Nature Medicine doi:10.1038/nm · immunoprecipitated with Ncl-specific monoclonal antibody (MS-3,...

Nature Medicine doi:10.1038/nm.2444

CAR

Integrin β1

Figure S1

100-‐

40-‐

50-‐ 60-‐

80-‐

MW

Figure S1. Adenovirus VOPBA and Western blot for Intergrin β1 and CAR. Bands about 46kDa and 100 kDa were visualized by adenovirus (AdV) VOPBA using 1 in 2000 (v/v) rabbit polyclonal Adenovirus Type 5 antibody (Abcam, San Francisco, CA, cat#6982). The 46 kDa band was the same size as a well known receptor for AdV, CAR. Western blot for CAR is 1 in 1000 (v/v) mouse CAR-specific mAb (clone RmcB), [Upstate (now Millipore), Millipore Headquarters, Billerica, MA cat# 05-644]. The 100 kDa band is integrin β1 another well known receptor for AdV, which was also detectable by Western blot [1 in 1000 (v/v) Rabbit polyclonal Integrin β1 Antibody, Cell Signaling Technology, Danvers, MA, cat# 4706]. The identity of the Integrin β1 band was further confirmed by MS.


1 2 Ncl (nM) RSV stock 10 0.1

100

150

75

MW

75

50

37

25

Ncl

F1

N P

M G

100

4 – 12 % gradient gel

Figure S2

Figure S2. Contaminating nucleolin cannot be detected in stocks of RSV. Soluble nucleolin (Vaxron, USA) and RSV stock were resolved by polyacrylamide gel electrophoresis and RSV antigens and nucleolin were detected by Western blot using polyclonal RSV-specific and nucleolin-specific antibodies, respectively. Note the lack of nucleolin in the RSV stocks and the lack of RSV antigen in soluble nucleolin.


+ + -‐ + + +

RSV A2

1° Ab

2° Ab

-‐ + +

MW 120 100 80

60 50

40

-‐ -‐ -‐ -‐

-‐ -‐

Figure S3

Figure S3. RSV VOPBA Controls. Ladder: 5 µL MagicMark™ XP Western Protein Standard. Biotinylated membrane proteins were probed with RSV, 1 in 1000 (w v-1) Goat RSV-specific antibody (Meridian Life Science Inc, Saco, ME cat# B65860G), and secondary antibody donkey goat-specific IgG-HRP (Santa Cruz Biotechnologies, cat# sc-2020). Note the ˜100 kDa band visible in VOPBA is absent when either virus or RSV-specific antibodies are omitted.


mAb19 -‐ -‐ + -‐ -‐ mAb29 -‐ + -‐ -‐ -‐ Goat RSV-‐specific Ab -‐ -‐ -‐ + + Goat mouse-‐specifc HRP Ab -‐ + + -‐ -‐ Donkey Goat-‐specific HRP Ab + -‐ -‐ + +

130-‐ 95-‐

Figure S4

MW

Figure S4. Additional controls for RSV A2 VOPBA. There is no band visible in the absence of a primary antibody. Less intense signals were obtained with mAb 29 (against RSV G) or mAb 19 (against RSV F) than with Goat RSV-specific antibody. To test the possibility that interaction of nucleolin with RSV might be similar to that of the virus with heparin14,15,16,18 we pre-incubated a Nitrocellulose strip blotted with polyacrylamide gel electrophoresis-resolved membrane lysate with a solution of heparin (0.1 USP mL-1 in PBS). Heparin did not interfere with the VOPBA signal (last lane). Goat RSV-specific antibody was used at 1 in 1000 (w/v) (Meridian Life Science Inc, cat# B65860G). Monoclonal ab19 and mAb29 are gifts from Dr. Geraldine Taylor.


No hep

arin

10 µg

/mL

100 µ

g/mL

1 mg/m

L0

20

40

60

80

GFP

-pos

itive

cel

ls (%

)

