Experimental Procedures

1. HEK293T cells are maintained in complete DMEM/F12 (1:1) media in a humidified cell culture incubator at 37°C, 5% CO₂. Transfection efficiency is higher when cells are between 3 weeks and 3 months age, post-thaw.
2. Passage cells one day before transfection, plating approximately 1.25 x 10⁶cells per 6 cm dish, or an amount that yields 60-90% confluence the next day. The procedure may be scaled: 6 cm dishes yield 2 bead aliquots, 10cm dishes yield 5 bead aliquots, and 15 cm dishes yield 12 bead aliquots.
3. Change medium 2-3 hours before transfection.
4. Transfect each plate with half pCDNA-3FHP and half pCMV-HE (by weight) using calcium phosphate transfection (or any other suitable method). Use 10 μg total weight of DNA for 6 cm dishes, 20 μg for 10 cm dishes, and 50 μg for 15 cm dishes. Include any desired controls.
5. Calculate volumes for water, DNA, calcium solution, and 2X HBS phosphate solution. Total volume should be 500 μL for 6 cm dishes, 1 mL total for 10 cm dishes, or 2.5 mL for 15 cm dishes.
6. Add in the following order: sterile water, DNA, and calcium chloride (calcium chloride volume is 31 μL for 6 cm, 62 μL for 10 cm, or 155 μL for 15 cm dishes).
7. To the DNA-calcium tube, add 2X HBS dropwise (250 μL for 6 cm, 500 μL for 10 cm, or 1.25 μL for 15 cm dishes) while agitating the receiving tube by flicking or agitating on a vortexer set low enough that no splashing occurs.
8. After 5 to 20 minutes, apply transfection reagent dropwise onto labeled plates.
9. Incubate at 37°C, 5% CO₂for 8 hours to overnight with transfection reagent. Wash cells once with 1X PBS and apply fresh medium.
10. Allow cells to grow for two days, then lyse according to the procedure below. Alternatively, cells can be frozen at -80°C in parafilm-wrapped dishes after removing growth medium and a PBS rinse.

Preparation of FLAG antibody-bound beads

1. Resuspend immobilized protein A/G beads by inverting several times. Transfer 20 µL of the bead suspension per 6 cm plate (50 µL per 10 cm plate, 125 µL per 15 cm plate) into a single tube, which will be split into bead groups later.
2. Pellet beads by centrifugation and remove storage buffer. Bead centrifugation steps should be at 350 x g for 2 minutes. Wash beads three times with TN buffer. Wash by adding the buffer to resuspend the beads, then centrifuge and remove the buffer.
3. After washing, resuspend beads in TN buffer at half the original bead volume but at least 200 µL.
4. Bind anti-FLAG IgG antibody onto washed beads by adding 2.8 µL of anti-FLAG antibody per each 6 cm dish (7 µL per 10 cm dish, or 17.5 µL per 15 cm dish).
5. Rotate at room temperature for 3 to 4 hours. Proceed to cell lysis during incubation.
6. After the anti-FLAG antibody is bound to beads, spin and remove unbound antibody. Wash beads three times with 500 μL FLAG lysis buffer (protease inhibitors are not necessary in this buffer). When adding wash buffer the final time, resuspend beads with low retention tips, and divide equal volumes into separate tubes for each condition used in the transfection. Place tubes on ice.

HBV polymerase immunoprecipitation and purification

1. Wash cells once with 2 mL 1X PBS per 6 cm plate (4 mL per 10 cm plate, or 10 mL per 15 cm plate), being careful not to detach cells.
2. Wash cells once with 2 mL cold 1X PBX with protease inhibitors per 6 cm plate (4 mL per 10 cm plate, or 10mL per 15 cm plate). From here, keep samples RNase-free and on ice.
3. To each 6 cm plate, add 0.4 mL cold FLAG lysis buffer with inhibitors, (1 mL to 10 cm plates, or 2.5 mL to 15 cm plates). Free cells from the dish by scraping with a cell scraper or spraying with the buffer from a pipet tip.
4. Collect cells from the same treatment condition into a single chilled tube, and rotate for 20 minutes at 4°C.
5. Centrifuge lysate at 4°C for 10 minutes at maximum speed in a microcentrifuge ~18,000 x g. Supernatants represent the cytoplasmic fraction, which contains HBV polymerase.
6. Transfer supernatants to chilled tubes of prepared anti-FLAG antibody-bound beads. The pellet and some lysate (~100 μL) may be frozen together for troubleshooting.
7. Rotate antibody-bound beads and cell lysate supernatant at 4°C overnight to allow immunoprecipitation to occur.
8. The next day, spin beads at 4°C for 2 minutes at 350 x g. Remove unbound supernatant (supernatant may be saved for troubleshooting).
9. Wash beads five times with 500 μL FLAG wash buffer. When adding wash buffer the final time, resuspend beads with low retention tips, and divide equal volumes into separate chilled labeled tubes for each assay to be performed. Assays usually include a western blot (polymerase protein levels), northern blot (epsilon RNA levels), and the priming assay itself.
10. Store bead aliquots at -80°C, removing wash buffer before further experiments. Approximate bead volume is 10 μL per tube.

