Supplementary Table 1. mAbs analyzed

mAb Antigenic Domains

Critical Binding Residues1 by Alanine Scanning2

Citations

CBH-4B Domain A NA (Hadlock, Journal of Virology, 2000), (Keck, Journal of Virology, 2004)

HC-1 Domain B 529, 530, 535, 536, 537, 540 (Keck, Journal of Virology, 2008)

CBH-2 Domain B 425, 426, 431, 434, 436, 523, 529, 530, 535, 536

(Hadlock, Journal of Virology, 2000), (Keck, Journal of Virology, 2008)

CBH-5 Domain B 412, 416, 417, 418, 420, 421, 422, 423, 483, 484, 485, 488, 523, 525, 527, 530, 533, 535, 538, 540, 550

(Hadlock, Journal of Virology, 2000), (Owsianka, J. Gen. Virol., 2008)

CBH-7 Domain C 538, 540, 547, 549 (Hadlock, Journal of Virology, 2000), (Owsianka, J. Gen. Virol., 2008)

HC84.22 Domain D 420, 426, 428, 429, 437, 441, 442, 443, 530, 616

(Keck, PLOSPathogens, 2012)

HC84.26 Domain D 429, 441, 442, 446, 616 (Keck, PLOSPathogens, 2012)

HC33.4 Epitope "1" 413, 418, 420 (Keck, Journal of Virology, 2013)

HC33.8 Epitope "1" 413, 418, 420 (Keck, Journal of Virology, 2013)

AR1A Antigenic Region 1

416, 417, 484, 485, 538, 540, 546, 549

(Law, Nature Medicine, 2008)

AR2A Antigenic Region 2

540 (Law, Nature Medicine, 2008)

AR3A Antigenic Region 3/Domain B

424, 523, 525, 530, 535, 538, 540

(Law, Nature Medicine, 2008)

AR3B Antigenic Region 3/Domain B

412, 416, 418, 423, 424, 523, 525, 530, 535, 540

(Law, Nature Medicine, 2008)

AR3C Antigenic Region 3/Domain B

424, 488, 523, 525, 530, 535, 538, 540

(Law, Nature Medicine, 2008)

AR3D Antigenic Region 3/Domain B

412, 424, 523, 530, 535 (Law, Nature Medicine, 2008)

AR4A Antigenic Region 4

201, 204, 205, 206, 487, 657, 658, 692, 698

(Giang, PNAS, 2012)

AR4B Antigenic Region 4

NA (Giang, PNAS, 2012)

AR5A Antigenic Region 5

201, 204, 205, 206, 639, 657, 658, 665, 692

(Giang, PNAS, 2012)

1Numbering based on polyprotein position

2Mutation of critical binding residues reported for HC84.22, HC84.26, HC33.4, HC33.8 to alanine reduce mAb

binding by at least 60%. Mutation of critical binding residues reported for the remaining mAbs reduce binding by at least 50%.

NA: Not Available

Lowest RelativeInfection(most sensitive)

Highest RelativeInfection(most resistant)

AR3D

AR3B

AR3A

AR3C

HC84

.22

CBH-

5

HC84

.26

CBH-

2

CBH-

4B

AR4B

AR1A

AR5A

AR4A

HC33

.8

HC33

.4

AR2A

CBH-

7

HC-1

1a09 0.487 0.529 0.515 0.432 0.655 0.564 0.494 0.673 0.858 0.643 0.695 0.439 0.389 0.459 0.229 0.649 0.607 0.5981a31 0.315 0.205 0.015 0.066 0.145 0.650 0.074 0.737 0.743 0.591 0.457 0.058 0.087 0.523 0.435 1.171 0.434 0.1481a38 0.730 0.742 0.620 0.544 0.620 0.618 0.012 0.577 0.990 0.906 0.887 0.645 0.601 0.309 0.198 0.718 0.751 0.8061a53 0.187 0.151 0.089 0.014 0.159 0.169 0.009 0.635 0.931 0.301 0.728 0.204 0.023 0.033 0.041 0.147 0.848 0.8221a72 0.397 0.355 0.376 0.270 0.336 0.317 0.137 0.430 1.041 0.448 0.698 0.372 0.094 0.799 0.437 0.902 0.489 0.7871a80 0.551 0.543 0.470 0.376 0.998 0.615 1.419 1.079 1.090 0.532 0.855 0.342 0.108 0.381 0.179 0.834 0.795 0.489

