EnvCT 144

CT 124FS

CT 124

CT 118

CT 90

CT 66

CT 59CT 43

CT 28

WT

YW_SL

LL_RQ

705

WT 855

CT144

CT118

CT90CT66

CT59 CT43CT28

CT124

HIR LLP-1 ECLLP-3EC1 150

LLP-2

Selective Cell-Free or Cell-to-Cell HIV-1 Infection by gp41 Cytoplasmic Tail Mutants

Natasha D. Durham and Benjamin K. Chen

Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mt. Sinai, New York, NY 10029

AbstractHIV-1 gp41 CT Mutants

(a)

(b)

(a) Cell-free Infection (b) Cell-to-cell Infection

Infectivity Assays

The gp41 transmembrane subunit of the HIV-1 envelope (Env) has a cytoplasmic tail (CT) ~150 amino acids (aa) long. The CT has several functions during the viral life cycle, including the i) endocytosis of cell-surface Env ii) packaging Env into viral particles and iii) controlling the fusogenic potential of Env. Full truncation of the gp41 CT (∆CT144) does not directly impair viral fusion and can generate infectious virus in a cell-type dependent manner. Smaller deletions result in non-infectious virus particles, although Env is still expressed and packaged. Only the full truncation, ∆CT144, has been tested in the context of cell-to-cell infection. We therefore set out to systematically examine which domains of the CT are required for cell-to-cell infectivity in comparison to those needed for cell-free infection.

Virus Target Cell

MT-4 cell

INFECT

HIV+

VIROLOGICALSYNAPSE

Donor Cell Target Cell

HIV+ Jurkat

cell

MT-4 cell

HIV+

Single & multi-round infectivity assays: Cell-free vs. Cell-associated inocula

env

CT1

44

CT1

24FS

CT1

24

CT1

18

CT9

0

CT6

6

CT5

9

CT4

3

CT2

8

WT0

50

100

150

env

CT1

44

CT1

24FS

CT1

24

CT1

18

CT9

0

CT6

6

CT5

9

CT4

3

CT2

8

WT0

50

100

150

200

HIR LLP-1 ECLLP-3EC

1 150

LLP-2

705

WT 855

CT144

CT118

CT90CT66

CT59 CT43CT28

CT124

% In

fect

ion

% In

fect

ion

(a) 293T cell lysates

env

CT1

44C

T124

CT1

18

CT9

0

CT6

6C

T59

CT4

3

CT2

8

WT

1 2 3 4 5 6 7 8 9 10 11

unin

fect

ed

α-gp120

α-gp41

α-HIV

gp120

gp41

gp160

p24

(b) Virus particle lysates

env

CT1

44C

T124

CT1

18

CT9

0

CT6

6C

T59

CT4

3

CT2

8

WT

1 2 3 4 5 6 7 8 9 10 11

unin

fect

ed

α-gp120

α-HIV

gp120

gp41

gp160

p24

α-gp41

HIV-1 Envelope Expression

(c)

(d) Cell-surface envelope

Rel

ativ

e gp

120

leve

l

WT

CT1

44

CT1

24

CT1

18

CT9

0

CT6

6

CT5

9

CT4

3

CT2

80

1

2

3

4

gp120/p24 in 293T cell lysategp120/p24 in virus lysate

% o

f Max

.

HIV Env (APC)

WT ΔCT144 ΔCT124 ΔCT118 ΔCT90

ΔCT66 ΔCT59 ΔCT43 ΔCT28

(a)

% In

fect

ion

(e) Cell-free infection (f) Cell-to-cell infection

WT 792 KYWWN L LQYWSQEL 805 YW_SL - - - - - - - - S L - - - - LL_RQ - - - - - RQ - - - - - - -

% In

fect

ion

(b) 293T cell lysates (c) Virus particle lysates

∆env

WT

unin

fect

ed

α-gp120

α-gp41

α-HIV

YW

_SL

LL_R

Q

∆env

WT

1

unin

fect

ed

YW

_SL

LL_R

Q

α-gp120

α-gp41

α-HIV

2 3 4 51 2 3 4 5

WT

YW_S

L

LL_R

Q

env0

20

40

60

80

100

WT

YW_S

L

LL_R

Q

env0

20

40

60

80

100

WT

YW_S

L

LL_R

Q

0.0

0.5

1.0

1.5

gp120/p24 in 293T cell lysategp120/p24 in virus lysate

(d)

Rel

ativ

e gp

120

leve

l

gp120

gp41

gp160

p24

gp120

gp41

gp160

p24

HIR LLP-1 ECLLP-3EC1 150

LLP-2

LLP-3 Point Mutants

(a) Cell-to-cell transferWT ΔCT100 ΔCT28

Targ

et c

ells

HIV (mCherry)

(b) (d)

WT ΔCT100 ΔCT28

(c) Cell-to-cell viral membrane fusion

Unc

l. C

CF2

(CFP

)

Cleaved CCF2 (Pac. Blue)

% F

usio

n

% T

rans

fer

Cell-to-cell Transfer and Fusion assays ConclusionsSeveral gp41 CT mutants have striking selective deficiencies in cell-free but not cell-to-cell infectivity, or vice versa.

Functional properties of Env required for cell-free and cell-to-cell infection are genetically distinct.

Infectivity differences may reflect a different intrinsic regulatory influence of the CT on cell-associated vs. particle-associated Env, or differential interaction with host or viral proteins.