Figure S5

a

c

50-‐ RSV-‐F

Ncl IP

1 2 3 4

200 µm

10 ug/mL 313

234

156

78

0

Log FL1 100 101 102 103 104

Coun

ts

100 ug/mL 343

257

171

85

0

Log FL1 100 101 102 103 104

Coun

ts

104

1 mg/mL 315

236

157

78

0

Log FL1 100 101 102 103

Coun

ts

Heparin

No heparin

Heparin

No heparin

Heparin

No heparin

b

*

Figure S5. Addition of heparin to cell culture inhibits viral infection but not nucleolin/F protein interaction. (a) Cells and virus were treated with the indicated concentrations of heparin for 1 hr at 37 ºC. Virus and heparin mixture were added to the cells treated with the same dose of heparin, detached the following day using trypsin-EDTA, fixed and enumerated by flow cytometry. (b) Graphical representation of flow cytometry data displayed in panel (a). *: “no heparin” control group is significantly different to all other heparin dose groups used (P < 0.001). (c) Western analysis of immunoprecipitation experiments to test Ncl- F protein interaction in the presence of a large excess of heparin. Twenty nM soluble nucleolin protein was incubated with 105 infectious units of RSV and immunoprecipitated with an nucleolin-specific monoclonal antibody. In the first three lanes sample was immunoprecipitated with Ncl-specific monoclonal antibody (MS-3, cat. no. sc-8031, Santa Cruz Biotechnologies). Samples were resolved by polyacrylamide electrophoresis and Western blotted for RSV proteins using a goat RSV-specific polyclonal antibody (cat. no. B65860G, Biodesign International). Lanes 2, 3 and 4 all contain 20 nM recombinant Ncl. In the last lane an isotype control IgG1 antibody (cat. no. 550878, BD Pharmingen) was used instead of the Ncl-specific antibody. Note that even though heparin is known to interact with RSV F-protein15,16,17,19 addition of heparin to the IP reaction does not interfere with the co-immunoprecipitation of F-protein with Ncl.


Cytoplasm

Nucleus

XZ

XY

a

b

Apical surface

Figure S6

XZ

DIC, XY

c

Nucleus (DAPI) Zona occludens (green)

basal

apical 2 µm

2 µm

2 µm

Figure S6. Confocal microscopy of 1HAEo- cells showing nucleolin staining at the cell surface. Confocal microscopy of 1HAEo- cells was done under conditions where cells remain polarized27 . (a) We stained 1HAEo- cells as in (b) and (c) but with a monoclonal antibody specific for Zona Occludens-1 (ZO-1) (Zymed, Invitrogen, cat# 33-9100). Note that the pattern of the cellular staining indicates cells are polarized. DIC = Differential interference contrast microscopy. (b) Image in the XY plane showing punctate staining of nucleolin at the cell surface (white arrows). (c) Image of the same cell in the XZ plane again showing nucleolin staining at the cell surface. (b and c) We fixed 1HAEo– cells with methanol acetone and stained with polyclonal rabbit nucleolin-specific primary antibody (H-250; Santa Cruz) and goat rabbit-specific Alexa 594 (Invitrogen) secondary antibody.


Isotype Control Ab

An^-‐Ncl Ab

Control

Figure S7

200 µm

Figure S7. Epifluorescence images of RSV-GFP show loss of GFP fluorescence when virus entry is inhibited with a polyclonal Ncl-specific antibody. One-HAE cells were treated with 4 µg ml-1 of nucleolin-specific antibody and infected with virus 1 hr later. The next day, cells were fixed and imaged with a Nikon spot camera mounted on a Nikon E600 fluorescence microscope, using a blue filter.


siNclΔ3

siDAF siNcl

Uninfected

381

285

190

95

0

Log FL1 100 101 102 103 104

Coun

ts

Figure S8

a

b

sTrf No treatment

No infec^on sNcl

261

195

130

65

0

Log FL1 100 101 102 103 104

Coun

ts

Figure S8. (a) Flow cytometry of RSV-GFP infection of 1HAEo– cells in the presence of soluble nucleolin (sNcl) or soluble transferrin (sTrf). Virus incubated with soluble nucleolin showed significantly decreased cellular infection. (b) Flow cytometry of nucleolin silenced 1HAEo– cells. We transfected cells with siRNA oligonucleotides against human nucleolin and infected up to 4 days later with RSV-GFP. The following day cells were detached with trypsin-EDTA, fixed and infection was enumerated by flow cytometry.


Figure S9. Infection of 1HAEo- cells by adenovirus type-5 GFP is not affected by nucleolin-specific antibody and control antibody treatments, or soluble nucleolin or transferrin. a, We treated 1HAEo– cells with 4 µg ml-1 of nucleolin-specific or control isotype antibody and infected with virus 1 hr later. The following day cells were detached with trypsin-EDTA, fixed and infection was enumerated by flow cytometry. Adenovirus type-5 GFP (AdV-GFP) neg - white, AdV and 4 ug ml-1 isotype control antibody – black, AdV and 4 ug ml-1 Ncl-specific pAb – grey. b, Stocks of AdV-GFP were treated with soluble nucleolin (Ncl) or soluble transferrin (Trf) protein and incubated at 37 ºC for 1 hr. Virus-protein mixture was added to Hep2 cells, and cells were detached, fixed and enumerated by flow cytometry the following day. AdV neg - white, AdV 30 nM sTrf – grey, AdV 30 nM sNcl – black.

a b NS NS

Figure S9

391

293

195

97

0

Log FL1 100 101 102 103 104

Coun

ts

297

222

148

74

0

Log FL1 100 101 102 103 104

Coun

ts


Figure S10. Flow cytometry of nucleolin silenced 1HAEo– cells infected with adenovirus type 5 GFP. We transfected cells with siRNA oligonucleotides against human nucleolin and infected them up to 4 days later with AdV-GFP. The following day we detached the cells with trypsin-EDTA, fixed and infection was enumerated by flow cytometry.

