Transcriptional transactivation of parvovirus B19 promoters in nonpermissive human cells by adenovirus type 2

Abstract

The pathogenic human parvovirus B19 contains a promoter at map unit 6 (B19p6) of the viral genome, expression from which is largely restricted to human cells in the erythroid lineage, whereas a putative promoter at map unit 44 (B19p44) is inactive during a natural viral infection. Although nonerythroid human cells, such as HeLa and KB, allow expression from the B19p6 promoter but not from the B19p44 promoter following DNA-mediated transfection, little expression from the B19p6 promoter occurs following recombinant virus infection (submitted for publication). However, significant expression from the B19p6 promoter as well as the B19p44 promoter could be detected in a human 293 cell line that expresses the adenovirus early gene products, suggesting that coinfection with adenovirus might mediate transcriptional transactivation of the B19 promoters in nonpermissive cells. Expression of the firefly luciferase reporter gene from the B19 promoters was evaluated either following plasmid transfection or following infection with the recombinant adeno-associated virus type 2 vectors. Both B19p6 and B19p44 promoters could be transactivated by coinfection with adenovirus in nonpermissive human cells, although the extent of transactivation of the B19p44promoter was significantly lower than that of the B19p6 promoter. Expression of the adenovirus E1A proteins was necessary and sufficient for the observed transactivation of the B19 promoters. These studies further illustrate that the underlying molecular mechanisms of transactivation of parvovirus promoters in general by the adenovirus early proteins have similarities with those of the well-documented transactivation of the adeno-associated virus type 2 promoters.

The prevalence of human parvovirus B19 DNA and antibodies in blood donors from four Chinese blood centers

“…Hot‐start amplification was performed under the following conditions: one cycle of 95°C for 10 minutes; 45 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds; and a final cycle of 95°C for 15 seconds, 60°C for 15 seconds, and then gradually increased to 95°C over 30 minutes at a ramp rate of 2%. The quantitation standards used in the real‐time PCR assay were dilutions of plasmid pYT103, which contains a B19V Genotype 1 strain (Au) genome 23 . A quantitative standard curve was used to assign values (copies/mL) to test samples.…”

Rescue and Autonomous Replication of Adeno-Associated Virus Type 2 Genomes Containing Rep-Binding Site Mutations in the Viral p5 Promoter

“…Autonomous replication of AAV in 293 cells was not detected during infection studies when a multiplicity of infection of 20 (approximately 2,000 viral genomes/cell) was used (65). We suspect that we detected a low-level replication of AAV in the absence of adenovirus because substantially larger numbers (Ͼ500,000 viral genomes/ cell) were delivered in 293 cells during plasmid-mediated transfections (39,40). Abundant expression of viral transcripts occurs from transfected recombinant plasmids, even in the absence of adenovirus, which does not lead to efficient replication of viral genomes (25), suggesting suboptimal transport to the cytoplasm and/or inefficient translation of these transcripts.…”

Enhanced Transduction of Human Hematopoietic Stem Cells by AAV6 Vectors: Implications in Gene Therapy and Genome Editing

“…[22][23][24] More recently, AAV6 vectors were reported to lead to successful genome editing of sickle mutation in primary human HSCs from patients with sickle cell disease. 25 However, multiplicities of infection (MOIs) of 100,000-200,000 vgs/cell were required to achieve transduction efficiencies ranging between 45%-55% in those studies, although different strategies have been explored to improve the transduction efficiency of AAV6 vectors in human HSCs, such as the use of (1) self-complimentary AAV vectors, [26][27][28] (2) tropism specific promoters, 29,30 and (3) capsid-mutagenesis of AAV vectors. [31][32][33][34][35] Recently, human serum albumin (HSA) was shown to increase the transduction efficiency of all AAV serotype vectors, 36 and more recently, polyvinyl alcohol (PVA), was reported to be a superior replacement for HSA for ex vivo expansion of HSCs.…”