Abstract The major homology region (MHR) is a highly conserved sequence in the Gag gene of all retroviruses, including HIV-1. Its role in assembly is unknown,

AbstractThe major homology region (MHR) is a highly conserved sequence in the Gag gene of all retroviruses, including HIV-1. Its role in assembly is unknown, but deletion of the motif significantly impairs membrane binding and viral particle formation. Preliminary studies in transfected cells indicated that GagΔMHR was defective in formation of an RNA-containing,membrane-bound replication intermediate. To determine the role of the MHR in formation of this complex, fluorescence-based binding studies were conducted in vitro, using tRNA and large unilamellar vesicles of 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS-LUVs) as model membranes. As previously reported, GagΔMHR bound to lipid bilayers with slightly reduced affinity compared to wild-type Gag (GagWT) and no differences in tRNA binding between GagWT and GagΔMHR were detected. From our studies, we were able to determine the change in free energy (ΔG) for all of these associations and find that, the energies for membrane binding and for tRNA binding are within room temperature indicating that in vitro Gag will not preferenitally bind to either substrate. However, we speculate that, due to proximity, in vivo RNA binding would be preferred. Interestingly, GagWT showed a strong tendency to self-associate on both tRNA and membranes whereas self-association of GagΔMHR on tRNA was not detectable. These results suggest that, during infection, the MHR plays a key role in promoting productive protein-protein interactions on RNA.

• What?– Determine if the interaction between HIV-1 Gag and

RNA is a prerequisite for membrane binding .– Determine the assembly event sequence of HIV-1 viral

particles.– Gain an insight into the role of the MHR in this

assembly.• Why?

– The steps that precede the formation of viral budding structures at the plasma membrane are not well defined.

– MHR is highly conserved throughout all retroviruses. – The MHR’s function is unknown.

• How?– Experiments designed to determine the change in free

energy for GagWT and GagMHR.

Introduction

Figure 1: Sucrose Gradient Method and

Rationale

Figure 1: Sucrose Gradient Method and

Rationale

Gag proteins were expressed in

mammalian cells

20% 60%

20%

60%

Cell lysates were subjected to sucrose

density gradient centrifugation

Fractions were separated by gel electrophoresis

Figure 2: Event Sequence Determination for Particle Formation

(G = -RT ln(K) )

Figure 2: Event Sequence Determination for Particle Formation

(G = -RT ln(K) )

G1+G2+G3 = G4+G5+G6G1+G2+G3 = G4+G5+G6

Gag (mono) + Membrane Gag (mono) + RNA

Gag (mono)* Membrane Gag (mono)* RNA

Gag (associated)* Membrane Gag (associated)* RNA

RNA *Gag * Membrane Membrane *Gag *RNA

Intrinsic Fluorescence


Fluorescein Homotransfer

Fluorescein Homotransfer





Fluorescein HomotransferFluorescein

Homotransfer



G Diagram

G Diagram

Methods

Methods

G1G1

G2G2

G3G3

G4G4

G5G5

G6G6

Binding

Assembly

Binding

Figure 6: Binding of Gag to POPS and tRNA

Figure 6: Binding of Gag to POPS and tRNA

Intrinsic Fluorescence - The substrate (POPS or tRNA) was titrated into 200nM protein in buffer (0.5M

NaCl, 40mM HEPES, 1mM DTT), 83M POPS or 33M tRNA . Intrinsic fluorescence was followed by ex. 280nm, em. 320-400nm.

Gag Binding to POPS

0

0.2

0.4

0.6

0.8

1

1.2

0 20 40 60 80 100 120

[POPS uM]

No

rmal

ized

FI

WT Kd= 6.0 +/- 1.5

dMHR Kd=11.01 +/- 2.11

Gag Binding to tRNA

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20 25 30 35 40 45

[tRNA uM]

No

rmal

ized

FI

WT Kd=3.99 +/- 0.32

dMHR Kd=4.51+/- 0.58

Figure 7: Monitoring Gag Assembly on POPS and tRNA by Homotransfer

Figure 7: Monitoring Gag Assembly on POPS and tRNA by Homotransfer

Homotransfer - Fluorescein labeled protein was titrated into 83M POPS, 33M tRNA, or buffer. Energy homotransfer was followed by anisotropy ex. 490nm, em. 516nm.

