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Supplementary Information for Mutations in the HIV-1 envelope glycoprotein can broadly rescue blocks at multiple steps in the HIV-1 replication cycle Rachel Van Duyne, Lillian S. Kuo, Phuong Pham, Ken Fujii, Eric O. Freed

Eric O. Freed Email: efreed@nih.gov This PDF file includes:

Supplementary text Figs. S1 to S11 Table S1 References for SI reference citations

www.pnas.org/cgi/doi/10.1073/pnas.1820333116

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Supplementary Information Text

Supplemental Materials and Methods

Virus titering and infections with high MOI

293T cells were transfected with pBR43IeG WT and Env mutant constructs using

Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Virus-containing

supernatants were filtered through a 0.45 µm membrane 48 h post-transfection and virus was

concentrated by ultracentrifugation. Virus pellets were resuspended in 10x less volume RPMI-10

post-ultracentrifugation. Concentrated virus was used to inoculate TZM-bl cells and Jurkat cells,

and to spinoculate Jurkat cells. One day before inoculation, 1x104 TZM-bl cells/well and 5x104

Jurkat cells/well were plated in a 96-well plate in 150µl media. Immediately prior to inoculation,

concentrated virus stocks (300µl) were two-fold serially diluted in media containing 10µg/mL

DEAE-Dextran (12x dilutions for TZM-bl and 6x dilutions for Jurkat) for a final volume of

150µl. Media was removed from TZM-bl cells, virus dilutions were added, incubated for 2 h at

37C, and media was changed. Virus dilutions were added to Jurkat cells and incubated at 37C. 48

h post-infection, TZM-bl cells were harvested in PBS + 5mM EDTA and fixed in 4% PFA; Jurkat

cells were harvested and fixed in 4% PFA.

One day before spinoculation, 5x104 Jurkat cells/well were plated in a 96-well plate in

150µl media. Immediately prior to spinoculation, concentrated virus stocks (300µl) were diluted

as above. Cells and virus were incubated (total volume approximately 300µl) and spun at

1,200xg for 2 h at 25C. Cell pellets were resuspended in RPMI-10 and incubated at 37C. 48 h

post-infection, Jurkat cells were harvested and fixed in 4% PFA. GFP+ cells were analyzed by

flow cytometry. Infectious titers and MOI were calculated as previously described (1). Infection

3

of Jurkat cells with an MOI of 10 by spinoculation was performed as above, except with 1x104

cells/condition.

Virus release, Env incorporation, expression, and processing, and pol product

incorporation

The radioimmunoprecipitation-based virus release assay has been described previously

(2, 3). Briefly, Jurkat cells were infected with the indicated RT-normalized VSV-G-pseudotyped

Env mutant virus stocks for 2-3 h. 24 h post-infection, cells were metabolically labeled with [35S]

Met/Cys-Pro-mix (Perkin Elmer) overnight (~18 h). Virus-containing supernatants were filtered

and pelleted by ultracentrifugation, cell and virus pellets were lysed, and were

immunoprecipitated overnight with HIV-IgG (obtained from the NIH ARP). Samples were

subjected to SDS-PAGE, followed by exposure to a PhosphorImager cassette and quantification

of bands by Quantity One or ImageLab (Bio-Rad) software. Virus release efficiency (VRE) was

calculated as the amount of virion p24 divided by total Gag (cell Pr55Gag + cell p24 + virion p24);

Env expression was calculated as the ratio of cellular Env (gp160+gp120) to cellular p24; Env

incorporation was calculated as the ratio of virion gp120 to virion p24; Env processing was

evaluated based on the ratio of cellular gp120 to cellular gp160. The incorporation of pol gene

products – p66 (RT), p51 (RT), and p32 (IN) – into virions was calculated relative to virion p24

levels. All data shown are normalized to pNL4-3 WT.