1a116 0.214 0.265 0.528 0.307 0.303 0.480 0.477 0.644 0.922 0.496 0.710 0.357 0.453 0.387 0.264 0.700 0.546 0.4671a123 0.778 0.768 0.425 0.507 0.392 0.388 0.002 0.762 0.829 0.151 0.513 0.120 0.023 0.169 0.023 0.587 0.537 0.6911a129 0.617 0.837 0.798 0.646 0.739 0.592 0.063 0.579 0.982 0.989 1.010 0.846 0.402 0.662 0.459 1.024 0.743 0.5421a142 0.508 0.603 0.538 0.540 1.045 0.883 1.489 1.215 1.071 0.412 0.675 0.130 0.064 0.206 0.045 0.172 0.761 0.7841a154 0.329 0.372 0.231 0.131 0.174 0.670 0.003 0.646 0.887 0.343 0.638 0.086 0.046 0.043 0.004 0.172 0.448 0.3271a157 0.407 0.412 0.310 0.233 0.410 0.722 0.082 0.557 0.887 0.586 0.754 0.452 0.230 0.168 0.065 0.530 0.493 0.3281b09 0.222 0.161 0.121 0.047 0.092 0.059 0.000 0.094 1.214 0.528 0.913 0.184 0.074 0.448 0.142 0.614 0.741 0.7781b14 0.443 0.408 0.536 0.154 0.310 0.376 0.004 0.363 0.935 0.851 1.024 0.453 0.373 1.003 0.695 0.967 1.138 0.9691b21 1.611 1.679 0.691 0.204 0.063 0.096 0.022 1.337 1.480 1.203 1.206 0.143 0.176 0.094 0.062 0.641 0.675 0.4821b34 0.479 0.312 0.310 0.125 0.159 0.119 0.000 0.110 0.845 0.909 0.977 0.700 0.195 0.684 0.352 0.843 0.572 0.4371b38 0.312 0.188 0.144 0.109 0.256 0.242 0.013 0.249 0.935 0.781 0.907 0.439 0.196 0.846 0.674 1.032 0.939 0.9071b52 0.644 0.601 0.595 0.408 0.370 0.388 0.002 0.352 0.797 0.873 0.959 0.879 0.377 0.560 0.384 0.954 0.885 0.7821b58 0.706 0.765 0.733 0.529 0.647 0.525 0.054 0.571 1.031 0.782 0.812 0.607 0.312 0.892 0.618 0.737 0.907 0.873

HCVp

p

mAbs

Supplementary Figure 1

Supplementary Figure 1. Each neutralizing mAb produces a “neutralization 1

fingerprint” across the HCVpp library. Eighteen previously-characterized HCV-2

specific mAbs were tested for neutralization of each of the 19 clonal genotype 1a and 3

1b HCVpp. Four representative mAbs are shown in Figure 1, and neutralization results 4

for all 18 mAbs are shown here. Relative infection is calculated as infection in the 5

presence of 10 µg/ml of neutralizing mAb relative to infection in the presence of 6

nonspecific IgG. Each value is a mean of duplicate wells. For each mAb, relative 7

infection values are colored on a gradient, with lower relative infection values (greatest 8

neutralization) darker green, and higher relative infection values (less neutralization) 9

darker red. Neutralization of pseudoparticles with MLV envelope was measured as a 10

negative control (values not shown). 11

0.1 1 10 1000.001

0.01

0.1

1

CBH5CBH2AR3AHC84.22HC33.4HC84.26

HCVcc IC50 (µg/mL)

Rel

ativ

e in

fect

ion

(HC

Vpp)

r=.93p<.02

Supplementary Figure 2

Supplementary Figure 2. Significant correlation between HCVcc IC50 and HCVpp relative infection. 50% inhibitory concentrations (IC50’s) of the six indicated mAbs against full-length replication competent HCV (HCVcc) bearing H77 E1E2 were compared by Spearman correlation to the relative infection of HCVpp with H77 E1E2, measured using the same antibodies. Two mAbs that did not achieve 50% neutralization of HCVcc at the highest mAb concentration tested, 20 µg/mL, were assigned an IC50 of 40 µg/mL.

