Chipmunk parvovirus is distinct from members in the genus Erythrovirus of the family Parvoviridae

Abstract

The transcription profile of chipmunk parvovirus (ChpPV), a tentative member of the genus Erythrovirus in the subfamily Parvovirinae of the family Parvoviridae, was characterized by transfecting a nearly full-length genome. We found that it is unique from the profiles of human parvovirus B19 and simian parvovirus, the members in the genus Erythrovirus so far characterized, in that the small RNA transcripts were not processed for encoding small non-structural proteins. However, like the large non-structural protein NS1 of the human parvovirus B19, the ChpPV NS1 is a potent inducer of apoptosis. Further phylogenetic analysis of ChpPV with other parvoviruses in the subfamily Parvovirinae indicates that ChpPV is distinct from the members in genus Erythrovirus. Thus, we conclude that ChpPV may represent a new genus in the family Parvoviridae.

Figure 1. Replication of the construct pC1ChpPV in COS-7 cells.

COS-7 cells were transfected with pC1ChpPV. Hirt DNA sample was extracted from the cells at 48 hrs post transfection, and were digested with DpnI (DpnI +) vs. without DpnI (DpnI -). Samples were subjected to Southern blotting analysis using the ChpPV NSCap probe (nt 1–5205). Ten ng of pC1ChpPV plasmid DNA were treated in parallel as a DpnI-digestion control (lane 2).

Figure 2. Transcription mapping of ChpPV RNA by RNase protection assays.

(A) Schematic diagram of the ChpPV genome and the probes used for analysis by RNase protection assays. The transcription units, with the promoter (P3), splice donor (D1 and D2) and acceptor (A1-1, A1-2, A2) sites, and polyadenylation signals, (pA)p at nt 2841 and (pA)d at nt 5107, are indicated. All the ChpPV probes are shown with their respective nucleotide numbers, along with the designated bands they are expected to protect and their predicted sizes. Spl, spliced RNAs; Unspl, unspliced RNAs; RT, RNAs read-through the (pA)p site. (B) Mapping of the ChpPV transcription units by RNase protection assays. Ten µg of total RNA isolated two days after the transfection of COS-7 cells with plasmid pC1ChpPV were protected by ChpPV probes P1-P7. Lanes 7&9, ³²P-labeled RNA markers with sizes indicated.

Figure 3. Transcription profile of ChpPV RNA.

(A) Northern blotting analysis. Total RNA was isolated from COS-7 cells transfected with pC1ChpPV, and was used for Northern blotting analysis. The blots were hybridized to three ChpPV DNA probes (NSCap, NS1 and Cap) that spanned various regions of the ChpPV genome, as diagramed at the bottom of panel B. Sizes of the RNA bands detected by each probe are indicated in kb to the right side of each lane. The RNA marker ladder is shown in lane 1. (B) Transcription map of ChpPV. The ChpPV genome is shown to scale, with transcription landmarks indicated. All of the RNA species detected in our assays are diagrammed to display their identities and respective sizes in the absence of the polyA tail. The putative ORFs that each can encode are also diagramed, and the predicted sizes (kDa) of translated proteins are indicated to the right.

Figure 4. Phylogenetic analysis of ChpPV with the other 17 representative parvoviruses in each genus of Parvovirinae.

Genome sequences (A), NS1 (B) and VP1 (C) amino acid sequences of representative members in each genus of the subfamily Parvovirinae were used to determine the neighbor-joining phylogenetic tree using BioEdit Version 7. Genbank accession numbers of the sequences used follow the name of the virus.

Figure 5. Transfection of ChpPV NS1 induces apoptosis in B19V-permissive cells.

UT7/Epo-S1 cells were transfected with pGFPHA (as negative control), pGFP-B19VNS1HA (positive control), and pGFP-ChpPVNS1HA. (A) Cells were harvested at 24 hrs posttransfection and subjected to Western blotting analysis using an anti-HA monoclonal antibody (Sigma, Clone HA-7). Untransfected cells were used as control (lane 1). A molecular marker is shown to the right with sizes in kDa. (B) Cells were stained with AnnexinV and propidium iodide (PI) at 24, 48 and 72 hrs posttransfection, respectively, and were then subjected to flow cytometry analysis. GFP-positive populations were gated for plotting by PI vs. AnnexinV staining. Numbers represent average percentage with standard deviation from three independent experiments. A representative experiment is shown. The number shown in the upper right quadrant and the number shown in the lower right quadrant in each plot are percentages of AnnexinV+/PI+ population and AnnexinV+/PI- population, respectively.