4

References 1. Janas AM & Wu L (2009) HIV-1 interactions with cells: from viral binding to

cell-cell transmission. Curr Protoc Cell Biol Chapter 26:Unit 26 25. 2. Demirov DG, Ono A, Orenstein JM, & Freed EO (2002) Overexpression of the

N-terminal domain of TSG101 inhibits HIV-1 budding by blocking late domain function. Proc Natl Acad Sci U S A 99(2):955-960.

3. Waheed AA, Ono A, & Freed EO (2009) Methods for the study of HIV-1 assembly. Methods Mol Biol 485:163-184.

C1 V1 V2 C2 V3 C3 V4 C4 V5 C5 HR1 HR2 MSD CT

Days post-transfection

RT A

ctiv

ity (c

pm/µ

l)C)

RT A

ctiv

ity (c

pm/µ

l)

D)

Days post-transfection

0

5,000

10,000

15,000

20,000

25,000

0 10 20 30 40

WT

p6-L41A

p6-L41A/Vpu-K31stop

p6-L41A/Env-R166I

mock

0

5,000

10,000

15,000

20,000

25,000

30,000

0 5 10 15

WT

p6-Y36A

p6-Y36A/Env-A327T

p6-Y36A/Env-P81S/A327T

mock

Env (-)

RT A

ctiv

ity (c

pm/µ

l)

B)

Days post-transfection

0

10,000

20,000

30,000

40,000

50,000

60,000

70,000

80,000

0 6 12 18 24

WT

p6-Y36S/L44R

p6-Y36S/L44R/Vpu-M1I

p6-Y36S/L44R/Env-I744V

p6-Y36S/L44R/Env-R786K

mock

p6 WT 35LYPLASLRSLF45 Vpu WT 1MQP3 … 30RKI32 Env WT 165IRD167 … 326QAH328 … 743SIR745 … 785RRG788

Y36A -A-------- A327T --- -T- --- ---

Y36S/L44R -S-------R M1I I-- --- I744V, R786K --- --- -V- -K-

L41A ------A--- K31stop --- -STOP R166I -I- --- --- ---

MA CA NC p6polSP1 SP2

vifvprA)

vpu

rev revtat tatgp120

gp41

FPnef

Fig. S1. Analysis of inactivating mutations in Vpu, and substitutions in Env that do not rescue replication-defective p6-Alix binding site mutants. A) Schematic of the HIV-1 genome as shown in Fig. 1A. Mutations in p6, Vpu, and Env are indicated by underlined residues and amino acid positions (NL4-3 numbering). Location of the Vpu mutations and non-rescuing Env mutations within the genome are indicated by dashed regions. Domains are defined as in Fig. 1A. B-D) Jurkat T-cells were transfected with the indicated pNL4-3 p6, p6/Vpu, and p6/Env mutant proviral clones and replication kinetics were monitored by measuring RT activity. Individual panels/graphs represent one p6 mutant with its corresponding second-site change(s): B) p6-Y36A C) p6-Y36S/L44R, D) p6-L41R. WT NL4-3 replication kinetics are indicated by a solid black line, p6 mutants by solid colored lines, and p6/Env or p6/Vpu mutants by colored line markers of varying symbols. Data are representative of at least two independent experiments.

% Infe

ctivity (

RLU

s)

0

20

40

60

80

100

120

mock WT Env Env (-) Env-Y61H Env-P81S Env-A556T

Jurkat à TZM-bl

Fig. S2. The Env compensatory mutants exhibit severe defects in cell-free, single-cycle infectivity independent of the producer cell. A) 293T-derived, GFP-expressing pBR43IeG virus-containing supernatants were concentrated 10x by ultracentrifugation and were used to infect TZM-bl cells. GFP+ cells were quantified by flow cytometry approximately 48 h post-infection; data are plotted against total amount of virus used to infect cells, normalized to RT cpm. Data from two independent experiments are shown. B) Jurkat-derived, virus-containing supernatants were collected from days of peak replication (Fig. 3A), RT normalized, and used to infect TZM-bl cells. Luciferase activity was measured approximately 36 h post-infection; data are normalized to WT. Data from three independent experiments are shown as means ± SD. C) Virus was prepared and concentrated as in panel A, and was used to infect Jurkat cells by direct inoculation (“I”) or spinoculation (“S”). GFP+ cells were quantified by flow cytometry approximately 48 h post-infection; data are plotted against total amount of virus used to infect cells, normalized to RT cpm. Data from three independent experiments are shown.

