我的學思歷程～從冠狀病毒到肝炎病毒～

賴明詔 Michael M.C. Lai

成功大學校長April 17, 2009

成功大學成立於 1931 於台南 （鄭成功 的故鄉 )

工學院，理學院，文學院， 電機資訊，規劃設計，管理學院，醫學院，社會科學，生物科技學院

學生 : 20538 名 (10462 名大學生， 6948 名碩士， 3128 名博士

教授 : 1789 名職員 : 3673 名 (1520 名於大學部， 2153 名於附設醫院 )

台灣第二大大學 企業界最愛

我的研究生涯大學時代 ( 台大 醫學院醫科 )研究生時代 ( 加大柏克萊 )大學教授時代 ( 南加大 )　　腫瘤病毒　　冠狀病毒　 肝炎病毒 (D and C 型）

回國 (2003) 後 中央研究院副院長 成功大學校長

病毒形狀

病毒形狀 (二 )

廿面體的基本構造

Triangulation numbers

5-, 3-, 2x symmetry

Quasisymmetry

Structure of Calicivirus

病 毒 的 基 因

☼ 人 類 細 胞 有 3

萬 個 基 因

☼ 細 菌 有 5 千 個 基 因

☼ 病 毒 有 10~100 個 基 因

The Central Dogma

DNA RNA Proteins

RNA

Retroviruses (RNA tumor viruses)(Viruses with reverse transcriptase)

Rous Sarcoma virus (Peter Duesberg, Harry Rubin) RNA, glycoproteins, phosphoproteins Oncogenes: src. Rel (NFkB), erb, etc. (Peter Vogt)

Demyelinating diseases

Multiple sclerosis: central nervous system demyelination (virus-induced autoimmune reaction) Theiler’s encephalomyelitis virus

(picornavirus) Measles virus Experimental allergic encephalitis (EAE) Mouse hepatitis virus (MHV): Coronavirus

Coronavirus （冠狀病毒 )

Multiple sclerosis 多發性神經硬化

Common colds 傷風感冒

SARS

Genome structure of Coronavirus:31-kb, single-strand, (+)-sense RNA

MHV

IBV

SARS

L 12 2-1 3 4

56 7

(Proteases and RNA polymerase)

HE S E M N

I

a

L 12 3 54 6

SE

M Nab

ab

L 12 3 54 6

SE

M N

Ib

a ba

HE S E M N(A)n 31 kb

POL

MHV Genomic and Subgenomic RNAs

1 2 3 4 5 6 7

(Leader

Intergenic Sequence

3'-UTR

RNA recombination

台灣 SARS 病例

肝 炎 病 毒

A 型 : 急性肝炎 Picornavirus

B 型 : 慢行肝炎 ( 肝癌 ) Hepadnavirus

C 型 : 慢性肝炎 ( 肝癌 ) Flavivirus

D 型 : 慢性肝炎 Deltaviridae

E 型 : 急性肝炎 Calicivirus

Hepatitis Delta Virus

RNA

Nucleocapsid(HDAg)

HBsAg

Majorprotein

Middleprotein

Largeprotein

Phospholipid

Challenges of Hepatitis Delta Virus

Viroid-like circular RNA with a protein

Make both full-length RNA replication and subgenomic mRNA transcription

Carry out RNA-dependent RNA transcription, but do not encode RNA-dependent polymerase: Cellular RdRP?

Hepatitis C virus

170 million carriers in the world (2% of population)

Tendency to cause persistent infection, leading to chronic hepatitis, liver cirrhosis, hepatoma

A flavivirus with single-stranded RNA

Hepatitis C virus

Relative risks of hepatocellular carcinomain non-Asians of Los Angeles County

HBV (-), HCV (-) 1.0

HBV (+) 5.1

HCV (+) 22.3

HCV (+), HBV (+) 56.2

(Adapted from Yuan et al. [1999])

HCV Genes and Gene products

Lindenbach et al, Nature 933, 2005

ER

Life cycle of HCV

Nature 436, 933-938 (18 August 2005 )

ER or Golgi ?

rER ?

Endosome

Receptor

Golgi ?

