Figure 1.

Structure of the human bocavirus 1 (HBoV1) genome and trans complementation of viral proteins for recombinant adeno-associated virus 2 (rAAV2)/HBoV1 production. (a) Schematic illustration of structures of the HBoV1 genome and the cap mRNAs. The HBoV1 genome (GenBank accession no. JQ923422³²) harbors three sets of open reading frames (ORFs) that encode: the nonstructural (NS) proteins NS1–4 and NP1, and the capsid proteins VP1–3. Also, an intragenic promoter is used to transcribe the non-coding RNA BocaSR. The ORFs of capsid proteins VP1–3 overlap. All of the viral mRNA transcripts are generated from a single precursor transcribed from the P5 promoter, through alternative splicing at the marked donor (D) and acceptor (A) sites. Also, alterative polyadenylation occurs at both proximal polyadenylation (pA)p sites within the unique sequence of VP1/VP2 and distal polyadenylation (pA)d sites. mRNA R6, R7, and R8 behave similarly in terms of VP1–3 expression.⁴³ NP1 is crucial to the generation of these cap mRNAs and to overcoming premature transcription caused by (pA)p. (b) Comparison of the effectiveness of HBoV1 helper plasmids. Left: Schematic illustration of helper plasmids. pHBoV1KUm630 is the replication-incompetent HBoV1 genome used in the prototype production system. pCMVR7cDNAm(pA)p encodes a modified HBoV1 R7 mRNA in which the (pA)p sites were eliminated by incorporating silent mutations. pCMVNSCap, pCMVNS*Cap, and pCMVNP1Cap are derivatives of pHBoV1KUm630 in which the P5 promoter is replaced with the CMV promoter. pCMVNScap expresses all of the structural and NS proteins of HBoV1, whereas pCMVNS*Cap lacks NS1/NS2 and pCMVNP1Cap lacks NS1-4. Right: Efficiencies of helper plasmids at left in producing rAAV2/HBoV1. Bars represent mean yield (N as indicated; error bars represent ±standard error of the mean [SEM] or difference [in the tests of N = 2]) relative to that of the prototype system, which typically yields 2.9 ± 0.5 × 10¹¹ DNase I-resistant particles (DRP) of purified rAAV2/HBoV1 from forty 150 mm dishes of transfected HEK293 cells.