A3H II EXPRESSED IN SF9 CELLS

GST affinity purification

Optimization of the expression and purification of APOBEC3H Maria Klarizza Panaligan, Karen Siu, Jeffrey E. Lee

Department of Laboratory Medicine & Pathobiology, University of Toronto

Introduction A3H proteins are restriction factors that contribute to the innate immune response against retroviruses, such as the human immunodeficiency virus type 1 (HIV-1).

FUNCTION• DNA cytidine deaminase that generate C-to-U mutations on the viral (–) ssDNA strand

RationaleThe structure of A3H has not yet been solved, and little is knownof its haplotypes (II and VII) that are active against HIV-1. In our study, we focused on isolating sufficient amounts of stable A3H that will be useful for future downstream experiments.

Methods & ResultsPROTEIN EXPRESSION AND PURIFICATION• A3H forms inclusion bodies when expressed from E.coli• Recombinant protein expression in Sf9 and S2 insect cells

• After PEI precipitation,nanodrop 260/280 readings dropped from ~1.51 to ~0.5

Discussion

Target Cell

CA3H

C UA

reverse transcripted

(-) strand ssDNA

C-to-U deaminationby A3H

Deaminated(-) ssDNA

(+) ssDNA(G-to-A)

ANTAGONIST• Antagonized by an HIV-1 protein called Vif

A3H +Vif

• A3H can be expressed in S2 cells, but in lower amounts compared to Sf9 cells• PEI precipitation is an efficient method for removing nucleic acid contaminants from A3H• A3H becomes “sticky” and is difficult to elute fromthe resin after removal of nucleic acids

Future Directions

• A260/280 readings indicate nucleic acid contamination in purified A3H II and VII from both Sf9 and S2 cells. • Needed to remove nucleic acids in order to study A3H

• Intracellular expression of A3H confirmed in S2 cells

(+) (+) media

A3H II and VII from S2 cellswestern blot

S2 cellmedia

western blot

hapII

hapVII

ANTIVIRAL ACTIVITY

infected cell

healthycell

• Hypermutates viral DNA, resulting in a genome that can nolonger be used for viral replication in the next host cell.

unsuccesfulinfection

Human cells have developed multiple mechanisms to inhibit viral replication, and viruses in turn have evolved diverse strategies to antagonize these efforts. Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins are a key factor in an intracellular defense strategy for inhibition of replication of HIV. While seven haplotypes of APOBEC3H are present in humans, only haplotypes II, V and VII are active against HIV-1. These haplotypes are packaged into newly assembled virions and induce C-to-U mutations on the viral minus-strand DNA, which results in a hypermutated genome that would be unable to infect the next target cell. HIV-1 disrupts this host defense mechanism by encoding a viral infectivity factor (Vif) that effectively targets APOBEC3 proteins for E3 ubiquitin ligase-mediated proteasomal degradation. Stable APOBEC3H haplotypes have been shown to show some catalytic activity even in the presence of Vif. The study of the HIV-1 Vif/APOBEC3 axis represents an attractive target for HIV/AIDS research. Biochemical and high-resolution structural analysis of the interplay between HIV-1 Vif, APOBEC3s and other cellular factors provide an integrated approach to understand their function and to provide a master blueprint to design new classes of HIV drugs that unleash the potent anti-viral activity of host innate immune factors for AIDS prevention/therapy. Here, we present our preliminary work towards expressing and purifying various full-length APOBEC3H haplotypes for future functional and structural studies.

HIV-1 Vifclose-updiagram

Abstract

Method A260/280 for A3H II

High salt washes

Benzonase + RNase A treatment

PEI precipitation 0.5

1.52

1M NaCl

1M LiCl

2M NaBr

1.471.591.46

haplotype# 15 18 105 121 178IIIIIIIVVVIVII

N R G K EN R R D D R R D D L R D DN R R D E L G K DN R R K E

anti-HIV

activity

SF9/BACULOVIRUS DROSOPHILA S2Method used virus infection transfectionViability declines after

infectionconstantly

high

Cell line

GST-affinity purification

GS

T

GS

H

A3H

GS

T

A3H

GS

H

GS

HG

ST

+thrombin

A3H

• A3H II and VII expressed in and purified from Sf9 cells

GST tag (~26kDa)

A3H+GST tag (~48kDa)

REMOVING CONTAMINATING NUCLEIC ACID FROM A3HII

4.) Nucleic acid content was monitored through 260/280 NanoDrop 2000 readings

PEI

A3H

A3H

A3H

PEI

PEIA3H

A3H

A3H

PEI

A3H

A3HA3H

PEIA3H

A3H

A3H

+PEI+ non-saturating

ammoniumsulfate

rock 4C for

20minand spin

resuspend pellet for dialysis and

GSTPURIFICATION

PROTOCOL

PEIPEI

PEI

+ saturated ammonium

sulfateand spin

PEI

PEI

1.) High salt washes during GST-affinity purification. 2.) Benzonase and RNase A treatment3.) PEI precipitation

A3H+GST tag

A3H

PEI

1A1 1A2 1A3

Ion Exchange ChromatographyGST affinity purification

coomasie gel

A3HIIlysate

A3HIIbeads

A3HVIIlysate

A3HVIIbeads

MW(kDa)

MW(kDa)

1A1 1A2 1A3

1A1 1A2 1A3

A3H VIIMono S runA3H IIMono S run

A3H IIsize column run

Size Exclusion Chromatography

1A6-1A10

MW 1A6 1A7 1A8 1A9 1A10

1A1-1A8

1A1-1A8

MW(kDa)

A3H VIIsize column run

1A1 1A2 1A3

BIOCHEMICAL PROFILE

• 7 haplotypes: I II III IV V VI VII

UbUb

proteasomaldegradation

degradedA3H

A3H

Ub

VifEloB

EloC

CUL5RBX2 E2 Ub ACKNOWLEDGEMENTS:

Prof. Linda Chelico,University of Saskatchewan

coomasiegel

coomasiegel

coomasiegel coomasie

gel

thrombin-cleaved

A3H

1) Search for additives or detergents that help stabilize A3H after nucleic acid removal.2) If insect cell expression remains unpromising, move on to expressing in mammalian cells2) Study the binding of A3H with ssDNA substrate3) Study the binding of A3H with its viral antagonist, Vif4) Understand the structure of A3H through X-ray crystallography and other biophysical methods

coomasiegel

45

2535

20

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45

2535

20

45

2535

20

MW(kDa)

45

2535

20

45

2535

20

45

25

35

20

45

2535

20

(kDa)

MW(kDa)