HCV Genome and HCV Replicon

R. Bartenschlager et al. / Antiviral Research 60 (2003) 91–102

Clinical manifestations of HCV

Liver: Asymptomatic or acute hepatitis chronic hepatitis liver cirrhosis hepatocellular carcinoma

B cells:Mixed cryoglobulinemia (B-cell oligoclonal proliferation) non-Hodgkin’s B-cell lymphoma

Autoimmune diseases:Sjogren’s syndromePseudomembranous glomerulonephritis

Purpura, Diabetes mellitus, thyroid disease

HCV

HCV: A non-retroviral oncogenic RNA virus

Frequent Mutations of p53(Teramoto et al., Cancer Res.)

B cell Lymphoma

Hepatocellular Carcinoma

Hypermutation of Ig

Ivanovski et al., Blood)

Chronic Infection

Liver Cirrhosis

LymphoproliferativeDiseases (mixed Cryoglobulinemia)

Establishment of a B-cell lymphoma cell line (SB cells)

• Established from the spleen of an HCV-positive patient with B-cell lymphoma and mixed cryoglobulinemia: proof that HCV infects B cells in vivo.

• Produces infectious virus particles that are capable of infecting B cells but not

hepatocytes.

• The immunoglobulin gene is monoclonal but undergoes continuous evolution

•

Molecular mechanism of viral lympho- vs hepatotropism

Harvest

Detection (qRT-PCR)

In vitro Transcribed RNA

Electroporation into Huh7.5 and Raji

SB virus RNA, but not JFH-1 Virus, Replicates in Raji Cells

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08

1.0E+09

4 8 12 16 20 24 28 42 56 80 96

Day

HC

V R

NA

(c

op

ies

/ug

RN

A) JFH-1

JFH-1 GND 1b SB SB GND

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08

1.0E+09

4 8 12 16 20 24 28 42 56 80 96

Day

HC

V R

NA

(co

pie

s/u

g R

NA

)

JFH-1

JFH-1 GND

1b

SB

SB GND

Raji

JFH-1 Virus, but not SB Virus RNA, Replicates in Huh7.5 Cells

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08

1.0E+09

4 8 12 16 20 24 28 42 56 80 96

Day

HC

V R

NA

(co

pie

s/u

g R

NA

) JFH-1 JFH-1-GND

1b

SB SB GND

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08

1.0E+09

4 8 12 16 20 24 28 42 56 80 96

Day

HC

V R

NA

(co

pie

s/u

g R

NA

) JFH-1 JFH-1-GND 1b SB SB GND

Huh7.5

•SB virus is lymphotropic.•JFH-1 virus is hepatotropic.

HCV induces a mutator phenotype

HCV infection causes a 5-10-fold increase in mutation frequency of cellular genes, including immunoglobulin (Ig) and somatic genes, e.g., p53 or -catenin genes.

Increased mutations are seen in in vitro HCV-infected B cells and in PBMC from HCV-infected individuals.

Mutations are amplified in the HCV-infected lymphoma and hepatoma.

Amplified oncogene mutations in HCV(+) B-cell tumors and hepatoma

B celllymphomas

Hepatocellularcarcinomas(HCC)

30 3.9

35 5.9

3.90

2.5

HCV-associatedNon-HCV

HCV tumor nontumor

HBV tumor nontumor

Nonviral tissues

Mutation frequency(mutation/bp) x10-4

p53 -catenin

33 0

30 11

7.4 7.4

4.4

MK 0 2 4 8 16 2 4 8 16 Day p.i.

Raji JT

DSBs

VH

HCV RNA

Mock HCV (+) HCV (-) DW

MK - HCV+ - HCV+

D

C

B

A

E

F

DSBs in VH

DSBs in p53

DSBs

872 603 234

872 603

234

Double-stranded DNA breaks in HCV-infected cells (by linker-ligation PCR)

Challenges of Hepatitis C Virus

VaccinesTherapy (interferon and ribavirin)Mechanism of HCV pathogenesis

(persistent infection) and oncogenesis The mechanism of immune escape The role of B and T cell infection

Challenges of Infectious Diseases

Antibiotics Small pox Polio

Hepatitis B virus Human papillomavirus

HIV Malaria Drug-resistant tuberculosis

Emerging and reemerging infectious diseases

Courtesy of Dr. Anthony Fauci, NIAID

SARS

Avian flu

HIV

Marburg

Measles

大學生要學什麼

追求自己的興趣，不管「熱」或「冷」門不怕選「錯」門多嘗試新的領域重視通識教育，培養人文藝術素養及品德養成表達的能力