Regulasi dan Ekspresi gen

pada Bakteriofaga/Virus

• dr. Fera Ibrahim, MSc., Ph.D, SpMK(K)

• Departemen Mikrobiologi Klinik FKUI-RSCM

Virus adalah parasit obligate intracellular

- Bacteriophages
- Human dan animal
viruses
 What is the difference between a cells
and a virus ?

Foundations in Microbiology, 8th ED. 2008, McGraw-Hill,New York

 SIZE OF MICROBES
Virus is an agent smaller
than bacterium, that most
cannot be seen by light
microscopy

The smallest viruses have a

diameter of 10 nm
whereas the largest are
approximately 300 nm in
diameter, which is about
the size of the smallest
bacterial cell

Comparison of the size of selected viruses to E. coli and a human red blood cell

Baumann, RW. Microbiology with Diseasesby taxonomy 3rd ed. Pearson International . 2011
VIRUS
DNA VIRUSES
RNA VIRUSES
• Genetic stages in the
multiplication of double
stranded DNA viruses
(simplified)
The virus penetrates the host cell
and release the DNA
1. DNA enters nucleus
2. DNA transcription
3. Viral RNA is translated into protein in
cytoplasma, proteins enter nucleus
4. Viral DNA replicate repeatedly in
nucleus
5. Viral DNA and proteins assembled
into a mature virus
6. Some DNA viruses integrated to host
chromosomes
REPLICATION OF
DNA VIRUS

→ Herpes simplex virus

Murray PR, et al, Medical Microbiology 5th eds, 2005

• Replication of
positive-strand single
stranded RNA
(simplified)
1. Penetration and uncoating of
viral RNA
2. Positive-strand RNA is
tranlated into proteins
3. A negative genome is
synthetized against the positive
template to produce large
numbers of (+) strand RNA
4. Synthesize (+) strand RNA
5. Assembly of RNA strands and
proteins into mature virus
 REPLICATION OF
RNA (+)VIRUS

→ Picornavirus

Murray PR, et al, Medical Microbiology 5th eds, 2005

REPLICATION OF
RNA (-)VIRUS

→ Rhabdovirus

Murray PR, et al, Medical Microbiology 5th eds, 2005

REPLICATION OF
RETROVIRUS

→Human Immunodeficiency
Virus (HIV)
 A Common Strategy for Viral
Propagation
• All viral genomes are packaged inside particles that
mediate their transmission from host to host.
• The viral genome contains the information for initiating
and completing an infectious cycle within a susceptible,
permissive cell.
• An infectious cycle includes attachment and entry of
the particle, decoding of genome information,
translation of viral mRNA by host ribosomes, genome
replication, and assembly and release of particles
containing the genome.
• All successful viruses are able to establish themselves in
a host population so that virus survival is ensured.
Mechanisms of Viral RNA Synthesis
• The genomes of all RNA viruses except retroviruses
encode an RNA-dependent RNA polymerase to
catalyze the synthesis of new genomes and mRNAs
• Genome and mRNA synthesis of most RNA viruses
occurs in the cytoplasm of the cell
• The virions of RNA viruses with (−) strand and double-
stranded RNA genomes must contain the RNA
polymerase, because the incoming viral RNA can be
neither translated nor copied by the cellular machinery.
• Mechanism of viral RNA synthesis meets two essential
requirements common to the infectious cycles of all
these viruses :
• (i) during replication the RNA genome must be copied from
one end to the other with no loss of nucleotide sequence;
• (ii) viral mRNAs must be produced that can be translated
efficiently by the cellular protein synthetic machinery
Replikasi virus
 The virion attaches to permissive monkey cells upon
Single-cell reproductive binding of VP1 to a major histocompatibility complex
cycle of simian virus 40 (MHC) class I molecule on the surface. The virion is
then endocytosed in caveolae (1 and 2), transported to
the endoplasmic reticulum, and enters that organelle
(3). Subsequently, it is transported to the nucleus and
uncoated by unknown mechanisms (4). The viral
genome packaged by cellular nucleosomes is found
within the nucleus (5). The early transcription unit is
transcribed by host cell RNA polymerase II (6). After
alternative splicing and export to the cytoplasm (7),
the early mRNAs are translated to produce the early
proteins LT and sT (8). The former is imported into the
nucleus (9), where it binds to the simian virus 40 origin
of replication to initiate DNA synthesis (10). Apart from
LT, all components needed for viral DNA replication are
provided by the host cell. As they are synthesized,
daughter viral DNA molecules associate with cellular
nucleosomes to form the viral nucleoproteins often
called minichromosomes. LT also stimulates
transcription of the late gene from replicated viral DNA
templates (11). Processed late mRNAs are exported to
the cytoplasm (12), and translated to produce the
virion structural proteins VP1, VP2, and VP3 (13).
These structural proteins are imported into the
nucleus (14) and assemble around viral
minichromosomes to form progeny virions (15).
Virions are released by an unknown mechanism (16).
Single-cell reproductive cycle
of Picornaviridae
The virion binds to a cellular receptor (1); release of the
poliovirus genome occurs from within early endosomes
located close (within 100 to 200 nm) to the plasma
membrane (2). The VPg protein, depicted
as a small orange circle at the 5′ end of the virion RNA, is
removed, and the RNA associates with ribosomes (3).
Translation is initiated at an internal site 741 nucleotides
from the 5’ end of the viral mRNA, and a polyprotein
precursor is synthesized (4). The polyprotein is cleaved
during and after its synthesis to yield the individual viral
proteins (5). Only the initial cleavages are shown here.
The proteins that participate in viral RNA synthesis are
transported to membrane vesicles (6). RNA synthesis
occurs on the surfaces of these infected-cell-specific
membrane vesicles. The (+) strand RNA is transported to
these membrane vesicles (7), where it is copied into
double-stranded RNAs (8). Newly synthesized (−) strands
serve as templates for the synthesis of (+) strand genomic
RNAs (9). Some of the newly synthesized (+) strand RNA
molecules are translated after the removal of VPg (10).
Structural proteins formed by partial cleavage of the P1
precursor (11) associate with (+) strand RNA molecules
that retain VPg to form progeny virions (12), which are
released from the cell upon lysis (13)
Strategies of transcription of
viral DNA templates
 Eukaryotic RNA polymerases
synthesize different classes of
cellular and viral RNA
Prerequisites for transcription of
viral genes by RNA polymerase II
Initiation of transcription by RNA polymerase II
Transcription of Viral DNA Templates by
the Cellular Machinery Alone