NS

Figure S10

siNcl

siNclΔ3

150

112

75

37

0

Log FL1 100 101 102 103 104

Coun

ts


Figure S11. (a) RSV infects Sf9 insect cells that express human nucleolin. Sf9 cells were transfected with vector (grey histogram) or human nucleolin (black histogram) and infected with RSV-A2-GFP. Cells were detached the following day with trypsin/EDTA, fixed and infection was enumerated by flow cytometry. (b) Adenovirus does not infect vector or nucleolin transfected Sf9 insect cells. Sf9 cells were treated as in (a) but infected with AdV type-5-GFP, and detached the following day with trypsin/EDTA, fixed and infection was enumerated by flow cytometry.

RSV-‐GFP 540

0 100 104

FL1 101 102 103

Coun

ts na^ve hNcl-‐transfected

Figure S11 a

387

290

193

96

0

Log FL1 100 101 102 103 104

Coun

ts

Vector

hNcl-‐transfected

AdV-‐GFP b


Figure S12. Immunostaining of mouse lung sections with Ncl-specific (H-250, Santa Cruz) antibody (left) and isotype-matched, irrelevant primary antibody (right). In airway epithelial cells, note positive nuclear and apical immunostaining (brown) similar to that observed with another nucleolin-specific rabbit polyclonal (Abcam; ab22758) shown in Fig. 4b, and no immunostaining observed using isotype-matched antibody. Hematoxylin counterstain.

Polyclonal Ncl-‐specific Ab

Irrelevant Primary Ab

10 µm

20 µm

Figure S12


Figure S13. Effect of siRNA reduced Ncl expression on lung viral titer 4 days post-infection. Unpaired t test, two-tailed, P < 0.05, n = 8 per group.

*

Figure S13


Table S1

1HAE HEp-2 CHO MDCK

Code species descripiton score queries score queries score queries score queries

Q8ZTN5_PYRAE Pyrobaculum aerophilum AAA family ATPase, possible cell division control protein cdc48 52 1 78 1 75 1 N/D N/D

Q7SYE2_BRARE Danio rerio (Zebrafish) Actinin, alpha 4 849 22 687 11 804 15 953 26

Q7ZY34_XENLA Xenopus laevis ACTN1 protein 929 24 592 11 858 15 1224 32 Q6DFU3_XENLA Xenopus laevis Actn3-prov protein 550 16 486 8 N/D N/D 739 18 Q6DCS8_XENLA Xenopus laevis Actn4-prov protein 820 21 622 11 783 14 958 24 Q6P786_RAT Rattus norvegicus (Rat) alpha Actinin 4 1259 28 N/D N/D 1011 20 1441 35 AAH05033 human Alpha-actinin-4, ACTN4 1321 30 1319 24 1020 18 1522 36 C1TC_HUMAN human C-1-tetrahydrofolate synthase, cytoplasmic 299 7 136 2 N/D N/D 266 5

Q1N554_9GAMM Bermanella marisrubri Chaperone protein htpG 69 1 86 1 100 1 90 1

Q4STW7_TETNG Tetraodon nigroviridis (Green puffer) Chromosome undetermined SCAF14091 657 19 459 8 651 12 819 24

Q7ZVM3_BRARE Danio rerio (Zebrafish) Eukaryotic translation elongation factor 2, like 518 13 491 10 878 23 N/D N/D

Q9VAY2_DROME Drosophila melanogaster Glycoprotein 93 129 2 158 2 157 2 N/D N/D Q1FI35_9CLOT Clostridium phytofermentans GTP cyclohydrolase I 56 2 60 1 59 1 60 4

Q27HW6_SCOMX Psetta maxima (Turbot) (Pleuronectes maximus) Heat shock protein 90 60 1 189 3 151 3 N/D N/D

Q6DD73_XENLA Xenopus laevis MGC79035 protein 333 12 389 6 418 7 465 13 Q6NRW6_XENLA Xenopus laevis MGC81191 protein 926 24 N/D N/D 836 15 1108 31

