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Supplementary Materials for
Virion incorporation of integrin α4β7 facilitates HIV-1 infection and
intestinal homing
Christina Guzzo, David Ichikawa, Chung Park, Damilola Phillips, Qingbo Liu,
Peng Zhang, Alice Kwon, Huiyi Miao, Jacky Lu, Catherine Rehm, James Arthos,
Claudia Cicala, Myron S. Cohen, Anthony S. Fauci, John H. Kehrl, Paolo Lusso*
*Corresponding author. Email: [email protected]
Published 12 May 2017, Sci. Immunol. 2, eaam7341 (2017)
DOI: 10.1126/sciimmunol.aam7341
The PDF file includes:
Fig. S1. Schematic representation and validation of the
immunomagnetic virion capture assay.
Fig. S2. Role of HIV-1 Gag and Env components in virion
incorporation of integrin α4β7.
Fig. S3. Effect of RA treatment on cell surface expression and virion
incorporation of different lymphocyte markers.
Fig. S4. Correlation between α4β7 cell surface expression and α4β7
incorporation into HIV-1 progeny virions.
Fig. S5. Validation of engineered α4β7+ and α4β7− HIV-1 THRO viral
stocks.
Fig. S6. Validation of engineered α4β7+ and α4β7− HIV-1 BaL
pseudovirus stocks.
Legend for movie S1
Other Supplementary Material for this manuscript includes the following:
(available at immunology.sciencemag.org/cgi/content/full/2/11/eaam7341/DC1)
Movie S1 (.mp4 format). A three-dimensional image reconstruction showing
α4β7+ HIV-1 virions captured along the endothelial surface of HEVs in Peyer’s
patches.
Raw data tables with statistical analyses (Microsoft Excel format).
immunology.sciencemag.org/cgi/content/full/2/11/eaam7341/DC1
Figure S1
Fig. S1. Schematic representation and validation of the immunomagnetic virion-capture assay.
(A) To evaluate virion incorporation of cellular proteins, we used a previously described
immunomagnetic virion-capture assay (22-24) schematically illustrated in this cartoon. Briefly,
magnetic beads pre-coated with anti-mouse IgG antibodies are incubated with each monoclonal
antibody (mAb), extensively wahsed to remove unbound mAb, and then incubated with HIV-1
virions. After extensive washing to remove unbound virions, the bead-captured virions are lysed
by detergent treatment, and the absolute amount of captured p24Gag antigen in the lysate is
quantified. (B) The assay was validated using a panel of anti-HIV-1 envelope (Env) human mAbs
recognizing different antigenic regions of gp120 (2G12, PG9, PG16, VRC34), as well as an Fc-
chimeric form of soluble CD4 (CD4-Ig) on a defined viral stock of the CCR5-tropic (R5) HIV-1
isolate BaL produced in primary human peripheral blood mononuclear cells (PBMC) activated
with anti-CD3 mAb (OKT3). The efficiency of virion capture (expressed as the fraction of captured
p24Gag antigen) is shown in a representative experiment out of several performed with similar
results. The efficiency of virion capture is typically below 10% of the total input virus, even using
broadly-reactive anti-Env mAbs. Background levels of virion capture were consistently low, as
assessed using beads coated with an isotype-control mAb (blue bar).
Isoty
pe contro
l
CD4-Ig
2G12
PG9PG16
VRC340
2
4
6
8
Cap
ture
eff
icie
ncy
(% o
f tot
al)
A
B
Legend to Supplementary Movie
Movie S1. A three-dimensional image reconstruction showing α4β7+ HIV-1 virions
captured along the endothelial surface of HEVs in Peyer’s patches.
Twenty µm superimposed image was generated with 21 slices of a single section image (same as
in Fig. 7C, left panel). Sections were stained with anti-CD4 (blue) and anti-PNAd (red). PNAd
staining specifically indicated locations of high endothelial venules (HEVs) in a Peyer’s patch.
Specific binding of α4β7+ virions (green) was detected in HEVs in a Peyer’s patch. The scale bar
denotes 10 µm.