1
Supporting Materials and Methods
Plasmids and vector construction. The GalT-RFP, ssKDEL-RFP, Flag-MxA,
Flag-MxAK83A and Flag-MxAL612K plasmids were from Jennifer
Lippincott-Schwartz (National Institutes of Health, Bethesda, MD). The plasmid
pcDNA3.0-1.3HBV was from State Key Laboratory for Diagnosis and Treatment of
Infectious Diseases (Zhejiang University, China). Flag-HBc was kindly provided by
Dr. Dong Jiang (Peking University Hepatology Institute, Peking University People’s
Hospital, Beijing, China). For CFP-MxA and Myc-MxA construction, the MxA coding
region was amplified by PCR from Flag-MxA, and was inserted into pECFP-C1 and
pCMV-Tag3A vector respectively. The MxA truncations were constructed from
CFP-MxA or Myc-MxA and confirmed by sequencing. The primers used for cloning
are listed in Supporting Table 1.
ELISA analysis. Cells were seeded in 6-well plates. Twenty-four hours after
transfection, cell culture supernatants were collected and analyzed for HBsAg using
ELISA kits (InTec, Xiamen, China) following the manufacturer’s recommendations.
Analysis of viral DNA in the culture medium. The viral DNA in the culture
supernatants was extracted using a MiniBEST Viral DNA Extraction kit (TaKaRa,
Dalian, China) and quantified by real-time PCR with a SYBR® Premix Ex TaqTM kit
(TaKaRa) as previously described.1 Primer sequences are listed in Supporting Table 2.
2
Southern blotting. The cytoplasmic DNA was electrophoresed in a 0.7% agarose gel,
transferred to a nylon membrane, then hybridized with a digoxigenin-dUTP (DIG)
labeled HBV DNA probe and detected using a DIG-High Prime DNA Labeling and
Detection Starter Kit (Roche, Basel, Switzerland) according to the manufacturer’s
protocol. The molecular weight markers, namely 3.2kb and 2.4kb HBV DNA , and the
HBV DNA hybrid probe were generated from the plasmid pcDNA3.0-1.3HBV by
PCR . The primers are listed in Supporting Table 3.
Viral RNA extraction and real-time analysis. Total cellular RNA was extracted with
Trizol (Invitrogen) following the manufacturer’s instructions. The purified products
were treated with RQ1 RNase-Free DNase (Promega, Madison, WI) to remove the
DNA prior to reverse transcription. Cytoplasmic RNA and nuclear RNA were
extracted with a Cytoplasmic and Nuclear RNA Purification Kit (Norgen Bioteck,
Thorold, ON) as recommended by the manufacturer. Encapsidated pregenomic HBV
RNA was purified according to the published protocols,2 with modifications, briefly,
after precipitating capsids with polyethylene glycol (PEG)-8000, encapsidated pgRNA
was extract with Trizol from the pellets, then treated with RQ1 RNase-Free DNase
prior to reverse transcription. For quantification analysis, total HBV RNAs,
cytoplasmic HBV RNAs, nuclear HBV RNAs and pgRNA underwent reverse
transcription using M-MLV Reverse Transcriptase (Promega) and were quantified by
real-time PCR. GAPDH was used as an internal standard for HBV RNAs
quantification. Serial dilutions of plasmid pcDNA3.0-1.3HBV were used as external
3
standards for HBV pgRNA quantification. The primers are listed in Supporting Table
3.
Live-cell imaging and photobleaching. For live-cell imaging, cells in chambers were
imaged in buffered medium on a heated stage adjusted to 37°C and contained in a
moisturized and CO2-controlled box. Photobleaching was performed using an
appropriate laser line at full power. For fluorescence recovery after photobleaching
(FRAP) analysis, a selected region of a cell was photobleached and the fluorescence
recovery of the region was monitored at low intensity illumination. For fluorescence
loss in photobleaching (FLIP) analysis, a selected region was repetitively
photobleached and the loss of fluorescence from regions outside of the photobleached
region was monitored. Fluorescence resonance energy transfer (FRET) experiments
were performed as described previously.3 In cells expressing YFP-HBcAg and
CFP-MxA, the acceptor (YFP-HBcAg) was bleached using the 514 nm laser line. The
whole cell was imaged at low laser intensity for both donor (CFP-MxA) and acceptor
immediately before and after bleaching. FRET efficiency was calculated from the
increase of the donor fluorescence intensity.