A)

B)

% G

FP

+ C

ells

C)

S

I

% G

FP

+ C

ells

293T à TZM-bl

Virus Input (RT cpm x 106)

Virus Input (RT cpm x 106)

0

20

40

60

80

100

120

0 2 4 6 8

293T à JurkatWT, N=1

Env-A556T, N=1

WT, N=2

Env-A556T, N=2

WT, N=3

Env-A556T, N=3

WT, N=1

WT, N=2

WT, N=3

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10

WT, N=1

Env-A556T, N=1

WT, N=2

Env-A556T, N=2

*******

********

****

Days post-transfection

0

5,000

10,000

15,000

20,000

25,000

0 5 10 15

WTEnv-P81SEnv-P81S/A327TEnv-A327TEnv (-)mock

% In

fect

ivity

(RLU

s)

0

20

40

60

80

100

120

140

160

mock WT Env-P81S Env-P81S/A327T Env-A327T

293T à TZM-bl

A)

B)

RT

Activ

ity (c

pm/µ

l)

Fig. S3. The Env-A327T mutation does not affect the Env-P81S phenotype. A) Jurkat cells were transfected with the indicated pNL4-3 Env mutant proviral clones and replication kinetics were monitored by measuring RT activity. B) Single-cycle infectivity of the indicated mutants was analyzed in TZM-bl cells as in Fig. 3B. Data from at least three independent experiments are shown as means ± SD; “ns” indicates “not significant”.

ns****

0

10000

20000

30000

40000

50000

60000

0 10 20

Donor 1

0

20000

40000

60000

80000

100000

120000

0 10 20

Donor 2

05000

100001500020000250003000035000400004500050000

0 10 20

Donor 3

0

50000

100000

150000

200000

250000

0 10 20

Jurkat

RT A

ctiv

ity (c

pm/µ

l)

Days post-infection

B) C) D)A)

Fig. S4. Replication kinetics of Env mutants in primary cells recapitulate their phenotypes in cell lines. A-C) 293T-derived, VSV-G pseudotyped, Env mutants were used to infect PBMCs from three independent donors (Donor 1-3) in duplicate (Fig. 4). Jurkat cells (D) were included for comparison; replication kinetics were monitored by measuring RT activity. 0

500

1000

1500

2000

2500

3000

3500

4000

4500

0 5 10 15 20

WTEnv-Y61HEnv-P81SEnv-A556TmockEnv (-)

A)

0

50

100

150

200

250

300

WT Env (-) Env-Y61H Env-P81S Env-A556T

Virus Release EfficiencyEnv ExpressionEnv IncorporationEnv Processing

% o

f WT

NL4

-3

B)

C)

0

50

100

150

200

250

300

350

WT Env (-) Env-Y61H Env-P81S Env-A556T

p66/p24

p51/p24

p32/p24

% o

f WT

NL4

-3

gp120

Fig. S5. Env compensatory mutations do not increase virus release, Env expression, or virion incorporation of Env or pol products. A) 293T-derived, VSV-G-pseudotyped Env mutant viruses

were RT-normalized and used to infect Jurkat cells that were metabolically labeled 24 h post-infection with 35S-Met/Cys overnight (~18 h). Cell and virus lysates were immunoprecipitated with

HIV-IgG, run on SDS-PAGE, and quantified by PhosphorImager analysis. A representative gel image is shown. Major viral gene products are indicated by arrows in both the cell and virus

fractions. Migration positions of mass standards are indicated in kDa. B, C) Data shown in panel a were quantified as indicated in the Materials and Methods. All values are shown normalized

to WT. Data from at least three independent experiments are shown as means ± SD.

