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Review
. 2022 Mar 8;14(3):557.
doi: 10.3390/v14030557.

Imaging of Hepatitis B Virus Nucleic Acids: Current Advances and Challenges

Luisa F Bustamante-Jaramillo  1 , Joshua Fingal  1 , Marie-Lise Blondot  2 , Gustaf E Rydell  1 , Michael Kann  1   3
Affiliations
Review

Imaging of Hepatitis B Virus Nucleic Acids: Current Advances and Challenges

Luisa F Bustamante-Jaramillo et al. Viruses. .

Abstract

Hepatitis B virus infections are the main reason for hepatocellular carcinoma development. Current treatment reduces the viral load but rarely leads to virus elimination. Despite its medical importance, little is known about infection dynamics on the cellular level not at least due to technical obstacles. Regardless of infections leading to extreme viral loads, which may reach 1010 virions per mL serum, hepatitis B viruses are of low abundance and productivity in individual cells. Imaging of the infections in cells is thus a particular challenge especially for cccDNA that exists only in a few copies. The review describes the significance of microscopical approaches on genome and transcript detection for understanding hepatitis B virus infections, implications for understanding treatment outcomes, and recent microscopical approaches, which have not been applied in HBV research.

Keywords: CRISPR/cas9; ClampFISH; MS2; OR protein; PP7; RNA transcripts; Sun-Tag; anchor; aptamer; branched chain DNA (bDNA); cccDNA; click chemistry; fluorescent in situ hybridization (FISH); hepatitis B virus; imaging; in situ hybridization (ISH); molecular beacon; peptide nucleic acids (PNA); quantum dots; sandwich nucleic acid hybridization; single cell analysis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Classical in situ hybridization (ISH); (A) Deproteinization of dsDNA; (B) Addition of fluorophore-labelled dsDNA probe; (C) Melting of dsDNA and probe; (D) Hybridization. More information on the graphical elements is given in the box within the figure.
Figure 2
Figure 2
Branched chain DNA (bDNA) assay for detecting target sequences on dsDNA: (A) Hybridization of two different target probes to dsDNA after melting; (B) Hybridization of the pre-amplifier to the free nucleotides of the two adjacent target probes; (C) Hybridization of identical amplifier molecules to the pre-amplifier; (D) Binding of labelled probes to the amplifier molecules. More information on the graphical elements is given in the box within the figure.
Figure 3
Figure 3
CRISPR/dCas9 in genome visualisation. (A) dsDNA detection. dCas9 binds to PAM and sgRNA. dCas9 is fused to a single fluorescent protein or conjugated to multiple fluorescent proteins through Sun-Tag. (B) RNA detection. As in A., but the PAM is extended by an oligonucleotide (PAMmer). More information on the graphical elements is given in the box within the figure.
Figure 4
Figure 4
STRIDE for sequence detection on dsDNA: (A) CRISPR/Cas9-mediated induction of double-strand breaks (DSB); (B) Modified deoxynucleotides are enzymatically conjugated to 3′ DNA ends by Pol I; (C) Two different primary antibodies, directed against the modified nucleotides bind in close proximity; (D) Secondary antibodies conjugated with oligonucleotides bind to primary antibodies; (E) Another oligonucleotide binds to the two oligonucleotides from D., forming a circular DNA template; (F) One oligonucleotide from D. acts as primer for elongation in a rolling circle mechanism by a DNA polymerase using the circular DNA from E. as template thereby generating a ssDNA with repetitive motifs; (G) Hybridization of fluorophore-labelled oligonucleotides to the ssDNA from the rolling circle. More information on the graphical elements is given in the box within the figure.
Figure 5
Figure 5
Simplified schematic presentation of CRISPR-Sunspot system: (A) Transduction of a cell line with a lentivirus expressing TRE3G-dCas9-24xGCN_V4; (B) The transduced cells are with a second lentivirus expressing scFv-sfGFP lentivirus; (C) Co-transfection of two plasmids of the double-transduced cells; the first coding for two sets of three different sgRNAs each, the second plasmid expresses the PAMmers; (D) Detection and visualisation of the target sequence. sgRNAs and PAMmer bind to the target sequence allowing recruitment of Sun-tagged Cas9. More information on the graphical elements is given in the box within the figure.
Figure 6
Figure 6
Schematic representation of aptamer systems for the detection of mRNA and ssRNA. The stem-loop formed (purple) by the aptamer sequence of chimeric mRNA provides a binding platform for fluorescently-tagged proteins in aptamer–protein systems (A) or conditional fluorophores in light-up aptamer systems (B). In light-up aptamer systems, fluorescence emission only occurs after aptamer-binding of the conditional fluorophore which makes these systems background-free. More information on the graphical elements is given in the boxes within the figure.
Figure 7
Figure 7
Schematic representation of mRNA detection with molecular beacons: (A) The self-complimentary sequence of the molecular beacon forms a stem (black), thereby keeping the fluorophore and fluorescence quencher in close vicinity effectively quenching the fluorophore; (B) Upon hybridization of the molecular beacon’s complimentary sequence (purple) with the target, the quencher is distanced from the fluorophore which consequently regains its fluorescent properties. More information on the graphical elements is given in the boxes within the figure.
Figure 8
Figure 8
Schematic representation of the most common modes PNA-based probes hybridise with dsDNA: (A) Fluorescent PNA probes (green) can be designed to directly label dsDNA by forming a triple helical structure (triplex); (B) As in A., but by double duplex invasion; or (C) by duplex invasion, among other binding modes. Alternatively, as duplex invasion (C) leads to strand displacement, a PNA opener can make hybridization sites available for secondary probes without sample denaturation or a fixation step.
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