GANAB_HUMAN human Neutral alpha-glucosidase AB 286 6 874 20 146 4 N/D N/D

Q8STW7_ENCCU Encephalitozoon cuniculi (Microsporidian parasite) NON-MUSCLE ALPHA ACTININ 72 1 58 1 N/D N/D 72 1

DNMS or Q8CE30_MOUSE mouse nucleolin 218 5 456 9 574 11 199 6 NUCL_HUMAN human nucleolin 469 9 843 15 N/D N/D 374 10

Q6BEQ3_CAEEL Caenorhabditis elegans Protein ZK1151.1f, partially confirmed by transcript evidence 80 2 70 2 N/D N/D 72 1

Q3UZ14_MOUSE or Q8C153_MOUSE mouse Putative uncharacterized protein, Eef2

602-652 16

1058-1086 21

1636-1670 39 N/D N/D

TERA_HUMAN human Transitional endoplasmic reticulum ATPase,VCP 52 1 303 7 330 7 N/D N/D

score = total protein score queries = number of peptides matched N/D = not detected


1

Supplementary Methods Cells. We maintained subconfluent HEp-2, MDCK (American Type Culture Collection

(ATCC), and 1HAEo- cells (gift from Dr. D. Gruenert, University of California, San

Francisco, California) at 37 ºC in Dulbecco's modified essential medium (Invitrogen)

supplemented with 1% L-glutamine (Invitrogen) and 10% (v v-1) heat-inactivated fetal

bovine serum (FBS) (Invitrogen). We maintained CHO-K1 and pgsA-745 cells (ATCC)

at 37 ºC in Nutrient Mixture F-12 Ham (Sigma, St. Louis, MO) containing 1% L-

glutamine. We maintained Sf9 cells (Invitrogen) at 27 ºC in Grace’s supplemented media

(Invitrogen) containing 10% (v v-1) heat-inactivated FBS. We kept cell cultures in a

humidified incubator containing 5% CO2.

Viruses. We prepared working stocks of RSV A2 (ATCC; VR-1540) and RSV B

(ATCC; VR-2579). RSV lacking G protein (RSV ΔG) (gift of Dr. P.L. Collins, National

Institutes of Health, Bethesda, MD), RSV expressing GFP (rgRSV224) (gift of Dr. M.E.

Peeples, Children’s Research Institute, Columbus, OH), and community isolates of RSV

A (HLI1) and RSV B (HLI2) (gift of Drs. R. Tan and E. Thomas, Children’s and

Women’s Health Centre of British Columbia, Vancouver, BC) as previously described1.

Preparation of cell membrane extracts. We biotinylated and extracted cell surface

proteins from subconfluent flasks of HEp-2 cells using Pierce® Cell Surface Protein

Isolation Kit (Thermo Scientific) and according to manufacturer’s protocol.


2

Gel electrophoresis. We loaded biotinylated cell surface proteins in 10 µL volumes per

well of 4–12% Novex® Tris-Glycine Gels (Invitrogen) and ran for 100 min using a

constant voltage of 125 V.

Adenovirus VOPBA. After electrophoresis, we blotted biotinylated cell surface proteins

as mentioned for RSV VOPBA, above. We blocked membranes in 5% milk TBS

containing 0.1% Tween 20 (TBS-T) for 1 h on shaker at room temperature. We then

incubated membranes with adenovirus type-5 (AdV5) in a solution of 2.5% milk TBS-T

containing 1012-1013 PFU mL-1 of virus for 3 h on a shaker at room temperature. We

washed membranes with TBS-T three times 10 min each on a shaker at room temperature

and then incubated with 1 in 2,000 (v v-1) Rabbit polyclonal to Adenovirus Type 5

(ab6982) (Abcam) in 2.5% milk TBST overnight at 4 ºC. After three 10 min TBS-T

washes on a shaker at room temperature, we incubated membranes 1 in 5,000 (w v-1)

Goat rabbit-specific IgG-HRP (sc-2004) (Santa Cruz Biotechnology) in 2.5% milk TBS-

T on a shaker at room temperature for one hour. Membranes underwent three 10 min

TBS-T washes and we then exposed them to Amersham™ ECL™ Western Blotting

Analysis System (GE Healthcare) as recommended by manufacturer. We then exposed

blots to autoradiography film (Hyperfilm ECL, GE Healthcare) and developed

immediately.

CAR and Integrin β1 Western Blots. Electrophoresis, blotting, and blocking

nitrocellulose membranes were as explained for RSV VOPBA. We performed Western

blot using either 1 in 1,000 (v v-1) mouse CAR-specific mAb (clone RmcB) (Upstate,


3

now part of Millipore) or 1 in 1,000 (v v-1) rabbit Integrin β1 Antibody (#4706) (Cell

Signaling Technology, Inc.). We washed membranes, exposed them to corresponding

secondary antibodies, and imaged as explained in RSV VOPBA.

