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REGULATION OF TRANSLATION DURING VIRAL INFECTION
Interferons are produced in response to viral
infection as part of the rapid innate immune
response
Interferons diffuse to neighboring cells and bind
to cell surface receptors to activate
transcription of antiviral genes
Two interferon induced genes are:
RNase L - degrades RNA
protein kinase RNA-activated (Pkr)phosphorylates eIF2a, inhibiting
translation initiation
dsRNA activated protein kinase (Pkr)
Pkr is a serine threonine kinase composed of
an N-terminal regulatory domain and a Cterminal catalytic domain
Pkr is activated by the binding of dsRNA to two
dsRNA binding motifs at the N-terminus of the
protein.
Activation leads to autophosphorylation of Pkr
Phosphorylation of eIF2a inhibits recycling of eIF2
Model of activation of Pkr
Viral regulation of Pkr
Viruses use at least five different mechanisms to
block Pkr activation or to stop activated Pkr from
inhibiting translation
• inhibition of dsRNA binding- adenovirus VA
RNA binds Pkr blocks its activation by dsRNA
• binding and sequestering dsRNA- vaccinia
virus E3L protein
• inhibition of Pkr dimerization- hepatitis C virus
NS5A
• inhibitors of kinase function- vaccinia virus K3L
protein has homology to N-terminus of eIF2-a
Regulation of eIF4F activity by different viruses
1) Phosphorylation of eIF4E 2) Cleavage of eIF4G
Rapamycin
Regulation of poly A binding
protein activity
Poly A binding protein interaction
with eIF4G, bring together the
ends of the mRNA
Rotaviruses inhibit host translation
by blocking the function of Pab1p
Viral protein nsP3 occupies the
binding site of Pabp1p on eIF4G
It prevents binding of Pab1p to
eIF4G preventing formation of the
circular complex, allowing
translation of viral RNAs
What a Typical Virus Must Do to Survive
 Find the right cell and enter the cell.
 Uncoat to activate the viral genome.
 Translate the viral genes.
 Replicate the viral genome.
 Assemble new virus particles.
 Exit from the cell.
 Find a new host to infect.
Every virus must be able to exploit
specific processes of its host for
gene expression and replication, and
must be able to overcome host
defenses.
Genomes of RNA viruses:
 unimolecular
 segmented
 single stranded of (+) polarity
 single stranded of (-) polarity
 double stranded
 circular
Common requirement:
They must be copied within the infected cell
to provide new genomes and mRNAs
Genomes of RNA viruses
The genomes of all RNA viruses except retroviruses
encode an RNA-dependent RNA polymerase to
Catalyze the synthesis of new genomes and mRNAs
RNA viruses with (-) strand and double stranded
RNA genomes must contain the RNA polymerase
RNA viruses with (+) strand RNA genome lack a
virion polymerase
Questions: Are RNAs (-) strand viruses infectious?
Are RNAs of (+) strand viruses infectious?
Replication and Expression of RNA Virus
Genomes
 Plus-strand RNA viruses do not have a polymerase in their virions.
RNAs of these viruses are infectious, but they must first be
translated in cells to produce a viral RNA-Dependent-RNAPolymerase (RDRP) is synthesized for genome replication.
 After the genomic RNA is copied into a negative strand, it serves
as a template for replication of progeny plus-strand RNAs. There
is no DNA phase to replication of these viruses.
 Picorna-like viruses, such as Poliovirus and some plant viruses,
that express their genomes by proteolytic processing translate
their genomes as large polyproteins from their genomic RNAs that
is processed into individual viral proteins.
 Alpha-like viruses, such as sindbis virus and tobacco mosaic virus
synthesize subgenomic mRNAs from the negative strand, which
contains promoters that are recognized by the replicase.
Replication of Plus-Sense Viral RNA
(+)
(-) (+)
(+) (-)
(-) (+)
*
Synthesis of
Minus-Strand
By RDRP
*
or
Synthesis of
Progeny PlusStrand RNA
By RDRP
Genomic RNA Replicative
(+ssRNA)
intermediate
*
Synthesis of
Minus-Strand
Intermediates
…..
*
*
*
*
*
Replicative
Form I
Replicative
Form II
Intermediates are Double-stranded RNA; Not DNA Intermediates.
No Proof Reading as in DNA replication so replication is error prone.
Plus Strand RNA Viruses Replicate in the Cytoplasm
Replication of an Enveloped, Plus Strand RNA Virus
 After Plus Strand RNA
viruses enter cells, the next
in step replication is release
of the genomic RNA.
 The viral RNA must then be
translated for expression of
the RNA-Dependent-RNA
Polymerase (RDRP).
 The RDRP functions in
transcription of mRNAs and
replication of genomic RNAs.
 Viral capsid proteins are
translated and assemble in
the cytoplasm with the viral
RNA.
 Unenveloped viruses are
released by lysis, enveloped
viruses bud from the plasma
membrane and are released
from the cell.
Endocytosis or fusion
Genome RNA
Translation of replicase proteins
Replication
Synthesis of
subgenomic mRNA
mRNA
Translation
Capsid protein
NUCLEUS
Translation
and modification
Glycoproteins
EUKARYOTIC HOST CELL
Viral RNA synthetase
Host factor(s)
Several Variations of this theme Occur
Budding
Nonsegmented Negative Strand RNA Viruses
Minus sense RNA genomes are
complementary to their mRNAs.
Viral genomic RNA not infectious.
All negative strand viruses are
enveloped.
Virus particles have nucleocapsid
cores consisting of the minussense genomic RNA
encapsidated by core-associated
proteins.
mRNAs
Genomic RNA
Antigenomic RNA
Many Progeny
Genomic RNAs
Negative strand RNA Replication
Nucleocapsids have RNA-dependent- RNA polymerase activity.