1. Radiolabeled ε RNA is transcribed using SP6 MAXIscript kit components. The template used is a DNA oligonucleotide coding for the ε RNA sequence annealed to an SP6 promoter. Other constructs such as plasmid-based expression constructs should work as well. For a 20 μL reaction add 4 μL of DEPC-treated water; 1 μL each of 10 mM ATP, CTP, and GTP; 2 μL of 0.5 mM UTP (diluted from 10 mM stock 1:20 with DEPC-treated water); 1 μL of 1 μg/μL template DNA; 3 μL of SP6 enzyme; and 5 μL of [α-³²P] UTP. Controls deficient in binding may be included.
2. Incubate at 37°C for 5 hours total. Halfway through the incubation, spin briefly (~4,000 x g for one second) to collect condensate.
3. After incubation, add 1 µL DNase and incubate at 37°C for 30 minutes.
4. Inactivate the DNase by incubating at 70°C for 15 minutes.
5. Labeled samples can be stored at -80°C. If desired, transcribed labeled RNA can be visually verified on a 1.5% agarose gel in 1X TAE under RNase-free conditions. Use an appropriate RNA ladder to evaluate size.
6. Combine aliquots of immunoprecipitated HBV polymerase-bound beads with 0.5 µg in vitro-transcribed ³²P-labeled ε RNA (approximately 1 to 3 μL) and 20 μL RIPA buffer with inhibitors. Inhibitory compounds to be tested should be added prior to addition of ³²P-labeled ε RNA.
7. Incubate for three hours at room temperature with shaking to allow HBV polymerase to bind the ε RNA. Shake on a vortexer set to 3 or agitate by hand every 5-10 minutes to keep beads in suspension.
8. Pellet the beads and remove and save the supernatant at ‑80°C, which contains any unbound components.
9. Wash the beads five times with 200 μL RIPA wash buffer. Samples may be stored at ‑80°C for later processing. The first and fifth washes may be saved to troubleshoot binding levels.

• After removing the final wash buffer, elute bound materials by adding 60 µL of 2X SDS lysis buffer. As controls, washes, input RNA (at 1:100 dilution), and unbound RNA (at 1:100) may be included; prepared by adding 30 μL of sample to 30 μL of 2X SDS lysis buffer. Boil all samples for 5 minutes, vortex, and boil 5 more minutes. Load 30 μL of each sample onto a 15% SDS-PAGE gel.
• Cut the gel horizontally at approximately 50 kDa. The top portion of the gel which contains the HBV polymerase protein can be visualized by western blot using anti-FLAG antibody. The bottom portion of the gel containing the ³²P-labeled ε RNA can be dried and directly exposed to film to detect the labeled ε RNA, which was bound to (and then disassociated from) the purified RT.

1. An ε RNA-specific radiolabeled probe will be used in a northern blot. This probe can be made from the same pCMV-HE plasmid used to transfect, a 1,389 bp fragment purified from an NcoI digest under RNase-free conditions. This purified fragment is used in an SP6 in vitro transcription using radiolabeled [α-³²P] UTP (see step 1 above from “In vitro transcription of ³²P-radiolabeled ε RNA and in vitro RNA binding assay” for an in vitro transcription assay).
2. Using immunoprecipitated HBV polymerase (purified from cells that were transfected with both ε RNA-expressing and HBV polymerase-expressing constructs), perform a Trizol extraction of 20 μL lysate (see step 6 above from “HBV polymerase immunoprecipitation and purification”) and a ~10 μL bead aliquot. This will compare the amount of ε RNA expressed to the amount of ε RNA bound to HBV polymerase.
3. Resuspend the extracted RNA samples in a final volume of 20 μL DEPC-treated water. Add 20 μL of formamide gel loading buffer to each sample and heat to 95°C for 5 minutes by boiling or in a thermal cycler. Load the hot samples onto a 6% acrylamide 8 M urea-PAGE gel in TBE. Run the gel at 300V until Bromophenol dye front comes off (approximate time is 2hrs).
4. Rinse gel 20 minutes in 1X TBE, rinse a nylon membrane in water for 5 minutes then in 1X TBE for 5 minutes.
5. For electrophoretic transfer, run below 300 mA (6 to 7 V) for 1 hour.
6. While the transfer is running, preheat ULTRAhyb solution to 68°C to dissolve precipitated material.
7. Crosslink the RNA to the membrane by exposing to a UV transilluminator.
8. Prehybridize at least 30 minutes at 68°C. Use 10mL ULTRAhyb buffer per 100 cm²of membrane.

9. Add prepared ε RNA-specific radiolabeled probe to the ULTRAhyb and allow the probe to hybridize 2 hours to overnight.

1. Make sure wash buffers have no precipitate, if so, heat to 37°C. Pre-heat high stringency wash to 68°C.
2. Wash blot twice in room temperature low stringency wash solution (20 mL per 100 cm²blot) with agitation for 5 minutes, dispose of washes in ³²P waste.
3. Wash twice for 15 minute at 68°C with high stringency wash solution with agitation. Dispose of washes in ³²P waste.
4. Seal radiolabeled blot in plastic wrap and expose to film or a phosphorimager screen.