RSV VOPBA. After electrophoresis, we blotted biotinylated cell surface proteins onto

Amersham™ Hybond™- ECL (GE Healthcare) nitrocellulose membranes overnight at 4

ºC using constant voltage of 30 V. We blocked membranes in 5% milk TBS (no Tween

20 because RSV is an enveloped virus) for 1 h on a shaker at room temperature. We then

incubated membranes with RSV (A2 or ΔG) in a solution of 2.5% milk TBS containing

3–5 × 105 PFU mL-1 virus for 3 h on a shaker at room temperature. We washed

membranes with TBS three times 10 min each on a shaker at room temperature and then

incubated with 1 in 1,000 (w v-1) of either RSV-specific antibodies in 2.5% milk TBS on

a shaker overnight at 4 ºC. Mouse monoclonal antibodies mAb 19 (against RSV F

protein) and mAb 29 (against RSV G protein) were gifts from Dr. Geraldine Taylor.

Anti-Respiratory Syncytial Virus (A2 Strain) was a rabbit polyclonal (PA1-23413) from

Affinity BioReagents. Goat RSV-specific polyclonal antibody was from Biodesign. We

used an irrelevant goat antibody against Clathrin HC (SC-6580) from Santa Cruz as an

isotype control. After incubation with RSV-specific antibodies, we washed and incubated

membranes three times for 10 min in TBS with 1 in 5,000 (w v-1) of corresponding

secondary antibodies in 2.5% milk TBS. Goat rabbit-specific IgG-HRP (sc-2004) and

donkey goat-specific IgG-HRP (sc-2020) were from Santa Cruz Biotechnology and

Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) was from Jackson

ImmunoResearch Laboratories, Inc. After three 10 min TBS washes, we exposed the


4

membranes to Amersham™ ECL™ Western Blotting Analysis System as recommended

by manufacturer. We then exposed blots to autoradiography film and developed

immediately.

Heparin treatment. In experiments that involved herparin treatment, we incubated

membranes with Heparin Sodium Salt, cat. no. 210-6 (Sigma) diluted in PBS to obtain a

final concentration of 0.1 USP ml-1. Heparin treatment was for one hour at room

temperature on shaker after blocking with milk TBS and before incubation with virus.

MS Analysis. Extracted peptides underwent trypsin digestion and LC-MS/MS using an

Applied Biosystems QSTAR Pulsar I Quadrupole Time-of-Flight Mass Spectrometer

equipped with nanoflow high performance liquid chromatography. We separated

samples by reverse-phase chromatography over a minimum 120 min gradient while

spraying into the mass spectrometer. We analyzed MS/MS data using a protein

identification search engine algorithm (MASCOT) and searched data against all species

sequences in MSDB and IPI Human. We performed MS analysis on cut gel bands as

described in methods from four different cell lines (human, hamster and dog) and hits

with a score greater than 52 indicated identity or extensive homology (P < 0.05). We

submitted the bands obtained from HEp-2 cells for MS analysis twice; once with bands

from 1HAE and MDCK cell lines and another time with the CHOK1 cell line. Nucleolin

was always a common hit (See Table S1 for a complete list of hits, protein scores and

number of peptides obtained).


5

Immunoprecipitation. We incubated 1HAEo- cells with RSV at a multiplicity of

infection (MOI) = 1, on ice for 1 h. We washed cells with PBS and harvested with cell

lysis buffer (10mM HEPES (pH 7.4), 50mM Na4P2O7, 50mM NaF, 50mM NaCl, 5mM

EDTA, 5mM EGTA, 1mM Na3VO4, 0.5% Triton X-100, 10 µg mL-1 leupeptin, and 1mM

phenylmethylsulfonyl fluoride) on ice and spun the cells at high speed to remove nuclei

and debris. We removed an aliquot of supernatant as total cell lysate (“L”) for Western

blot alongside “IP” preparations. We added either 4 µg rabbit anti-nucleolin (H-250,

Santa Cruz Biotechnology), 7 µg anti-RSV F (NCL-RSV3, Novocastra), or 7 µg anti-

RSV G (R1600-17, US Biological) to samples and placed the samples on a rotator

overnight at 4 ºC. Then we added Sepharose protein A or G and placed samples on a

rocker for 2 h at 4 ºC. We washed the lysate four times (5 min each at RT) in cell lysis

buffer and analyzed by Western Blot for nucleolin, RSV F, RSV G or polyclonal goat

antisera to RSV A and RSV B (Biodesign International).

