Roles of E4orf6 and VA I RNA in Adenovirus-Mediated Stimulation of Human Parvovirus B19 DNA Replication and Structural Gene Expression

Abstract

Despite its very narrow tropism for erythroid progenitor cells, human parvovirus B19 (B19V) has recently been shown to replicate and form infectious progeny virus in 293 cells in the presence of early adenoviral functions provided either by infection with adenovirus type 5 or by addition of the pHelper plasmid encoding the E2a, E4orf6, and VA RNA functions. In the present study we dissected the individual influence of these functions on B19V genome replication and expression of structural proteins VP1 and VP2. We show that, in the presence of the constitutively expressed E1A and E1B, E4orf6 alone is able to promote B19V DNA replication, resulting in a concomitant increase in VP expression levels. The stimulatory effects of E4orf6 require the integrity of the BC box motifs, which target cellular proteins such as p53 and the Mre11 DNA repair complex for proteosomal degradation through formation of an E3 ubiquitin ligase complex with E1B. VA RNA also strongly induces VP expression but, in contrast to E4orf6, in a replication-independent manner. This stimulation could be attributed exclusively to the VA I RNA transcript and does not involve major activating effects at the level of the B19V p6 promoter, but the nucleotide residues required for the welldefined pathway of VA I RNA mediated stimulation of translation through functional inactivation of protein kinase R. These data show that the cellular pathways regulating B19V replication may be very similar to those governing the productive cycle of the helper-dependent parvoviruses, the adeno-associated viruses.

A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins

“…Extraction of viral DNA by a modified Hirt procedure and Southern blot analysis were performed as described previously (16). Samples of DpnI-digested Hirt DNA (6 g) were analyzed on Southern blots for AAV replication with a 1.6-kb fragment from the cap region of pTAV2-0 as probe labeled either with [ 32 P]dCTP for radioactive detection or with Biotin-11-dUTP for nonradioactive detection.…”

“…HEK 293-and HeLa cells were cultivated as described previously (16). Human adenovirus type 2 (Ad2) was propagated and assayed in 293 cells.…”

“…Luciferase assays and Western blot analysis were performed as described previously (16). Mouse monoclonal antibodies (MAbs) and rabbit polyclonal antibodies used for immunoblot analysis were anti-Rep MAb 303.9 (1:10; Progen), anti-VP MAb B1 (1:10; Progen), anti-VP1 MAb A1 (1:10; Progen), anti-VP1/2 MAb A69 (1:10; Progen) or rabbit and mouse anti-Flag (1:1,000; Sigma), respectively, followed by reaction with horseradish peroxidaseconjugated secondary antibodies and ECL detection.…”

Differential Contribution of Adeno-Associated Virus Type 2 Rep Protein Expression and Nucleic Acid Elements to Inhibition of Adenoviral Replication in cis and in trans

“…Immunoblot analysis of AAV-2 protein expression was performed with anti-Rep monoclonal antibody 303.9 diluted 1:10 or with anti-Cap monoclonal antibody B1 diluted 1:20 (both from Progen, Heidelberg, Germany), followed by incubation with horseradish peroxidase-conjugated secondary antibodies and ECL detection as described previously (23).…”

“…Extraction of viral DNA by a modified Hirt procedure was performed essentially as described previously (23).…”