0.0001

0.001

0.01

0.1

1

10

0.01 0.1 1 10 100

Rel

ativ

e In

fect

ion

IC50 (µg/mL)

r=0.91p<.0001

Supplementary Figure 3

IC50 Relative InfectionCBH-5

pp1a116 >50 0.4801pp1b09 0.24 0.0591HC84.26pp1a72 3.38 0.1369pp1b21 0.02 0.0222pp1b09 0.01 0.0003CBH-2

pp1a129 >50 0.5788pp1b09 0.35 0.0940

HC-1pp1b14 >50 0.9688

pp1a123 >50 0.6909pp1a31 0.36 0.1485HC33.4pp1b14 12.02 0.6946pp1a09 3.53 0.2287

pp1a154 0.04 0.0039AR3A

pp1a129 12.02 0.7978pp1a80 14.13 0.4698pp1a31 0.33 0.0145AR3C

pp1a129 15.05 0.6461pp1a72 6.93 0.2697pp1a53 0.28 0.0140AR4A

pp1a38 6.03 0.6010pp1b34 0.93 0.1947pp1a53 0.20 0.0227AR5A

pp1b52 21.12 0.8793pp1a72 7.26 0.3718

a

b

Supplementary Figure 3. Significant correlation between HCVpp IC50 and HCVpp relative infection. (a) All combinations for which both relative infection at 10 µg/mL of mAb and IC50 were measured in independent experiments. (b) Correlation between relative infection value and IC50 for the same HCVpp/mAb combination. Each point represents one mAb/HCVpp combination. MAbs that did not achieve 50% neutralization at the highest mAb concentration tested, 50 µg/mL, were assigned an IC50 of 100 µg/mL. Spearman correlation (r)=0.91 with p<0.0001.

1b09 0.091b34 0.111b38 0.251b52 0.351b14 0.361a72 0.43

1a157 0.561b58 0.571a38 0.58

1a129 0.581a53 0.64

1a116 0.641a154 0.651a09 0.671a31 0.74

1a123 0.761a80 1.08

1a142 1.211b21 1.34

CBH-2

HCVpp Relativ

e In

fecti

on

E1E2

HVR1 Stem TM dom

1b09 0.00031b34 0.00041b52 0.0016

1a123 0.00211a154 0.00261b14 0.00431a53 0.00891a38 0.01181b38 0.01261b21 0.02221b58 0.0540

1a129 0.06281a31 0.0739

1a157 0.08251a72 0.1369

1a116 0.47721a09 0.49431a80 1.4193

1a142 1.4892HC84.26

HCVpp Relativ

e In

fecti

on

E1E2

HVR1 Stem TM dom

1b09 0.061b21 0.101b34 0.121a53 0.171b38 0.241a72 0.321b14 0.381b52 0.39

1a123 0.391a116 0.481b58 0.521a09 0.56

1a129 0.591a80 0.611a38 0.621a31 0.65

1a154 0.671a157 0.721a142 0.88

CBH-5

HCVpp Relativ

e In

fecti

on

E1E2

HVR1 Stem TM dom

Supplementary Figure 4

Supplementary Figure 4. Sequence analysis reveals resistance-associated polymorphisms. Sequence analysis for NC1 mAbs not shown in Figure 3. Clones are grouped into the 5 most sensitive, 7 with intermediate resistance, and 7 with greatest resistance, separated by horizontal black lines. Gray vertical bars indicate positions with a substitution in any resistant E1E2 clone but in none of the five most sensitive E1E2 clones. Black vertical bars indicate CD81 binding sites in E2. Blue vertical bars indicate critical binding residues for the indicated mAb, determined by alanine scanning. Sites with substitutions in at least two resistant clones but in no sensitive clones are included in the summary panel in the bottom row. Sites marked with vertical red bars are predominantly polymorphic in the seven most resistant clones. Orange vertical bars indicate sites that are polymorphic in an equal number of highly resistant and intermediate resistant clones. Green vertical bars indicate sites that are predominantly polymorphic in the seven clones with intermediate resistance.