WT Gag Association vs. Aggregation

0

0.2

0.4

0.6

0.8

1

0 50 100 150 200

[WT Gag nM]

No

rmal

ized

An

iso

tro

py

WT associating on POPS Kd=26.23

WT aggregating in solution Kd=13.86

WT associating on tRNA Kd=15.85

dMHR Gag Association vs. Aggregation

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 20 40 60 80 100 120 140 160 180 200[dMHR Gag nM]

No

rma

lize

d A

nis

otr

op

y

dMHR aggregating in solution Kd=14.08dMHR associating on tRNAdMHR associating on POPS Kd=61.75

Figure 8: Gag Complex Binding to Second Substrate

Figure 8: Gag Complex Binding to Second Substrate

Intrinsic Fluorescence - The substrate (POPS or tRNA) was titrated into 200nM protein in buffer (0.5M

NaCl, 40mM HEPES, 1mM DTT), 83M POPS or 33M tRNA . Intrinsic fluorescence was followed by ex. 280nm, em. 320-400nm.

Gag/POPS Complex Binding to tRNA

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20 25 30 35 40 45

[tRNA uM]

No

rmal

ized

FI

WT Kd=3.37 +/- 1.19dMHR Kd=6.28 +/- 0.29

Gag/tRNA Complex Binding to POPS

0

0.2

0.4

0.6

0.8

1

1.2

0 20 40 60 80 100 120

[POPS uM]

No

rmal

ized

FI

WT Kd=5.13 +/- 4.95

dMHR Kd=21.3 +/- 9.5

Figure 9: GagWT G Diagram for Particle Formation

Figure 9: GagWT G Diagram for Particle Formation

-3763 cal/mol*K -3469 cal/mol*K-3763 cal/mol*K -3469 cal/mol*K


Binding


Assembly



G = -1075 cal/mol*KG = -1075 cal/mol*K





G = -981 cal/mol*KG = -981 cal/mol*KBinding

Figure 10: GagMHR G Diagram for Particle

Formation

Figure 10: GagMHR G Diagram for Particle

Formation

-5014 cal/mol*K -2739 cal/mol*K +N/A-5014 cal/mol*K -2739 cal/mol*K +N/A





G = N/AG = N/A






Binding

Assembly

Binding

Conclusions

• Gag binds initially to RNA, and this complex facilitates efficient membrane binding.

• The MHR plays a critical role in Gag-Gag interactions which enables the RNA/Gag complex to present a membrane binding face.

GagWT Particle Formation Model

GagWT binds to RNA

GagWT/RNA complex binds with higher

affinity to membrane

GagWT associates on RNA

Legend GagWT Trimer

MACANC

RNA

Membrane

GagMHR Particle Formation Model

GagMHR binds to RNA

GagMHR/RNA complex binds with less

affinity to membrane

GagMHR does not associate on RNA

Legend GagMHR Trimer

MACANC

RNA

Membrane

Acknowledgements

• We would like to thank Indralatha Jayatilaka, Fadila Bouamr, Lynn VerPlank, Louisa Dowal and Marjorie Bon Homme for technical assistance.

• This work was made possible by funding through the National Institutes of Health (NIH 53132)

1 Berthet-Colominas, C., et al., Head-to-tail dimers and interdomain flexibility revealed by the crystal structure of HIV-1 capsid protein (p24) complexed with a monoclonal antibody Fab. Embo J, 1999. 18(5): p. 1124-36.

2 Ebbets-Reed, D., S. Scarlata, and C.A. Carter, The major homology region of the HIV-1 gag precursor influences membrane affinity. Biochemistry, 1996. 35(45): p. 14268-75.

3 Lee, Y. M., B. Liu, and X. F. U. 1999. Formation of virus assembly intermediate complexes in the cytoplasm by wild-type and assembly defective mutant human immunodeficiency virus type 1 and their association with membranes. J. Virol. 73:5654-5662.

Figure 4: Intermediate Complexes Contain RNA and are Membrane

Bound

Figure 4: Intermediate Complexes Contain RNA and are Membrane

Bound

Cytoplasmic extracts were prepared from cells expressing Pr55Gag and treated with

Panel A: untreated

Panel B: RNase

Panel C: EDTA

Panel D: 1% nonionic detergent

Gradient fractions were subjected to density gradient centrifugation and analyzed by polyacrylamide gel electrophoresis and immunoblotting using an anti p6 antibody

(IGEPAL)

Figure 5: Formation of Assembly Intermediates Require the MHR

Figure 5: Formation of Assembly Intermediates Require the MHR

Cytoplasmic extracts were prepared from transfected cells containingPanel A: Pr55Gag

Panel B: Pr55Gag-MHR

Panel C: Pr55Gag-Myr

Supernatants were subjected to density gradient centrifugation and analyzed by polyacrylamide gel electrophoresis and immunoblotting using an anti CA antibody.

The graph above each gradient indicates the density in g/ml of each fraction.

Documents

Abstract The major homology region (MHR) is a highly conserved sequence in the Gag gene of all retroviruses, including HIV-1. Its role in assembly is unknown,