gp160 gp120

Pr55

p24 p24 p32

p51 p66

•

•

•

•

WT

Env

(-)

Env-

Y61H

Env-

P81S

Env-

A556

T

WT

Env

(-)

Env-

Y61H

Env-

P81S

Env-

A556

T

cell virus

•60

•80

50

40

25

20

0

5,000

10,000

15,000

20,000

25,000

30,000

35,000

40,000

45,000

0 5 10 15 20 25 30 35

WT + DMSO

Env-E209K

Env-H641Y

WT + 3nM DTG

Env-E209K + 3nM DTG

Env-H641Y + 3nM DTG

mock

Days post-transfection

RT

Activity (

cpm

/µl)

A)

B)

0

10,000

20,000

30,000

40,000

50,000

60,000

0 5 10 15 20 25

WT + DMSO

Env-E209K/A539V

Env-A539V/H641Y

WT + 3nM DTG

Env-E209K/A539V + 3nM DTG

Env-A539V/H641Y + 3nM DTG

mock

+ 3.0 nM

DTG

Days post-transfection

RT

Activity (

cpm

/µl)

+ DMSO

+ 3.0 nM

DTG

+ DMSO

Fig. S6. Env-E209K and Env-H641Y mutants are partially resistant to DTG. Jurkat cells were transfected with the indicated pNL4-3 proviral clones in the presence or absence of 3 nM DTG, and virus replication was monitored by measuring RT activity. Data are representative of at least four (A) or at least two (B) independent experiments.

DTG WT A556T A539V

IC50 0.52 ± 0.53 1.874 ± 1.44 2.331± 1.99

R2 0.977 ± 0.008 0.950 ± 0.009 0.872 ± 0.060

FC 4.56 ± 1.880 5.25 ± 1.50

-1 0 1 2 30

2,000

4,000

6,000

8,000

log DTG, [nM]

RT

activ

ity (c

pm/µ

l)

pp n=2 (DTG) Day 12

WT

A539V

A556T

Fig. S7. gp41 mutants Env-A556T and Env-A539V exhibit fold change values comparable to INSTI-resistant IN mutants in the presence of DTG. Jurkat cells were transfected with the indicated proviral clones in the presence of serial dilutions (500 nM-0.244 nM; 2-fold increments, 12 concentrations) of DTG and replication kinetics were monitored by measuring RT activity. Data were plotted from day 10 post-transfection; IC50s and R2 values were calculated using GraphPad PRISM. Fold change (FC) was calculated compared to WT. Data from two independent experiments are shown as means ± SD.

0

20

40

60

80

100

120

mock WT Env (-) Env-A539V

293T à TZM-bl

% In

fect

ivity

(RLU

s)

Fig. S8. The gp41 mutant Env-A539V exhibits near-WT levels of cell-free infectivity. Single-cycle infectivity of the indicated mutants was measured in TZM-bl cells as in Fig. 3B. Data from at least three independent experiments are shown as means ± SD.

*B)

% G

FP+

cells

0

20

40

60

80

100

120

mock WT Env (-) Env-A556T Env-A539V

pBR43IeG 293Ts

C)

mock WT Env (-) Env-A556T Env-A539V

Day of peak:

GFP+

GFP+0.95

10 0 10 1 10 2 10 3 10 4

FL1-H :: FL1-Height

0

200

400

600

800

1.0K

SSC

-H

J1 5_4.197Jurkats4831

GFP+21.2

10 0 10 1 10 2 10 3 10 4

FL1-H :: FL1-Height

0

200

400

600

800

1.0K

SSC

-H

J2 5_2.185Jurkats3962

GFP+0.84

10 0 10 1 10 2 10 3 10 4

FL1-H :: FL1-Height

0

200

400

600

800

1.0K

SSC

-H

J5 5_4.198Jurkats4403

GFP+82.4

10 0 10 1 10 2 10 3 10 4

FL1-H :: FL1-Height

0

200

400

600

800

1.0K

SSC-

H

J14 4_30.209Jurkats2840

GFP+94.9

10 0 10 1 10 2 10 3 10 4

FL1-H :: FL1-Height

0

200

400

600

800

1.0K

SSC-

H

J17 4_28.193Jurkats3569

SSC

-H

tfx2293T 293T Jurkat+293T

A)

