Page 15 - O. Microbiology

The role of palmitoylation of tegument protein pp28

during human cytomegalovirus infection

Hyejin Jeon and Jun-Young Seo
Jae Bong Lee, and Jun-Young Seo

Severance Biomedical Science Institute, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea.

Abstract

Human cytomegalovirus (HCMV) tegument protein pp28 is essential for the final envelopment during viral maturation. This protein undergoes lipidation modifications such as

myristoylation and palmitoylation, which affect the activity and subcellular targeting of proteins. The pp28 protein localizes to the assembly compartment (AC) late in infection and its

localization depends on myristoylation. Myristoylation of pp28 is also necessary for the production of infectious virions. Meanwhile, plamitoylation of pp28 remains poorly understood.

Here, we show that pp28 is palmitoylated at cysteine residues (aa 6, 10, 11). Inhibition of palmitoylation by site-directed mutagenesis or palmitoylation inhibitor 2-bromopalmitate

treatment disrupts the subcellular localization of pp28 and also reduces the stability of the protein in transiently transfected cells. The recombinant HCMV producing pp28 mutant in which

three cysteine residues (aa 6, 10, 11) are substituted with alanine residues, exhibits delayed viral growth kinetics and decreases viral yield, although the recombinant virus is able to produce

infectious virions. Our data indicate that palmityolation at the N-terminal cysteine residues of pp28 plays a critical role in its localization and stability for efficient viral replication.

Introduction (A) 6 *** (B) DMSO MG132 5μM DMSO MG132 5μM

pp28-WT + DW pp28-WT + DMSO pp28-WT + MG132 10μM pp28-WT + CQ 40μM pp28-WT + CQ 100μM pp28-WT + CQ 100μM pp28-TCA+ DW pp28-TCA+ DMSO pp28-TCA + MG132 1μM pp28-TCA + MG132 5μM pp28-TCA + MG132 10μM pp28-TCA + CQ 40μM pp28-TCA + CQ 100μM pp28-TCA + CQ 200μM Fold change 4 WT-Myc TCA-Myc G2A-Myc WT-Myc TCA-Myc G2A-Myc WT-Myc TCA-Myc G2A-Myc WT-Myc TCA-Myc G2A-Myc

2

0 pp28 pp28 pp28 pp28 pp28 pp28 pp28 pp28 pp28 pp28 pp28 pp28
WT -> WT+MG 132 WB: α-pp28 WB: α-pp28
WB : α -Grp94

WB : α-pp28 TCA -> TCA+MG132 Palmitoylated pp28 Input pp28

Figure 3. Palmityolation of pp28 prevents proteasomal degradation.

(A) 293T cells were transfected and treated with proteasome inhibitor MG-132(1-10uM) or autophagosome inhibitor

chloroquine (CQ) (40-200uM) for 24h and level of expresstion was detected using western blotting. Grp94 was used as

a protein loading control.
Buchkovich, N. J et al. Journal of Virology (2010)

(B) 293T cells were transfected and treated with proteasome inhibitor MG-132(5uM). Palmitoylated pp28 in 293 cells

was identified using CAPTUREome™ S-Palmitoylated Protein Kit.

(A) (B) (C)

WT AD169

Palmitoylation TCA AD169

ㆍ Identification

WB: α-Grp94
Covalent modification of proteins with palmitoyl groups

ㆍ Types of palmitoylation WB: α-pp65

S-palmitoylation : Covalent attachment to cysteines

WB: α-pp28
O-plamityolation : Covalent attachment to serine and threonine

ㆍ Function

Play a significant role in subcellular trafficking of proteins and protein stability

Figure 4. Palmitoylation of pp28 regulates its subcellular localization and protein stability during HCMV infection
Several viruses exploit extensively the host palmitoylation machinery to to modify their proteins

(A) A schematic diagram of BAC (bacterial artificial chromosome) mutagenesis and transfection using electroporation method.
essential for infection

