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 well-defined 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.

Fig 1

Adenoviral functions induce both B19V genome replication and structural protein expression in 293 cells. 293 cells were transfected with 1 μg of SalI-linearized wild-type (pB19-M20) or NS1-negative (pB19-M20dNS1) B19V plasmid DNA. Early adenoviral functions and the B19V NS1 protein were supplied by cotransfection of 1 μg of pHelper plasmid and 0.5 μg of HCMV- or p6-driven NS1 constructs as indicated. The total amount of DNA in each transfection was adjusted to 4 μg with plasmid pCATCH containing the HCMV promoter without any downstream coding sequences. As a negative control and for monitoring the transfection efficiency, cells were transfected with 4 μg of the yellow fluorescent protein (YFP) expressing plasmid pYFP-C2 (Clontech). (A) Southern blot hybridization of DpnI-digested Hirt DNA with a biotin-11-dUTP-labeled hybridization probe from the B19V NS1 region. Arrows indicate putative monomeric (RF_M) and dimeric (RF_D) replicative B19V intermediates. (B and C) Western blot analysis of whole-cell extracts with the monoclonal antibodies MAB1424 recognizing the NS1 protein (B) and MAB8293 (both from Millipore) recognizing the B19V capsid proteins (C). Arrows indicate the positions of the NS1 protein and the VP1 and VP2 capsid proteins, respectively. (D) Quantitative B19V VP Western analysis with infrared fluorescence-labeled secondary antibodies as described in Materials and Methods. The VP2 expression data were normalized for the β-actin loading control and are represented as relative values, with the wild-type B19V DNA in the absence of pHelper set as 1. The bars represent arithmetic means ± the standard deviations (SD) of two independent experiments with the corresponding B19V VP ECL analysis and the β-actin control for one of these experiments shown in panel C.

Fig 2

E4orf6 is required for induction of B19V DNA synthesis in 293 cells. 293 cells were cotransfected with 1 μg of SalI-linearized pB19-M20 DNA and increasing amounts (25 ng, 100 ng, 250 ng, and 1 μg) of either pHelper or the isolated pHelper functions E2a, E4orf6, or VA RNA as indicated. Adjustment of the total amount of DNA to 4 μg was performed with the pCATCH plasmid. (A) Southern blot detection of DpnI-resistant B19V replicative intermediates essentially as described in the legend to Fig. 1A, except that a [³²P]dCTP-labeled hybridization probe was used. (B) Immunoblot analysis for B19V VP expression with the monoclonal antibody MAB8293, as described for Fig. 1B.

Fig 3

VA RNA activates B19V capsid protein expression independent of DNA replication. 293 cells were cotransfected with 1 μg of SalI-linearized NS1-negative B19V plasmid pB19-M20dNS1 and then evaluated for different early adenoviral functions and a p6-driven NS1 expression construct as indicated. An adjustment of the total amount of DNA to 4 μg for each transfection was performed with pCATCH plasmid DNA. Plasmid pYFP-C2 expressing the YFP was transfected as a negative control. Whole-cell extracts were subjected to B19V capsid protein immunoblot analysis with MAB8293 as described for Fig. 1B. (A and B) Two different concentrations (100 and 250 ng) of either pHelper or the isolated pHelper functions E2a, E4orf6, or VA RNA were cotransfected either in the absence (A) or presence (B) of 1 μg of complementing p6-NS1 construct. The lower panels show the results of the quantitative B19V VP Western analysis performed in parallel, which was normalized for the β-actin expression shown at the bottom of the upper panels. The bars represent arithmetic means ± the SD of two independent experiments. (C) Cells were cotransfected with increasing amounts of p6-NS1 construct (0.5, 1.0, and 2.0 μg) in the presence of two constant amounts (100 and 250 ng) of VA RNA.

Fig 4

E4orf6-mediated induction of B19V DNA synthesis requires the BC box motifs involved in formation of a cullin 5-based ubiquitin E3 ligase complex. 293 cells were cotransfected with 1 μg of SalI-linearized pB19-M20 DNA and increasing concentrations (25, 100, and 250 ng) of wild-type E4orf6 or constructs with various point mutations in the E4orf6 BC box motifs, which are required for the formation of a functional E3 ligase complex. The upper panels show the Southern blot detection of DpnI-resistant B19V replicative intermediates after isolation of Hirt DNA's as described for Fig. 2A, whereas the immunoblot analysis for B19V VP expression with MAB8293 as described for Fig. 1B is displayed in the lower panels. For the lanes marked “no B19V-DNA,” 4 μg of pYFP-C2 was transfected as a negative control.

Fig 5

VA RNA-mediated stimulation of B19V capsid protein depends on the interaction of VA I RNA with protein kinase R (PKR). (A and D) 293 cells were cotransfected with 1 μg of either pB19-M20 plasmid DNA or pB19-M20dNS1 plasmid DNA in SalI-linearized form as indicated and either increasing concentrations (25, 100, and 250 ng) of constructs encoding either both VA I and VA II RNA or the individual VA RNAs (A) or two different concentrations (25 and 100 ng) of constructs encoding wild-type VA I RNA or various VA I RNA mutants as indicated (D). Plasmid pYFP-C2 expressing the YFP was transfected as a negative control in panel D. (B) Quantitative B19V VP Western analysis of the protein extracts from the experiment with the NS1-negative B19V genome shown in the lower part of panel A with the β-actin loading control shown below the B19V VP ECL detection. (C and E) 293 cells were cotransfected with low amounts (20 ng) of a p6-luciferase reporter construct and either four different concentrations (25 ng, 100 ng, 250 ng, and 1 μg) of constructs encoding either both VA I and VA II RNA or the individual VA RNAs as indicated (C) or two different concentrations (100 and 250 ng) of wild-type VA I RNA or various VA I RNA mutants (E). Total DNA amounts were adjusted to 4 μg with the Bluescript SK2 vector. At 40 h posttransfection, the luciferase activities were determined, and these are represented as relative values with the respective activities in the absence of VA RNA set as 1. The data are presented as means ± the SD. P values of the statistical significances for the changes in luciferase activity compared to those in the absence of VA RNA are indicated above individual bars (*, P < 0.05; **, P < 0.01; ***, P < 0.001).