Statistical analysis. Statistical analysis was performed using SPSS software (version
11.0; Chicago, IL). Nominal data were compared by the t-test. p-values <0.05 were
considered statistically significant.
4
Supporting Tables
Supporting Table 1 Primers used for Vector construction
Vector Name
Type
Sequence 5’-3’
CFP-MXA(1-662)
Sense
CGGAATTCGAAGATGGTTGTTTCCG
Anti-sense
CGGGATCCAGCGATTCTGAGGGCTG
Sense
CGGAATTCTATGGTTGTTTCCGAAGT
Anti-sense
CGGAATTCTTAACCGGGGAACT
Sense
CGGAATTCTATGAAGTATGGTGTCG
Anti-sense
CGGGATCCTTAACCGGGGAACT
Sense
CGGGATCCCATGAAGTATGGTGTCG
Anti-sense
CGGAATTCTTAACCGGGGAACT
Sense
CGGAATTCTATGGTTGTTTCCGAAGT
Anti-sense
CGGGATCCTTATTACATGGAAGAGTCT
Sense
CGGGATCCCATGGTTGTTTCCGAAGT
Anti-sense
CGGAATTCTCACATGGAAGAGTCTG
Sense
P - GAGGAGATCTTTCAGCACCTGATGGCCT
○
Anti-sense
P - GACACCATACTTTTGTAGCTCCTCTGTT
○
Myc-MxA(1-662)
CFP-ΔN(359-662)
Myc-ΔN(359-662)
CFP-ΔC(1-574)
Myc-ΔC(1-574)
ΔCID(Δ363-574)
Supporting Table 2
Primers used for real time PCR
Gene name
Type
Sequence 5’-3’
HBV DNA
forward
GTTGCCCGTTTGTCCTCTAATTC
reverse
GGAGGGATACATAGAGGTTCCTT
5
HBV RNAs
HBV pgRNA
GAPDH
Supporting Table 3
forward
TCTCTGCAATGTCAACGACC
reverse
CAGACCAATTTATGCCTACAGC
forward
CCATACTGCACTCAGGCAA
reverse
ACCTGCCTCGTCGTCTAAC
forward
GGAGCCAAAAGGGTCATCATCT
reverse
AGGGGCCATCCACAGTCTTCT
Primers used for synthesizing probes and molecular weight
markers by PCR
Name
Type
Sequence 5’-3’
HBV probe
forward
CCATACTGCACTCAGGCAA
reverse
CCACTGCATGGCCTGAGGAT
forward
TCTCTGCAATGTCAACGACC
reverse
CAGACCAATTTATGCCTACAGC
forward
ATGTGAACAGTTTGTAGGCC
reverse
CAGACCAATTTATGCCTACAGC
3.2kb DNA
2.4kb DNA
Reference
1. Peltekian C, Gordien E, Garreau F, Meas-Yedid V, Soussan P, Willams V, et al.
Human MxA protein participates to the interferon-related inhibition of hepatitis B
virus replication in female transgenic mice. J Hepatol 2005;43:965-972.
2. Guo JT, Pryce M, Wang X, Barrasa MI, Hu J, Seeger C. Conditional Replication
6
of Duck Hepatitis B Virus in Hepatoma Cells. J Virol 2003;77:1885-93.
3. Lippincott-Schwartz J, Snapp E, Kenworthy A. Studying protein dynamics in
living cells. Nat Rev Mol Cell Biol 2001;2:444-456.
Supporting Figure Legends
Supporting Fig. 1 The quantitative expression of Flag-MxA in HepG2.2.15 cells.
Suspended HepG2.2.15 cells were transfected with double amounts of Flag-MxA and
Lipofectamine according to an optimized protocol. Twenty-four hours
post-transfection, cells were fixed, stained with anti-Flag antibody and visualized by
confocal microscopy.
Supporting Fig. 2 MxA does not alter the total level and neucleo/cytoplasmic
distribution of HBV RNA. HepG2.2.15 cells were transfected with Flag-MxA,
Flag-MxAK83A or Flag-MxAL612K. Twenty-four hours post-transfection, the levels
of intracellular total HBV RNAs (A), cytoplasmic HBV RNAs and nuclear HBV
RNAs (B) were quantified by real-time PCR. Intracellular HBcAg level was
determined by western blot with a specific anti-HBcAg antibody (C).