(B) HFF cells were infected with HCMV at MOI of 0.1 for 5 days. Cells were lysed and immunoblots were performed with the

indicated antibodies. Grp94 was used as a protein loading control.
Methods and Results (C) HFF cells were infected with HCMV at MOI of 1 for 5 days. Cells were fixed and stained with antibodies against pp65,

(A) (B) pp28 wild type pp28 and DAPI. Note that pp28 TCA mutants localize to the cytoplasm not viral assembly compartment. Scale bars, 10um.
Self interaction
Myristoylation
G Acidic cluster
1aa 2 30 44 50 59 190aa (A) (B)

ERGIC localization AC localization/assembly Intracellular virus growth curve Extracellular virus growth curve
pp28 WT + ERGIC DAPI Merge
DMSO 6 6
Number of infectious virions
WT WT

5 5
pp28 cysteine mutants : Deleted amino acids : Cysteine -> Alanine TCA TCA

(Log scale) 3 Number of infectious virions (Log scale) 3

pp28 WT + 2BP ERGIC DAPI Merge TCA(6, 10-11) Myc His 4 4

6 1011

(C) WT TCA 2 2
1
1
pp28 pp28

0 0

0 2 4 6 8 10 0 2 4 6 8 10
WB: α-pp28 Palmitoylated pp28
Days post infection Days post infection

Input pp28
WB: α-pp28

Figure 1. pp28 is palmitoylated at three cysteine residues 6, 10, 11 of the N-terminal domain. Figure 5. Growth kinetics of recombinant HCMV.

(A) Single-step growth kinetics assays of wild-type HCMV AD169, Triple cysteine to alanine mutants viruses were performed
(A) HeLa cells were transfected and treated with palmitoylation inhibitor 2BP (10 μM) for 24 h or DMSO as a control. The

by infecting HFFs at an MOI of 0.05. Virus titer was quantified by a fluorescence-based virus infectivity assay. Infected cells
subcellular localization of pp28 was detected using immunofluorescence assay. Scale bar, 10 μm

were harvested, scraped into media, at different time points post infection as indicated. Samples were snap frozen at −80°C to
(B) Illustration depicting the pp28 mutants used in this study.

lyse the cells.
(C) Palmitoylated pp28 in 293 cells was identified using the commercially available CAPTUREome™ S-Palmitoylated

(B) supernatants of infected cell cultures were harvested at different time points post infection as indicated
Protein Kit. 293T cells were transiently transfected with plasimds expressing the indicated proteins. The methodology for

Conclusions
acyl-RAC, including blocking of free thiols, cleavage of thioester linkages, and capture of nascent thiols on Sepharose, was

carried out according to the manufacturer’s instructions.

1. pp28 is palmitoylated at the N-terminal cysteine residues (6, 10 and 11)

(A) (B) 2. Palmitoylation of pp28 regulates its subcellular localization and protein stability.

3. Palmityolation of pp28 prevents proteasomal degradation.

pp28 WT ERGIC DAPI Merge 4. The recombinant HCMV producing pp28 mutant in which three cysteine residues (aa 6, 10, 11) are substituted

WT TCA with alanine residues, exhibits delayed viral growth kinetics and decreases viral yield

pp28 pp28 5. Our data indicate that palmityolation at the N-terminal cysteine residues (aa 6, 10, 11) of pp28 plays a critical role

WB: α-Grp94 in its localization and stability for efficient viral replication.
pp28 TCA ERGIC DAPI Merge

WB: α-pp28
Acknowledgement

We would like to thank Dr. Jin-Hyun Ahn (Sungkunkwan University) for kindly providing reagents of BAC

mutagenesis

References

Figure 2. Palmitoylation of pp28 affects its subcellular localization and protein stability.

(A) Western blot analysis of 293T cells were transiently transfected with plasimds expressing the indicated proteins. 1. J. Y. Seo, W. J. Britt, Sequence requirements for localization of human cytomegalovirus tegument protein pp28 to the virus assembly compartment and for assembly of

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Scale bar, 10 μm 4. Sanchez, V., Sztul, E. & Britt, W. J. Human cytomegalovirus pp28 (UL99) localizes to a cytoplasmic compartment which overlaps the endoplasmic reticulum-golgi-

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