Impairment of Hepatitis B Virus Virion Secretion by Single-Amino-Acid Substitutions in the Small Envelope Protein and Rescue by a Novel Glycosylation Site

December 2, 2010

http://jvi.asm.org/cgi/content/abstract/84/24/12850

Journal of Virology, December 2010, p. 12850-12861, Vol. 84, No. 24

doi:10.1128/JVI.01499-10

, American Society for Microbiology. .

Impairment of Hepatitis B Virus Virion Secretion by Single-Amino-Acid

Substitutions in the Small Envelope Protein and Rescue by a Novel Glycosylation

Site

Kiyoaki Ito,1, Yanli Qin ,1,, Guarnieri,1,,¶ Tamako ,1,||

Kwei,1,# Masashi Mizokami,2 Jiming Zhang,3 Jisu Li,1 Jack R. Wands,1 and Shuping

Tong1*

Liver Research Center, Rhode Island Hospital, Warren Alpert School of Medicine,

Brown University, Providence, Rhode Island,1 Research Center for Hepatitis and

Immunology, National Center for Global Health and Medicine, Ichikawa, Japan,2

Department of Infectious Diseases, Huashan Hospital, Shanghai Medical College,

Fudan University, Shanghai, China3

Received 19 July 2010/ Accepted 21 September 2010

Mutations in the S region of the hepatitis B virus (HBV) envelope gene are

associated with immune escape, occult infection, and resistance to therapy. We

previously identified naturally occurring mutations in the S gene that alter HBV

virion secretion. Here we used transcomplementation assay to confirm that the

I110M, G119E, and R169P mutations in the S domain of viral envelope proteins

impair virion secretion and that an M133T mutation rescues virion secretion of

the I110M and G119E mutants. The G119E mutation impaired detection of secreted

hepatitis B surface antigen (HBsAg), suggesting immune escape. The R169P mutant

protein is defective in HBsAg secretion as well and has a dominant negative

effect when it is coexpressed with wild-type envelope proteins. Although the S

domain is present in all three envelope proteins, the I110M, G119E, and R169P

mutations impair virion secretion through the small envelope protein.

Conversely, coexpression of just the small envelope protein of the M133T mutant

could rescue virion secretion. The M133T mutation could also overcome the

secretion defect caused by the G145R immune-escape mutation or mutation at N146,

the site of N-linked glycosylation. In fact, the M133T mutation creates a novel

N-linked glycosylation site (131NST133). Destroying this site by N131Q/T

mutation or preventing glycosylation by tunicamycin treatment of transfected

cells abrogated the effect of the M133T mutation. Our findings demonstrate that

N-linked glycosylation of HBV envelope proteins is critical for virion secretion

and that the secretion defect caused by mutations in the S protein can be

rescued by an extra glycosylation site.

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* Corresponding author. Mailing address: Liver Research Center, 4th Floor, 55

Claverick Street, Providence, RI 02906. Phone: (401) 444-7365. Fax: (401)

444-2939. E-mail: Shuping_Tong_MD@...

Published ahead of print on 29 September 2010.

Present address: Research Center for Hepatitis and Immunology, National Center

for Global Health and Medicine, Ichikawa, Japan.

These authors contributed equally to this work.

Present address: Department of Infectious Diseases, Huashan Hospital, Shanghai

Medical College, Fudan University, Shanghai, China.

¶ Present address: University of Colorado at Denver, Denver, CO.

|| Present address: Uniformed Services University of the Health Sciences,

Washington, DC.

# Present address: Boston University School of Medicine, Boston, MA.

December 2, 2010