The 5′ Untranslated Region of Human Bocavirus Capsid Transcripts Regulates Viral mRNA Biogenesis and Alternative Translation

Figures

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  FIG 1. HBoV1 transcription map and probes. (A) Genome structure of HBoV1, the main ORFs, and the major species of viral mRNA transcripts that are alternatively processed. P5, P5 promoter; D, 5′ splice donor site; A, 3′ splice acceptor site; (pA)p, proximal polyadenylation sites; (pA)d, distal polyadenylation site; LEH, left-end hairpin; REH, right-end hairpin. (B) Probes for Northern blotting or the RNase protection assay (RPA). RT, read-through RNA.

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  FIG 2. 5′ UTR regulated HBoV1 capsid mRNA abundance and protein translation. (A) Diagram of HBoV1 capsid expression constructs with the T7 promoter for the in vitro assay. (B) In vitro coupled transcription/translation assay. In vitro assays were performed according to the manufacturer's instructions. Expressed proteins were run on a 15% SDS-PAGE gel, and the signal was detected with a Cyclone Plus system (PerkinElmer) and analyzed using OptiQuant software. The ratio of VP1 to VP3 is presented at the bottom of the gel. The experiment was repeated at least three times. (C) Diagram of HBoV1 VP cDNA constructs with the cytomegalovirus (CMV) promoter. (D) Northern blot. Ten micrograms of total RNAs prepared from transfected cells were resolved on 1.5% agarose gels, transferred to Hybond-N+ membranes, and hybridized with probes spanning nt 349 to 5167. The signal was detected using a ChemiDoc MP imaging system (Bio-Rad). Ethidium bromide (EB)-stained 18S RNA bands are shown as the loading control. (E) Western blot (WB). The lysates of HEK293T cells transfected with the plasmids described in panel C were analyzed using an anti-Flag antibody to detect capsid expression. β-Actin served as the…

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  FIG 3. Identification of the cis elements in the 5′ UTR that regulated RNA abundance and translation. (A) Diagram of green fluorescent protein (GFP) reporter plasmids. Group I plasmids contained either the entire 5′ UTR or VP1 unique sequences. Group II plasmids contained both the 5′ UTR and VP1u. Group III constructs harbored VP1u with deletions in exons 1 to 4. Exon 4 in the plasmids of group IV was deleted. SV40, simian virus 40. (B) Microscopy of the fluorescent signal when reporter plasmids were transfected. (C) Western blot. The lysates of transfected HEK293T cells were analyzed using anti-GFP antibody, and β-actin served as the loading control. (D) Northern blot. Total RNAs were harvested from transfected cells, and Northern blotting was carried out as described in the legend to Fig. 2D. Ethidium bromide (EB)-stained 18S RNA bands are shown as the loading control. The image in panel D was spliced from two different gels because the samples could not be run in one gel.

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  FIG 4. 5′ UTR cis elements regulated HBoV1 capsid protein expression and mRNA abundance in HEK293T cells. (A) Diagram of capsid expression plasmids. Various lengths of the 5′ UTR sequence from p3XFlag-R6 were deleted to construct p3XFlag-Tru1, p3XFlag-Tru2, p3XFlag-E3 and -E4, and p3XFlag-mini. Plasmids p3XFlag-mini-T1 to p3XFlag-mini-T10 were generated by sequence deletion in exon 2 or exon 3 based on p3XFlag-mini, as indicated. (B) Western blot. The transfected HEK293T cell lysates were analyzed using anti-Flag antibody, and β-actin served as the loading control. (C) Northern blot. Total RNAs were harvested from transfected cells, and Northern blotting was carried out as described in the legend to Fig. 2D. Ethidium bromide (EB)-stained 18S RNA bands are shown as the loading control.

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  FIG 5. The alternative translation of VP1 and VP2 was IRES independent. (A) Diagram of bicistronic fluorescent reporter constructs. The 3×Flag tag, the simian virus 40 poly(A) signal, and multiple-cloning sites (MCS) are denoted. (B) Microscopy images of fluorescent signals. Images of the green fluorescent protein (GFP) fluorescent signals are on the top, and images of the mCherry fluorescent signals are on the bottom (in images of the same visual fields). (C) Western blot. The lysates of transfected HEK293T cells were analyzed using anti-GFP and anti-Flag antibodies. β-Actin served as the loading control. (D) Northern blot. Total RNAs were harvested from transfected cells, and Northern blotting was carried out as described in the legend to Fig. 2D. Ethidium bromide (EB)-stained 18S RNA bands are shown as the loading control.

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  FIG 6. The alternative translation of HBoV1 VP proteins was based on ribosomal leaky scanning. (A) Diagram of plasmids used for in vitro or in vivo translation. (B) Western blot. The lysates of transfected HEK293T cells were analyzed using anti-Flag antibody, and β-actin served as the loading control. (C) In vitro coupled transcription/translation assay. In vitro assays were performed as described in the legend to Fig. 2B. (D) Northern blot. Total RNAs were harvested from transfected cells, and Northern blotting was carried out as described in the legend to Fig. 2D. Ethidium bromide (EB)-stained 18S RNA bands are shown as the loading control.

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  FIG 7. uATGs influenced HBoV1 capsid protein expression. (A) Diagram of HBoV1 uATG-mutated constructs. The positions of uATG1, uATG2, and uATG3 are denoted by their nucleotide sequence numbers. (B) Western blot. The transfected HEK293T cell lysates were analyzed using anti-Flag antibody, and β-actin served as the loading control. (C) Northern blot. Total RNAs were harvested from transfected cells, and Northern blotting was carried out as described in the legend to Fig. 2D. Ethidium bromide (EB)-stained 18S RNA bands are shown as the loading control. ncRNA, noncoding RNA.

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  FIG 8. uATG2 and uATG3 knockout mutations in an HBoV1 infectious clone decreased viral progeny production. (A) Southern blot. Hirt DNA was isolated from HEK293T cells transfected with plasmids, as indicated, at 48 h posttransfection. The DpnI-digested fragments were resolved on 1% agarose gels and transferred to a Hybond-N+ membrane, followed by hybridization with an HBoV1 probe spanning from nt 1 to 5543. dRF, double replication form; mRF, monomer replication form; ssDNA, single-stranded DNA. (B) Northern blot. Total RNAs were harvested from transfected cells, and Northern blotting was carried out as described in the legend to Fig. 2D. Ethidium bromide (EB)-stained 18S RNA bands are shown as the loading control. The images in panel B were taken from different areas of the same photo or different gels and then joined together. (C) Quantitative PCR (qPCR) for the HBoV1 genome. Cell lysates of uATG-mutated HBoV1 infectious clones were prepared by four cycles of freezing and thawing, followed by DNase I digestion. The progeny viruses were separated by cesium chloride (CsCl) density gradient centrifugation, and genomic DNA was prepared as the template for qPCR. Means and standard deviations…

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  FIG 9. uATG mutations in an HBoV1 infectious clone altered viral RNA processing. RNase protection assay (RPA) with analysis of viral RNA polyadenylation at the (pA)p site. Ten micrograms of total RNAs prepared from HEK293T cells transfected with plasmids, as indicated, was protected by the (pA)p site-specific probe. RT, read through RNAs. The ratios of RNA polyadenylated at the (pA)p site versus read-through RNA are indicated as the mean and standard deviation. The numbers on the left are molecular size markers.