ubiquitin (16). The role this targeted degradation plays in the life cycle of AAV has not yet been clarified; however, E4orf6 mutants that cannot function in this regard do not support AAV replication as well as wild-type E4orf6 (R. Nayak and D. J. Pintel, unpublished data). Degradation of Mre11 by the Ad5 E3 ligase has also been implicated in allowing efficient Ad5 and AAV replication (24). Ubiquitination of AAV Rep proteins during viral infection, however, has not previously been reported. (Fig. 1A, lanes 3 and 4). For Rep immunoprecipitations (IPs), cells were lysed by boiling in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 2 mM EDTA, 1% NP-40) plus 1% SDS, extracts were diluted to 0.2% SDS, and antibody was added, and following capture on protein G beads (#10-1243; Invitrogen, Carlsbad, CA), the cells were subsequently washed in R...

Alternative splicing of adeno-associated virus type 2 (AAV2) P19-generated pre-mRNAs generates the small Rep proteins Rep52 and Rep40, which differ in their carboxyl termini. Both proteins are required for optimal packaging of AAV2 genomes. AAV5 Rep-encoding P19-generated transcripts are primarily polyadenylated within the central intron and not efficiently spliced; however, surprisingly, AAV5 was found to generate high levels of a Rep40-like protein. The AAV5 Rep40-like protein was generated by internal initiation and has the same C terminus as Rep52. Although precluded from using alternative splicing to generate multiple Rep isoforms, AAV5 ensures the production of a Rep40-like protein by utilizing a novel internal translation initiation event.The adeno-associated virus type 2 (AAV2) P19 promoter generates pre-mRNAs that are alternatively spliced to yield the small nonstructural proteins Rep52 and Rep40 from unspliced and spliced mRNAs, respectively. These Rep proteins (as well as the overlapping large Rep proteins) share a common Walker-type domain common to the superfamily 3 (SF3)-type helicases (3, 4). Both small Rep proteins are required for efficient packaging of the AAV2 genome; Rep52 and Rep40 both possess 3Ј-to-5Ј helicase activity and are required for unwinding of the double-stranded replicative forms of AAV for insertion into preformed capsids (5). Rep52 and Rep40 possess some functional redundancy in their helicase activities, ATPase activities, and DNA binding activities; however, their precise mechanisms of action are different. Structural analyses have indicated that when bound to DNA, AAV2 Rep52 remains monomeric (14), while Rep40 assumes a hexameric ringlike structure (1, 4), which has been suggested to be important for its function. Although Rep52 and Rep40 both are capable of unwinding DNA substrates with single-stranded DNA ends in a 3Ј-to-5Ј fashion, Rep40, but not Rep52, efficiently unwinds double-stranded DNA (1). Therefore, it has been suggested that during AAV replication, the Rep40 hexamer may be necessary for the initial unwinding of the first few bases of the double-stranded DNA intermediate, and further unwinding may then be accomplished by Rep52 alone or by Rep52 in conjunction with Rep40 and other Rep proteins (1).In contrast to AAV2, pre-mRNA transcripts derived from the AAV5 and goat AAV P19 promoter are preferentially polyadenylated in the viral central intron and thus spliced at low efficiency (10-12). Thus, these viruses would not be expected to generate significant quantities of Rep40. However, immunoblot analyses have consistently demonstrated the presence of significant levels of a Rep40-like (but not a Rep 68-like) protein during AAV5 and goat AAV coinfection with adenovirus, as well as during transient transfection (7,8,11). This suggested that AAV5 may generate a Rep40-like protein using a mechanism distinct from the alternative splicing utilized by AAV2. Here we show that, unlike AAV2 Rep40, the AAV5 Rep40-like protein has the same C terminus as Rep52 and that these tw...

Adeno-associated viral (AAV) capsid proteins, thought to be a rate-limiting step in the production of recombinant AAV (rAAV), are translated from spliced mRNAs. Improvement of the native AAV nonconsensus donor sequence increases splicing yet leaves the relative levels of VP1- and VP2/3-encoding mRNAs unchanged, and thus provides a means to increase delivery of correct ratios of AAV capsid proteins. This effect is independent of the AAV serotype used, and occurs whether the rep and cap genes supplied in trans are on the same or separate expression vectors. In the split-vector system, replacement of the more traditionally used cytomegalovirus promoter with that of the AAV5 P41 promoter allowed for even greater levels of splicing, and together with an improved intron donor, led to a 10- to 15-fold increase in the levels of splicing, rAAV production, and transduction compared with levels achieved by traditional cotransfection methods. Thus, the enhancement of splicing presents a useful method to enhance rAAV production via transient transfection.

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is an autosomal recessive disorder that develops in infancy and arises from mutation of the immunoglobulin helicase μ-binding protein 2 ( IGHMBP2 ) gene. Whereas IGHMBP2 is ubiquitously expressed, loss or reduction of function leads to alpha motor neuron loss and skeletal muscle atrophy. We previously developed a gene therapy strategy for SMARD1 using a single-stranded AAV9- IGHMBP2 vector and compared two different delivery methods in a validated SMARD1 mouse model. An important question in the field relates to the temporal requirements for this or any potential treatment. To examine the therapeutic window, we utilized our recently developed SMARD1 model, FVB/NJ- Ighmpb2 nmd-2J , to deliver AAV9- IGHMBP2 at four different time points starting at post-natal day 2 (P2) through P8. At each time point, significant improvements were observed in survival, weight gain, and motor function. Similarly, treatment improved important hallmarks of disease, including motor unit pathology. Whereas improvements were more pronounced in the early-treatment groups, even the later-treatment groups displayed significant phenotypic improvements. This work suggests that an effective gene therapy strategy could provide benefits to pre-symptomatic and early-symptomatic individuals, thereby expanding the potential therapeutic window for SMARD1.