Immunoprecipitation of RSV virus with soluble nucleolin protein in the presence or

absence of heparin. We incubated virus (105 infectious units) with 20 nM soluble

nucleolin protein for 2 h at 37 ºC in the presence or absence of 100 µg mL-1 heparin, and

rotated overnight at 4 ºC with 4 µg of nucleolin-specific antibody MS3 (cat. no. sc-8031,

Santa Cruz Biotechnology) or IgG1 isotype control antibody (BD Pharmingen). We

treated antibody-virus-protein mixtures with 20 µL protein-G agarose (cat. no.

11243233001 Roche Applied Science) with rotation at 4 ºC for 2 h, the next day, spun

precipitates for 3 min at 1,000 × g and washed with 1 mL MOSLB lysis buffer [10mM


6

HEPES (pH 7.4), 50mM Na4P2O7, 50mM NaF, 50mM NaCl, 5mM EDTA, 5mM EGTA,

1mM Na3VO4, 0.5% Triton X-100, 10 µg mL-1 leupeptin, and 1mM

phenylmethylsulfonyl fluoride] on ice, repeated 5 times with intermittent 3 min, 1,000 ×

g centrifugation steps. We resolved precipitates by polyacrylamide electrophoresis and

Western blotted for RSV proteins using goat RSV-specific polyclonal sera (Biodesign

International).

Confocal Microscopy of RSV-Nucleolin Co-Localization and competition using a

nucleolin-specific neutralizing antibody. We treated 1HAEo- cells with control rabbit

IgG or polyclonal rabbit nucleolin-specific IgG antibody H-250 (1:100 dilution) for 1 h

and inoculated with RSV A in the presence of antibody, on ice for 1 h. We washed the

cells 4 times with ice cold PBS and fixed and stained for RSV F using RSV-3 primary

antibody (Novocastra) (green) and nucleolin using rabbit nucleolin-specific antibody H-

250, and goat rabbit-specific Alexa 594 (Invitrogen) secondary antibody (1:400 dilution),

imaged with a Leica SP2 confocal microscope and analyzed using Improvision

Volocity® software, with minor brightness and contrast adjustment not exceeding 20% of

original values. We assessed RSV-nucleolin binding on the cell surface by counting, at

high magnification (960x), the number of virus-associated green fluorescent dots (“virus

fluorescence units” (VFU), mean diameter ± SD: 0.27 ± 0.05 µm, within the size range of

RSV virions) per cell surface using Volocity® software (conducted on three dimensional

rendered images by counting 6 images per treatment (with 3–5 cells in each image) for

each virus and antibody treatment). We quantified virus-nucleolin co-localization by use

of Pearson’s correlation2, in which entire image stacks (512 × 512 × 136 pixels, 6-frame


7

average) were quantified for green (RSV) and red (nucleolin) pixels, with a greater value

reflecting more virus-nucleolin co-localization. We observed cells under a fluorescence

microscope and the percentage of positively staining cells was determined.

Confocal microscopy to verify polarization of 1HAEo- cells. We performed staining

and imaging as described above except mouse ZO-1-specific antibody (Zymed,

Invitrogen, cat# 33-9100) was added at 1:100 dilution and Alexa 488 conjugated

secondary goat mouse-specific secondary antibody was added at 1:400 dilution.

Treatment of RSV and RSV infected cells with Heparin. We diluted heparin stock

(cat. no. 210-6, Sigma Aldrich) to a concentration of 1 g mL-1 in 170 ul PBS and heparin

dilutions were made in PBS. We treated cells and RSV virus stocks with 10 ug mL-1, 100

ug mL-1 and 1 mg mL-1 of heparin or an equivalent volume of PBS for 1 h at 37 ºC.

We added virus and heparin mixtures to heparin treated cells of the same heparin

concentration, detached with trypsin-EDTA the following day, fixed and imaged on a

Nikon E600 fluorescent microscope equipped with Spot camera (Nikon). We

enumerated infection by flow cytometry.

Neutralization. We added to each well of ~80% confluent HEp-2 cells in 24-well plates,

200 µL of 10 µg mL-1 (1:50 dilution) nucleolin-specific antibody IgG C23 (H-250) or

control rabbit IgG (Santa Cruz Biotechnology) for 1 h at 37 ºC. RgRSV224 (MOI=1, 33

µL per well) was added to cells and after 24 h, cells underwent 0.25% trypsin digestion (5


8

min) and fixation in 10% formalin, followed by enumeration of infection by flow

cytometry.