The
transcriptional
control region
of an avian
retrovirus
Models for transcriptional activation by the
herpes simplex virus type 1 VP16 protein
Viral Proteins That Regulate RNA
Polymerase II Transcription
• Positive
autoregulatory
loop
• The Human Immunodefi
ciency Virus Type 1 Tat Protein
Autoregulates Transcription

• Transcriptional
cascade,
Life cycle of adenovirus
Known and suspected interactions
with Hsp70 are shown. 1, Hsp70 is
involved in the recycling of clathrin
during the formation of clathrin
coated pits and vesicles and
afterwards in the uncoating of clathrin
coated vesicles (Greene and Eisenberg
1990; Newmyer et al. 2003; Newmyer
and Schmid 2001; Ungewickell 1985).
2, Binding of Hsp70 to the hexon
capsid protein. 3, Hsp70-mediated
release of the viral genome into the
nucleus. 4, Interaction of Hsp70 with
the immediate early gene product
E1A, dissociation of pRB·E2F
complexes, regulation of viral and host
transcription by E1A with likely
participation of Hsp70. 5, interaction
of Hsp70 with newly synthesized coat
proteins and possible role in the virion
assembly
The cellular transcriptional
regulator Nf-B and its
participation in viral
transcription
Processing of a viral or cellular mRNA
synthesized by RNA polymerase II
The translation :
5’-cap-dependent assembly of
the initiation complex
5’-end-dependent initiation
5’-End-Independent Initiation : internal ribosome entry site (IRES)
Translation elongation and termination
The Diversity of Viral Translation Strategies
The Diversity of Viral Translation Strategies
 Regulation of Translation during
Viral Infection
• Inhibition of translation initiation after viral infection
• Phosphorylation of eIF2 alfa
• Viral regulation of Pkr
• Beneficial effects of elF2 alfa phosphorylation on viral
replication
• Regulation of elF4F
• Cleavage of elF4G
• Modulation of elF4E activity by phosphorylation
• Modulation of elF4E activity by specific binding proteins
• Regulation of poly (A)-binding protein activity
• Regulation of elF3
• Regulation by miRNA
 Bacteriophage
• While phages are the best understood of all
viruses, some of them are also among the most
complex.
• Research on phages led to the discovery that some
double-stranded DNA viruses can reproduce by two
alternative mechanisms: the lytic cycle and the
lysogenic cycle.
• In the lytic cycle, the phage reproductive cycle
culminates in the death of the host.
• In the last stage, the bacterium lyses (breaks
open) and releases the phages produced within
the cell to infect others.
• Virulent phages reproduce only by a lytic cycle.
Reproduksi Faga menggunakan siklus
litik atau lisogenik
 Bacteriophage
• In the lysogenic cycle, the phage genome replicates without
destroying the host cell.
• Temperate phages, like phage lambda, use both lytic and
lysogenic cycles.
• Within the host, the virus’ circular DNA engages in either
the lytic or lysogenic cycle.
• During a lytic cycle, the viral genes immediately turn the
host cell into a virus-producing factory, and the cell soon
lyses and releases its viral products.
• During the lysogenic cycle, the viral DNA molecule is
incorporated by genetic recombination into a specific site on
the host cell’s chromosome.
• In this prophage stage, one of its genes codes for a protein
that represses most other prophage genes.
• Every time the host divides, it also copies the viral DNA and
passes the copies to daughter cells.
• Occasionally, the viral genome exits the bacterial
chromosome and initiates a lytic cycle.
• This switch from lysogenic to lytic may be initiated by an
environmental trigger.
 • The lambda phage which infects E. coli
demonstrates the cycles of a temperate phage
 Cro protein has the highest
 repressor has the highest affinity to OR3 and simiar affinity
affinity to OR1 then OR2 then to OR2 then
OR3 OR1