1a31 0.011a53 0.091b09 0.121b38 0.14

1a154 0.231b34 0.31

1a157 0.311a72 0.38

1a123 0.431a80 0.47

1a116 0.531a09 0.511b14 0.54

1a142 0.541b52 0.591a38 0.621b21 0.691b58 0.73

1a129 0.80AR3A

HCVpp Relativ

e In

fecti

on

E1E2

HVR1 Stem TM dom

1a53 0.151b09 0.161b38 0.191a31 0.20

1a116 0.261b34 0.311a72 0.35

1a154 0.371b14 0.41

1a157 0.411a09 0.531a80 0.541b52 0.60

1a142 0.601a38 0.741b58 0.77

1a123 0.771a129 0.841b21 1.68

AR3B

HCVpp Relativ

e In

fecti

on

E1E2

HVR1 Stem TM dom

1a53 0.011b09 0.051a31 0.071b38 0.111b34 0.13

1a154 0.131b14 0.151b21 0.20

1a157 0.231a72 0.27

1a116 0.311a80 0.381b52 0.411a09 0.43

1a123 0.511b58 0.53

1a142 0.541a38 0.54

1a129 0.65AR3C

HCVpp Relativ

e In

fecti

on

E1E2

HVR1 Stem TM dom

1a53 0.191a116 0.211b09 0.221b38 0.311a31 0.32

1a154 0.331a72 0.40

1a157 0.411b14 0.441b34 0.481a09 0.49

1a142 0.511a80 0.55

1a129 0.621b52 0.641b58 0.711a38 0.73

1a123 0.781b21 1.61

AR3D

HCVpp Relativ

e In

fecti

on

E1E2

HVR1 Stem TM dom

Supplementary Figure 4 (Continued)

0

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53N

430D

D43

1EL4

33I

L438

IL4

38V

F442

IF4

42L

K44

6EP

453S

A47

5TE

531A

F560

Y0

4

8

12

16

20

pp1a

53N

430D

D43

1EL4

33I

L438

IL4

38V

F442

IF4

42L

K44

6EP

453S

A47

5TE

531A

F560

Y

Supplementary Figure 5C

BH

-2Fo

ld C

hang

e in

Res

ista

nce

CB

H-5

Fold

Cha

nge

inR

esis

tanc

e

HC

84.2

2Fo

ld C

hang

e in

Res

ista

nce

AR

3AFo

ld C

hang

e in

Res

ista

nce

AR

3BFo

ld C

hang

e in

Res

ista

nce

AR

3CFo

ld C

hang

e in

Res

ista

nce

HCVpp

HCVpp

HC

82.2

6Fo

ld C

hang

e in

Res

ista

nce

Supplementary Figure 5. Introduction of resistance-associated polymorphisms into a second neutralization sensitive E1E2 clone (1a53) confirms the resistance phenotypes of D431E and F442I. Eleven mutations of interest were introduced by site directed mutagenesis into an E1E2 clone that was sensitive to neutralization by each of the NC1 mAbs (clone 1a53). The dashed line indicates relative infection of HCVpp using wildtype 1a53 in the presence of the indicated mAb, adjusted to 1. Each bar indicates the fold change in neutralization resistance after the indicated mutation(s) were introduced into clone 1a53. Error bars indicate standard deviation between duplicate wells. Black triangles indicate HCVpp with D431E mutations. Open triangles indicate HCVpp with F442I or F442L mutations.

0

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1.2

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0.4

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0.8

1

1.2

ML

V

pp1b

21

D43

0N

E43

1D

D43

0N/E

431D

pp1a

142

I438

L

I442

F

E44

6K

I438

L,I4

42F

0

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0.6

0.8

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1.2

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V

pp1b

21

D43

0N

E43

1D

D43

0N/E

431D

pp1a

142

I438

L

I442

F

E44

6K

I438

L,I4

42F

CBH-2

HC84.22

AR3C

AR3A

AR3B

HC84.26

AR3D

Rel

ativ

eIn

fect

ion

Rel

ativ

eIn

fect

ion

Rel

ativ

eIn

fect

ion

Rel

ativ

eIn

fect

ion

Rel

ativ

eIn

fect

ion

Rel

ativ

eIn

fect

ion

Rel

ativ

eIn

fect

ion

Supplementary Figure 6. Back-mutation of resistance-associated polymorphisms in E1E2 clones where they appear naturally. Each bar indicates relative infection of the indicated HCVpp in the presence of the indicated mAb. 1b21 is an E1E2 clone containing naturally occurring N430D and D431E. The dashed blue line indicates relative infection of pp1b21 prior to introduction of D430N, E431D, or both mutations. 1a142 is an E1E2 clone containing naturally occurring L438I, F442I, and K446E. The dashed red line indicates relative infection of pp1a142 prior to introduction of I438L, I442F, E446K, or I438L/I442F. Error bars indicate standard deviation between duplicate wells.