Image-Pro Plus infected spot segmentation. We captured images using the Nikon

E600 fluorescent microscope equipped with Spot camera and analyzed the images using

Image-Pro Plus software (Media Cybernetics). We conducted spot counting of green

fluorescent cells with the spot counting feature in Image-Pro Plus which uses color

segmentation to isolate the RSV infected, GFP-expressing cells from the surrounding

unstained cells of each image. We set thresholds against uninfected cell images to

determine a negative signal threshold and the number of infected cells per unit area were

recorded and tabulated.

Competition. We incubated nucleolin (Vaxron Corp.) or transferrin (Sigma) (30 nM

each) with rgRSV224 (MOI = 1, 3 h, RT), added protein pre-incubated viruses to ~80%

confluent 1HAEo– monolayers in 24-well plates and kept them at 37 ºC for 90 min with

occasional rocking. After removal of unbound viruses, we kept cells an additional 24 h at

37 ºC. Then we digested the cells with 0.25% trypsin, formalin-fixed the cells and

performed flow cytometry as described above.

RNAi: We conducted RNAi of cellular nucleolin using Oligofectamine (Invitrogen), as

per manufacturer’s instructions. RNA oligonucleotides included: Decay accelerating

factor (siDAF) (5’-CCAUCUCCUUCUCAUGUAATTdTdT-3’); human nucleolin


9

(siNcl) (5’-UUUCUCAAACGAAGUAAGCUUdTdT-3’); and 3-nucleotide-substituted

human nucleolin (siNclΔ3) (5’-UUUCUCUAAGGAAGUAAGCGUdTdT-3’).

We transfected 1HAEo- cells with RNAi oligonucleotides, washed twice with fresh

growth media the following day and infected with virus or harvested for Western blot 24

h later. We detected expression of GFP by flow cytometry the following day to

determine infection by RSV-GFP.

Flow cytometry. For enumeration of RSV-GFP in neutralization, competition, RNAi

and reconstitution experiments, we loaded 3.7% formol saline-fixed cell suspensions into

a Beckman Coulter Epics-XL (Beckman Coulter) flow cytometer. The fraction of GFP-

positive infected cells was determined using Summit analysis software (Dako

Cytomation), gated against sham-infected cells. For quantification of cell surface

nucleolin in RNAi experiments, we incubated live cells with 1:100 diluted primary

monoclonal nucleolin-specific MS-3 antibody (Santa Cruz Biotechnology) (1 h, on ice),

followed by three 5-min washes in PBS and incubation with 1:400 diluted secondary

Alexa 594-conjugated mouse-specific antibody (Invitrogen) (1 h, on ice). After three

washes in PBS, we fixed cells with 3% formol saline and performed flow cytometry.

Results were reported as the percentage of cells expressing cell surface nucleolin.

Confocal Microscopy of Sf9 Cells. We fixed Sf9 cells at room temperature by addition

of 3:1 methanol acetone for 2 min, and blocked with 1% BSA in PBS for 10 min.,

incubated with rabbit-anti nucleolin polyclonal antibody H-250 (1:100 dilution) overnight


10

at 4 ºC and washed with PBS. We added Alexa 594 secondary antibodies (Invitrogen) at

a dilution of 1:400 (overnight, 4 °C), followed with three 5-min washes in PBS and

stained Nuclei with 4’, 6-diamidino-2-phenylindole (DAPI) contained within the

mounting media, Vectashield (Vector Laboratories). We conducted Fluorescence

confocal microscopy using an AOBS Leica Confocal TCS SP2 microscope and

performed lambda scans to confirm the specificity of Alexa fluor detection. We collated

image stacks (90–140 × 512 × 512 pixels; 3-frame average) using Improvision Volocity®

software. We determined transfection efficiency by counting the number of cells

showing positive fluorescence and expressed it as a percentage of total cells counted

under phase contrast microscopy, with concomitant determination of whether positively-

staining cells had surface nucleolin expression.

Transfection. We transfected Sf9 cells with Lipofectamine 2000 (Invitrogen) as per

manufacturer’s instructions. We transfected 1HAEo– cells with siRNA oligonucleotides

using Oligofectamine (Invitrogen), washed twice with fresh growth media the following

day and infected with virus 3 days later, or harvested for Western blot. We detected

expression of GFP by flow cytometry the following day to determine infection by GFP-

RSV.

RSV propagation. We routinely screened cells involved with RSV propagation for

Mycoplasma and LPS. We seeded 2 x 107 HEp-2 cells (ATCC) in 10% EMEM in a T-

150 tissue culture flask and grown at 37 ºC, in a 5% CO2 incubator overnight. The next

day (Day 0), we rinsed cells twice with clean PBS. We added RSV-A2 (ATCC) in 12


11

mL of serum free EMEM at an MOI ~ 0.1 and incubated for 2 to 4 h while rotating flask

every 15 to 30 min. We then added 28 mL 6% EMEM to flasks and incubated cells for 3

to 4 days. By day 4, 50% cells usually detached and virus was harvested and purified.