 repressor is a dimer cro protein is a dimer

This binding inhibits transcription from PR

So the lytic cycle is switched off This binding blocks transcription from PRM
via cooperative
interaction So the lysogenic cycle is switched off

PR is not needed
 repressor falls off OR3 in the later
first stages of the
lytic cycle
 Interchangeable RNA polymerase
subunits as a strategy to control gene
expression in a bacterial virus
Siklus hidup HIV 1

PROVIRUS
STRUKTUR LTR HIV-1

Di daerah promotor terdapat kotak

TATAA. Hanya bagian hulu (5') LTR yang
digunakan untuk melakukan inisiasi
dan transkripsi dimulai pada 5' akhir
dari R.
 LTR HIV
LTR memiliki sisi pengikatan faktor transkripsi
yang lain termasuk AP-2 dan Sp-1. Pada
daerah enhancer terdapat dua sisi pengikatan
NF-B (10 bp) dan AP-2.

Beberapa faktor selular seperti NFAT-1, USF-1,

COUPTF berinteraksi dengan daerah pengatur
Daerah hulu situs NF-B merupakan tersebut. Sekuens di dekat situs inisiasi RNA
daerah pengatur, negative regulatory mengandung elemen pengatur seperti putative
element (NRE) yang berperan dalam inducer of short transcripts (IST), inisiator dan
ekspresi gen virus. elemen transactivation response (TAR)
 EKSPRESI GEN HIV-1 TAHAP AWAL
Aktivasi provirus laten oleh NF-B
Posisi nuc-1 yang berada di hilir situs awal
transkripsi, nantinya akan dirusak ketika proses
remodelling aktivasi transkripsi provirus laten.

DNA virus yang terintegrasi ke dalam

DNA inang

Bagian 5’ LTR HIV tersusun membentuk 3 unik

nukleosom yaitu: nuc-0, nuc-1, nuc-2. Daerah
nucleosome-free terletak di antara nuc-0 dan nuc-1. Di
daerah tersebut terdapat situs awal transkripsi

NF-B menstimulasi aktifitas CTD kinase

yang akan mengkatalisis proses fosforilasi
CTD RNA pol II stimulasi proses elongasi
transkripsi

Aktivasi provirus laten oleh NF-B

 EKSPRESI GEN HIV-1 TAHAP AWAL :
Aktivasi provirus laten oleh tat

•Interaksi antara tat dan TAR juga melibatkan pengikatan kompleks protein kinase yang
disebut dengan TAK.
•Aktifitas enzim itu menyebabkan daerah yang berada di dalam subunit RNA pol II, C-
terminal domain (CTD), terfosforilasi.
•CTD yang hiperfosforilasi menginduksi perubahan konformasi pada RNA pol II sehingga
menyebabkan kompleks transkripsi, melalui daerah promoter dan memulai proses elongasi
molekul RNA virus.
Ekspresi gen HIV-1 tahap awal untuk memproduksi nef, tat dan rev
Peran protein tat dan rev
Protein gag dan gag-pro-
pol ditranslasi dari
seluruh genom RNA yang
tidak dipotong.

ORF pada gag dan pol

berada di posisi berbeda
dan pembentukan gag-pro-
pol terjadi dengan
mekanisme pergeseran
Frameshifting Translasi

Slippery sequence
Siklus Hidup HIV
 FMUI-CMH

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