Download กระบวนการย้อนการถอดรหัส (Reverse transcription)

กระบวนการย้อนการถอดรห ัส
(Reverse transcription)
ผศ.ดร. ม ัญชุมาส เพราะสุนทร
1
RNA-Dependent Synthesis of
RNA and DNA

the dogma had been revised
2

DNA can be synthesized using an RNA
template


reverse transcriptase in RNA viruses
RNA viruses containing reverse
transcriptase are also known as
“retroviruses”
(retro : latin word for “backward”)

Some viral RNAs are replicated by RNAdirected RNA polymerase (RNA
replicases)

Some E. coli bacteriophage have RNA
genomes
3
Life cycle of typical retrovirus

Starts when
infecting virions
bind to specific
receptors on the
surface of the
host and enter
the cell.
4
Life cycle of retrovirus
5
Reverse transcriptase carries
out 3 kinds of reactions



RNA-dependent DNA
synthesis
RNA degradation
DNA-dependent DNA
synthesis
6
General structure of an integrated
retrovirus genome

: required for
packaging
retroviral RNAs
into mature
virus particles
LTRs : needed for
the regulation
and initiation of
transcription
and integration
into the host
DNA
7
Reverse transcriptase converts
genomic RNA of a retrovirus into a
ds-proviral DNA


for DNA synthesis to begin, reverse
transcriptase requires a primer
RNA viral genome contains noncovalently
bound tRNA



this tRNA acts as a primer
new DNA strand is synthesized in the
5’
3’direction
do not have 3’
5’ proofreading exonuclease

generating high error rates faster rate of viral evolution
appearance of new strains of disease-causing retrovirus
8
9
Integration is an obligatory step in
the life cycle of retroviruses

transcription of retroviral DNA occurs only after it had
been integrated into the host-cell DNA
10
Some retroviruses cause cancer
and AIDS




do not kill their host cells
have an additional gene that can cause the cell
become cancerous
are classified as “RNA tumor virus”
the first retrovirus of this type
 Rous sarcoma virus (avian sarcoma virus)

the cancer-causing gene is called “oncogene”
11
src gene

ยีน src ทำหน ้ำทีส
่ ร ้ำงโปรตีนและเอนไซม์ท ี่
เกีย
่ วข ้องกับกำรเกิดมะเร็ง

tyrosine kinase หรือ tyrosine-specific protein
kinase



เอนไซม์ทม
ี่ ก
ี ำรทำงำนร่วมกับกระบวนกำรแบ่งเซลล์ กำร
เจริญเติบโตและกำรเปลีย
่ นรูปร่ำงของเซลล์
is found in the DNA of normal chickens and
eukaryotes including humans
when associated with virus, this oncogene is
expressed at abnormally high levels

unregulated cell division and cancer
12
Oncogenes in retroviruses

derived from normal cellular genes called “protooncogene”

by rare recombination events
13
Conversion of a regulatory gene
into a viral oncogene
14
Cancer can be induced without the
participation of a virus

mutations in the cellular
proto-oncogenes



chromosomal
rearrangements
chemical agents
radiation
15
Retrovirus that causes AIDS




นอกจำก retrovirus จะก่อให ้เกิดมะเร็งแล ้ว ยัง
ก่อให ้เกิดโรค AIDS (Acquired Immune
Deficiency Syndrome)
ื้ ไวรัสทีเ่ รียกว่ำ HIV (Human
โดยเชอ
Immunodeficiency Virus)
identified in 1983 by Luc montagnier
HIV has an RNA genome with standard
retroviral genes along with several other
unusual genes
16


่ เดียวกับ
HIV มี RNA เป็ นสำรพันธุกรรม เชน
retrovirus อืน
่ ๆ
แต่ retrovirus ทีก
่ อ
่ ให ้เกิดโรค AIDS จะ
ทำลำยเซลล์ทม
ี่ ันบุกรุกเข ้ำไป โดยเฉพำะที่
T-lymphocyte ทำให ้เซลล์เจ ้ำบ ้ำนมีระบบ
ภูมค
ิ ุ ้มกันบกพร่อง
17
Problems with HIV


the genes undergo mutations at a very rapid
rate
the reverse transcriptase is 10-fold less
accurate in replication



increase mutation rates
complicating the development of an effective
vaccine
however, HIV infection needs repeated cycles
of cell invasion and replication

inhibition of viral enzymes offers promise as an
effective therapy
18
Fighting AIDS with inhibitors of HIV
reverse transcriptase
 first drug to be approved “AZT”


structural analog of deoxythymidine
HIV reverse transcriptase has higher affinity for
AZT triphosphate than dTTP


incorporation of AZT triphosphate
causes premature termination of viral DNA synthesis
19




some other AIDS drugs : DDI
nonnucleoside reverse transcriptase inhibitors
(NNRTI)
protease inhibitors
combination use of reverse transcriptase
inhibitors and protease inhibitors
20
ELISA for detecting HIV envelope
protein
21
Polymerase chain reaction and
screening for HIV
22