Fig. S9. GFP+ donor cells and representative raw flow cytometry data for Fig. 8. A) Schematic of initiating spreading infections in Jurkat T cells by coculture with 293T cells transfected with the GFP-expressing clone pBR43IeG. B) 293T cells transfected with GFP-expressing clone pBR43IeG were measured for GFP+ cells 48 h post-transfection. 293Ts were used to inoculate Jurkat cells for spreading infections at a ratio of 103 293T:106 Jurkat cells. Data from three independent experiments are shown as means ± SD ; “ns” indicates “not significant”. C) Representative raw flow cytometry data are shown for days of peak replication corresponding to Fig. 8A

nsns

Fig. S10. Cell-free infection of Jurkat cells at high MOI with WT and Env-A539V correlates with high MFI. A) Jurkat-titered, RT-normalized virus (Fig. S2C) was used to infect Jurkat cells by spinoculation with virus equivalent to an MOI of 0.1 or 10 calculated from WT. GFP+ cells were quantified by flow cytometry approximately 48 h post-infection. Data from three independent experiments are shown as means ± SD ; “ns” indicates “not significant”. B) The geometric mean fluorescence intensity (MFI) of GFP+ cells from A) was calculated. Data from three independent experiments are shown as means ± SD.

%G

FP+

Gat

ed C

ells

Mea

n Fl

uore

scen

ce In

tens

ity (M

FI)

A) B)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

mock WT Env-A556T Env-A539V

Jurkat

0.110

0

20

40

60

80

100

120

mock WT Env-A556T Env-A539V

Jurkat

0.110

nsns

*** ns

Y61

P81A556

A539

D)

P81

Y61

B)

A) C)

A539

Fig. S11. Mapping the Env compensatory mutants on three-dimensional structures of HIV-1 Env. A prefusion Env monomer (A, C PDB: 5FYK) and a CD4-bound Env

trimer (B, D PDB: 5VN3) with positions of Env mutations highlighted. Gp120 is colored in pale green and gp41 is colored in wheat. Y61 is colored blue, P81 red,

A556 orange, and A539 magenta.

Table S1. PCR primers used for viral DNA amplification.

Name Sequence Length GC% TM DescriptionRVD_PRIME_251 ACAGTCAGACTCATCAAGCT 20 45 60.2 Fwd: pNL4-3 env @ 6011RVD_PRIME_252 GCATTGTCTGTGAAATTGGC 20 45 60 Rev: pNL4-3 env @ 7056RVD_PRIME_253 ACCAATAGTAGTAGCGGGAG 20 50 59.8 Fwd: pNL4-3 env @ 6638RVD_PRIME_254 CTTTTCCTACTTCCTGCCAC 20 50 60 Rev: pNL4-3 env @ 7511RVD_PRIME_255 CCAGGGAGAGCATTTGTTAC 20 50 60 Fwd: pNL4-3 env @ 7151RVD_PRIME_256 AATTTCTCTGTCCCACTCCA 20 45 60.1 Rev: pNL4-3 env @ 8119RVD_PRIME_257 TTTGTTCCTTGGGTTCTTGG 20 45 60 Fwd: pNL4-3 env @ 7765RVD_PRIME_258 GCTGCTGTATTGCTACTTGT 20 45 60 Rev: pNL4-3 env @ 8935RVD_PRIME_249 AATTGGTCAGTGCTGGAATC 20 45 60 Fwd: pNL4-3 pol @ 4198RVD_PRIME_250 GCAGACCCCAATATGTTGTT 20 45 60 Rev: pNL4-3 pol @ 5257