Define the block in cell-to-cell infection of large truncation muants.

Use these mutants to determine the contributions of one mode of infection in isolation.

Figure 1. Domains in the gp41 cytoplasmic tail (CT). (a) Schematic of the HIV-1 Env protein showing major structural and functional domains in gp120 and gp41. EC, endocytosis signal; HIR, highly immunogenic region; LLP, lentiviral lytic peptide. Adapted from Checkly et al., 2011. (b) gp41 CT mutants constructed by overlap PCR. Adapted from Jiang & Aiken, 2007.

Figure 2. HIV-1 envelope expression by gp41 CT truncation mutants. (a & b) Western blot of (a) 293T cell lysates or (b) virus particle lysates of WT NL-GI or gp41 CT mutants. (c) Quantification of gp120 levels expressed in 293T lysates and virus particle lysates. Env expression was calculated as a ratio of gp120/p24 and expressed relative to WT. (d) Cell surface HIV-1 Env on Jurkat cells expressing WT NL-GI or gp41 CT mutants (black line) compared to ∆env (filled grey). Env levels were detected on cells expressing high levels of HIV-1 infection with pooled polyclonal HIV-IG antibody.

Figure 3. Flow cytometry based assays used to measure HIV-1 infection. (a) In the cell-free infectivity assay, MT-4 target cells are incubated with NL-GI virus particles to allow infection. (b) In the cell-to-cell infectivity assay, Jurkat cells are nucleofected with NL-GI proviral DNA 24 h prior to the assay. Donor Jurkat cells and MT-4 target cells are labeled with Cell Proliferation dyes and co-cultured to allow infection. In single-round infection assays, cell culture media is replaced with media containing 10 µM AZT 18 h after co-culture. 40 h after initial infection, GFP expression levels are detected by flow cytometry, indicating HIV-1 infection.

Figure 6. HIV-1 envelope expression and single round infectivity of gp41 LLP-3 mutants YW_SL and LL_RQ in MT-4 cells. (a) Amino acid sequence alignment of gp41 LLP-3 mutants in relation to the full-length, wild type (WT) NL4-3 gp41 CT. Amino acid identity (-) and substitutions are shown.(b & c) Western blot of (b) 293T cell lysates or (c) virus particle lysates of WT NL-GI or gp41 LLP-3 mutants. (d) Quantification of gp120 levels expressed in 293T lysates and virus particle lysates. (e & f) Infection levels of MT-4 cells after (e) cell-free or (f) cell-to-cell infection by gp41 LLP-3 mutants, expressed as a percentage of WT NL-GI. Error bars represent the SEMs from the means of at least three independent experiments.

Figure 5. Cell-to-cell transfer and cell-to-cell viral membrane fusion of gp41 CT mutants into primary CD4+ T cells. (a) Representative flow cytometry plots and (b) cell-to-cell transfer levels of WT, ∆CT100, ∆CT28 or ∆env Gag-iCherry into primary CD4+ T cells. (c) Representative flow cytometry plots and (d) cell-to-cell viral membrane fusion levels of WT, ∆CT100, ∆CT28 or ∆env NL4-3 and BlaM-Vpr into primary CD4+ T cells. Error bars represent the SEMs from the means of two experiments performed in duplicate using CD4+ T cells from three blood donors.

WT

Bla

M-V

pr

CT1

00 B

laM

-Vpr

CT2

8 B

laM

-Vpr

env

Bla

M-V

pr

0

50

100

150

WT

Gag

-iChe

rry

CT1

00 G

ag-iC

herr

y

CT2

8 G

ag-iC

herr

y

env

Gag

-iChe

rry0

25

50

75

100

0 1 3 50.01

0.1

1

10

100

% G

FP P

ositi

ve

Days post infection

0 1 3 50.01

0.1

1

10

100

% G

FP P

ositi

ve

Days post infection

(c)

(d)

(e)

(f)

Figure 4. Infectivity of gp41 CT truncation mutants in MT-4 cells. (a & b) Representative flow cytometry plots of (a) cell-free or (b) cell-to-cell infection of MT-4 cells by WT, ∆CT118 or ∆CT28 NL-GI. (c & d) Single round infection levels after (c) cell-free or (d) cell-to-cell infection, expressed as a percentage of WT NL-GI. Error bars represent the SEMs from the means of at least three experiments. # indicates a single experiment performed in duplicate. (e & f) Multi-round infection of MT-4 cells initiated at Day 0 with (e) cell-free or (f) cell-associated WT, ∆CT118, ∆CT28 or ∆env NL-GI.

WT∆CT118∆CT28∆env

WT∆CT118∆CT28∆env

##

●

●

●

Future studies will:●

●

C2 C3 C4 C5

V1 V2 V3 V4 V5

Cytoplasmic Tail

TMD MPER

C1

Furin site

HR2HR1FP

gp120 gp41

29.7 16 17

11.4 14.6 19.5

This work was supported by grants from the NIH to BKC, NIAID AI074420/GM113885 and NIDA DP1DA028866-01.

(a) Cell-free Infection

(b) Cell-to-cell Infection

WT ΔCT118 ΔCT28

FL2-

H

HIV (GFP)

WT ΔCT118 ΔCT28

Targ

et c

ells

HIV (GFP)

0.5116.110.8

52.8 0.43 27.7

POSTER

220