RSV harvesting and sucrose gradient purification. We scraped infected HEp-2 cells

and collected them with media into 50 mL Falcon tubes, and then centrifuged at 820 × g

for 10 min. We kept all solutions and cells on ice or at 4 ºC unless otherwise stated. We

decanted supernatant and placed it in a cold 50 mL Falcon tube and resuspended the cell

pellet was with 5 mL medium, quick frozen in liquid nitrogen, and thawed at 37 ºC with

constant agitation. We repeated this freeze-thaw cycle once. We sonicated the

resuspended pellet on ice for 20 s twice and centrifuged at 820 × g for 10 min. We

discarded the cell pellet and combined the supernatant with the original decanted media

for ultracentrifugation (SW28 rotor) at 110,000 × g for 45 min, over a sucrose cushion

(30% sucrose in 0.1 M sodium chloride, 0.01 M Tris-HCl, 0.001 M EDTA, 1 M urea, pH

= 7.5). After this spin a virus pellet was clearly visible and we decanted the supernatant.

We resuspended the pellet in 100 µL PBS per T-150 flask (yield: 2 × 108 PFU RSV mL-1)

and stored it at –80 ºC or in liquid nitrogen.

RSV titer quantification. We propagated HEp-2 cells in 6-well plates in 10% DMEM-

F12 to >90% confluence, aspirated the medium and washed cells twice with PBS or

serum free medium. We added to each well of the 6-well plates serial dilutions of RSV

stock in cold PBS, and 400 µL of the diluted RSV solution with at least one well per plate

left uninfected as a negative control and plated samples in duplicate or triplicate. We


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incubated the plates for 90 min at 37 ºC in a 5% CO2-containing incubator, rocking

manually every 15 min. and removed inoculants. We then overlaid the wells with 4 mL

of 1:1 4% FBS DMEM-F12/1% agarose and left them at room temperature for several

minutes before putting them back into the incubator and incubated the plates for 4–7 days

at 37 ºC. We could see holes in the monolayer with a dissecting scope after 4–5 days. At

this point, we added 2 mL of 1% formaldehyde (made up in 0.15 M saline) to each well

for incubation overnight. Then we flicked off the agarose and rinsed the plate gently with

running tap water to remove remaining agarose, added 2–3 mL of 0.05% neutral red to

each well for 1 h, and then removed excess stain with running tap water. We dried the

plates and counted plaques under a dissecting scope or with scanner and computer.

Immunohistochemistry. We deparaffinised formalin-fixed, paraffin-embedded mouse

lung sections with xylene and rehydrated before performing antigen retrieval with 10 mM

Citrate buffer heated for 20 min in a microwave. We then treated the sections with 3%

hydrogen peroxidase for 30 min to block endogenous peroxidase activity and blocked

with Dako protein block (cat# X0909). The primary antibody was nucleolin-specific

rabbit polyclonal (Abcam; ab22758) used at 1:1,000 dilution and the secondary antibody

was a biotinylated goat rabbit-specific IgG used at 1:200 dilution. We used the “ABC

kit” from Vector Labs (PK=6100) for subsequent DAB staining as per manufacturer’s

instructions. For double staining, we did a second round of blocking and antigen retrieval

and substituted the RSV-specific rabbit polyclonal (Abnova; PAB13816) as the primary

antibody. We used the “ABC kit” for alkaline phosphatase permanent red color staining

and counterstained slides with hematoxylin.


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In vitro testing of mouse siRNA on mouse cells. In preparation for in vivo studies with

mouse nucleolin siRNA “oligo 3” and control “oligo 3∆3”, we grew NIH 3T3 cells in 6

well plates using standard methods and transfected with test siRNAs in the same method

described above and harvested for Western blot 4 days post transfection. We purchased

our highly purified siRNAs for in vivo work from Dharmacon, Inc. RNA oligos: mouse

nucleolin siRNA (oligo 3) (5’-GGCUCUGUUCGUGCAAGAAdTdT-3’); and 3-

nucleotide-substituted mouse nucleolin (oligo 3∆3) (5’-

GUCUCUGAUCGUGCAAGCAdTdT-3’).


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References

1. Kaan, P.M. & Hegele, R.G. Interaction between respiratory syncytial virus and particulate matter in guinea pig alveolar macrophages. Am J Respir Cell Mol Biol 28, 697-704 (2003).

2. Hibbs, A.R., MacDonald, G. & Garsha, K. Handbook of Biological Confocal Microscopy (Springer Science + Business, New York, 2006).


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