Supplementary Figure 6

0.01

0.1

1

10

0.01 0.1 1 10

0.01

0.1

1

0.01

0.1

1

>10pp1a31 wildtype (D431E)7.04pp1b09/D431E4.13pp1b21 wildtype (D431E)

pp1b09 wildtype .93

4.05pp1b09/D431Epp1b09 wildtype 2.07

1.62pp1b21 wildtype (D431E)1.00pp1a31 wildtype (D431E)

8.02pp1b09/D431E3.56pp1b21 wildtype (D431E)

pp1b09 wildtype .41.27pp1a31 wildtype (D431E)

IC50

IC50

IC50

AR3B

HC84.22

CBH-2

Supplementary Figure 7

b

c

a

Rel

ativ

e In

fect

ion

[mAb] (µg/mL)

Rel

ativ

e In

fect

ion

Rel

ativ

e In

fect

ion

Supplementary Figure 7. Resistance conferred by D431E is E1E2 context-specific. IC50’s were measured against HCVpp using two E1E2 clones with naturally-occurring D431E (1b21 and 1a31), a naturally occurring neutralization sensitive E1E2 clone (1b09), and 1b09 with D431E introduced by site-directed mutagenesis (1b09/D431E). (a) Mutation of D431 to E confers CBH-2 resistance to clone 1b09, and all E1E2 variants with D431E were CBH-2 resistant. (b) Mutation of D431 to E confers AR3B resistance to clone 1b09, and the 1b21 clone with naturally occurring D431E is resistant to AR3B as well. Clone 1a31, which also contains a naturally occurring D431E, is not resistant to AR3B. (c) Mutation of D431 to E also confers HC84.22 resistance to clone 1b09. However, clones 1a31 and 1b21, which also carry D431E, are not resistant to HC84.22. Dashed lines indicate 50% neutralization. Error bars indicate standard deviation between duplicate wells.

0

0.1

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0.3

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0.5

0.6

0.7

0.8

0.9

0 0.2 0.4 0.6 0.8 1 1.2

Rel

ativ

e In

fect

ion

AR

3A

Relative InfectionHC84.22

pp1a142(F442I)

pp1a129pp1b21(D431E)

pp1b09

pp1b09/D431E

pp1b09/F442I

Supplementary Figure 8

1

2

pp1a80(F442I)

Supplementary Figure 8. D431E and F442I polymorphisms are present in outliers on the neutralization correlation plot between mAbs HC84.22 and AR3A. Each diamond or colored circle indicates the neutralization of a single HCVpp by mAb HC84.22 on the x-axis and mAb AR3A on the y-axis. D431E confers relatively greater resistance to AR3A, while F442I confers relatively greater resistance to HC84.22. Solid purple circles indicate clones with naturally occurring D431E polymorphisms. Solid red circles indicate clones with naturally occurring F442I polymorphisms. Open purple and red diamonds indicate clone 1b09 with D431E or F442I, respectively, introduced by site directed mutagenesis. Two clones (1a142 and 1a80) with greater resistance to HC84.22 than to AR3A carry naturally-occurring F442I polymorphisms. A clone (1b21) with greater resistance to AR3A than to HC84.22 carries a naturally-occurring D431E polymorphism. Two other clones with naturally-occurring D431E (numbered “1” and “2”) are relatively sensitive to both AR3A and HC84.22. The E1E2 clone from the panel with highest combined resistance to both AR3A and HC84.22 (1a129) does not carry either D431E or F442I. E1E2 chimeras used in Figures 5 and 6 were generated between the circled clones on this plot.

