Supplementary Materials for · Virion incorporation of integrin α4β7 facilitates HIV-1 infection...

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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

Transcript of Supplementary Materials for · Virion incorporation of integrin α4β7 facilitates HIV-1 infection...

Page 1: Supplementary Materials for · Virion incorporation of integrin α4β7 facilitates HIV-1 infection and ... To evaluate virion incorporation of cellular proteins, we used a previously

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

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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

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CD4-Ig

2G12

PG9PG16

VRC340

2

4

6

8

Cap

ture

eff

icie

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(% o

f tot

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A

B

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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.