1E+04

1E+05

1E+06

1E+07

1E+08

moc

kpp

1b09

N43

0DD

431E

L433

IL4

38I

L438

VF4

42I

K44

6EP

453S

A47

5TE

531A

F560

YL4

38I,F

442I

LI/F

I/A47

5TLI

/FI/P

453S

/AT

HC

Vpp

entr

y (R

LU)

HCVpp

B

1E+04

1E+05

1E+06

1E+07

1E+08

moc

k1a

091a

311a

381a

531a

721a

801a

116*

1a12

31a

129

1a14

21a

154

1a15

71b

091b

141b

21*

1b34

1b38

1b52

1b58H

CVp

p en

try

(RLU

)

HCVpp

A

Supplementary Figure 9

Supplementary Figure 9. Additional polymorphisms can compensate for fitness cost of resistance polymorphisms. (A) The effect of site-directed mutations on 1b09 E1E2 fitness (ability to mediate HCVpp entry). RLU are relative light units, indicating productive entry of HCVpp into target cells. Values are means of two independent experiments performed in duplicate. Error bars indicate standard deviations. The dotted line indicates entry of HCVpp with wildtype 1b09 E1E2. (B) HCVpp entry mediated by naturally-occurring E1E2 clones in the HCVpp panel. Mock pseudoparticles with no E1E2 were used as a negative control. Solid triangles indicate HCVpp with naturally-occurring D431E polymorphisms. Open triangles indicate HCVpp with naturally-occurring F442I or F442L. Gray triangles indicated HCVpp with naturally-occurring F560Y. Values are the means of four to eleven independent experiments performed in duplicate. Error bars indicated standard deviations. The dotted line indicates median entry of all 19 HCVpp. HCVpp indicated with asterisks were never frozen prior to testing. The remaining HCVpp were freeze-thawed once prior to testing.

HC

Vpp

entr

y (R

LU)

pp1b09I538VQ546LT563V

pp1b09 pp1a129 pp1a129V538IL546QV563T

Supplementary Figure 10

1

10

100

1000

10000

100000

1000000

10000000

100000000 * *NS

Supplementary Figure 10. Fitness cost of I538V/Q546L/T563V polymorphisms is compensated in a resistant E1E2 clone where it arises naturally. Each bar represents entry by HCVpp with the indicated E1E2s. Values are means of two to six independent experiments performed in duplicate. RLU is relative light units. Error bars are standard deviations. An asterisk indicates p<.05 by T test. NS indicates p value not significant. Introduction of I538V/Q546L/T563V reduces fitness of clone 1b09, but clone 1a129, with naturally-occurring I538V/Q546L/T563V, does not exhibit reduced fitness, and reversion of these polymorphisms in clone 1a129 also reduces fitness. Therefore, fitness cost of these polymorphisms is E1E2 context dependent.

....|....|....|....|....|....|....|....|....1a31 ..N..A.D...FV.......L.............Y..V..A...1a53 A.N....D...FV.......L................A..A...1b09 TYTWGENETDVLILNNTRPPQGNWFGCTWMNSTGFTKTCGGPPC1b38 ..S.........L.......R..........G............1a154 ..S..A.D...FV.......L................V..A...1b34 ..N.........L.......L...........S...........1a157 ..N..A.D...FV.......L................V..A...1a72 ..S........FV.......L................V..A...1a123 ...........FV.......L...........S....V..A...1a80 ..N....D...FV.......L................V..A...1a09 ..N....D....V.......L................V..A...1a116 ..N..DSD...FV.......L...........S....V..A...1b14 ............L..................G............1a142 ..G....D...FV.......L.............Y..V..A...1b52 ............L..................G............1a38 ..S....D...FV.......L................V..A...1b21 ..S.........L...............................1b58 ..S.........L...............................1a129 ..S....D...FV.......L................V..A...

Supplementary Figure 11

526 569

Supplementary Figure 11. Neutralization resistance conferred by I538V/Q546L/T563V is E1E2 context dependent. Sequence alignment of amino acids 526-569 for all E1E2 clones in the HCVpp panel. Clones are listed by increasing resistance to mAb AR3A. Boxes indicate residues 538, 546, and 563. Dots indicate homology to sensitive clone 1b09 at that site. Arrows indicate genotype 1b clones. The I538V/Q546L/T563V combination of polymorphisms is common in genotype 1a isolates in the E1E2 panel and not independently associated with NC1 mAb resistance.