 Polymerase transcribes the Viral genomic RNA into mRNA and
plus-sense antigenomic RNA.
 Antigenomic RNAs are copied into progeny minus-sense
genomic RNAs.
 Progeny genomic RNAs synthesize more mRNAs and function to
form virion formation.
Replication of a Typical Minus Strand RNA Virus
Generalized Replication Strategy of Negative Strand Viruses
1) Virus enters cell by endocytosis.
2) The viral nucleocapsid core is
released.
3) Viral mRNAs are transcribed in
the core from the negative strand
genome.
4) Viral proteins are translated &
accumulate in cells.
5) As proteins increase in amount
they associate with the newly
synthesized plus sense RNAs to
form antigenomic nucleocapsids.
6) The antigenomic RNAs replicate
to form progeny genomic (minus
sense) nucleocapsids.
7) New rounds of mRNA synthesis
occur and replication cycles are
repeated.
8) The minus sense nucleocapsids
undergo morphogenesis and
virus is released from cells.
Endocytosis or fusion
mRNA synthesis
mRNAs
Replication
Translation
vcRNA
(+)
Capsid proteins
Replication
Translation
and modification
NUCLEUS
Progeny
RNA Genomes
(-)
Glycoproteins
EUKARYOTIC HOST CELL
Nucleocapsid with genome RNA(-)
Nucleocapsid with vcRNA (+)
Viral RNA Synthetase
Budding
Life Cycle of Double Stranded RNA Viruses
1) Viruses with dsRNA
genomes are complex,
contain several segments,
and an RNA-dependentRNA-Polymerase.
2) Particles enter cells via
endocytosis.
3) Proteolytic digestion results
in subviral particles.
4) Core particle moves into the
cytoplasm & begin
synthesis of early viral
mRNAs from the dsRNA
genomic RNAs.
5) Late or secondary mRNA transcripts begin to appear at six to eight
hours post infection, suggesting that an early protein is required for
secondary mRNA synthesis.
6) The mRNAs appear to be translated & assemble with viral proteins to
form the early nascent cores.
7) Double stranded RNAs are synthesized in the cores, which undergo a
series of steps to form mature virus particles that are released from
the cell.
A key point is that all mRNA transcription and genomic RNA
replication occurs in the cytoplasm in viral cores.
Reverse transcription is
the hallmark of the
retrovirus replication
cycle.
Viral Mediated Events:
• Virus enters cells by direct
fusion or endocytosis.
• Icosahedral viral particle is
released into the cytoplasm
and begins to transcribe
double stranded DNA from the
diploid RNA genome.
• An integration complex is
transported to the nucleus and
functions to integrate the viral
DNA into the host genome.
Host mediated Events:
• The integrated viral DNA is
transcribed by host RNA
polymerase II to produce full
length viral RNAs.
• These RNAs are differentially
spliced to produce viral
genomic and mRNAs and are
exported to the cytoplasm.
• Viral proteins are translated
and assemble to form virions.
Reverse transcription
DNA copy of genome
Integration
into host DNA
RNA Synthesis
genomic
RNAs
mRNAs
Translation
RT
Splicing
NUCLEUS
Gag
Budding
Maturation
Glycoproteins
EUKARYOTIC HOST CELL
Retrovirus Replication Cycle
Generalized Replication Strategy of DNA Viruses
General Replication Scheme for a DNA Virus
1) Virus enters cell and DNA is released.
2) DNA moves to the nucleus and early
mRNAs are transcribed using host
DNA dependent RNA polymerase.
3) Early mRNAs are translated and
proteins elicit progeny DNA
replication.
4) Late mRNAs are synthesized to
produce capsids for assembly of
progeny DNA.
5) Encapsidated DNA exits the nucleus
and is released by lysis of host cells.
Note: Many different strategies of
DNA replication have been
identified and several different DNA
virus families exist. Viral DNAs do
not integrate into the host genome
during lytic replication, but may
during abortive infections.
Uncoating
Genome (DNA)
Transcription
+
mRNAs
Translation
Modification
+
DNA replication
+
Assembly
NUCLEUS
Release
EUKARYOTIC HOST CELL
Host RNA polymerase
Host DNA polymerase
Viral-encoded factor
Strategies for replication of RNA viruses:
RNA Dependent RNA Polymerase (RDRP):
 first evidence in 1960s with mengovirus and
poliovirus
 can synthesize viral RNA in the presence of
actinomycin D
 poliovirus 3Dpol can copy polyadenylated
genomic RNA in the presence of an oligo (U)
primer and ribonucleoside triphosphates
(ATP, UTP, CTP and GTP)
 many RdRps are associated with membranes or
nucleocapsids in infected cells
 some require a primer, others can initiate RNA
synthesis without a primer
RNA-directed RNA synthesis follows
universal rules:
 RNA synthesis initiates and terminates at specific
sites on the viral RNA template
Catalyzed by virus encoded polymerase, but
viral accessory proteins and host proteins
may be required
 Some require a primer with a free 3’ –OH end
to which nucleotides complementary
to the template strand are added
 RNA primers may be protein linked or may
contain a 5’ cap
 RNA is synthesized by template directed
incorporation of NMPs into 3’ end
Common motifs have been identified in the
Sequences of all RNA polymerases
Motif C includes the Gly-Asp-Asp sequence
(GDD) conserved in RNA polymerases of
Most (+) strand RNA viruses part of the
active site of the enzyme
Poliovirus genome organization
• Poliovirus replication occurs on membranes.
• 2C and 3AB bring the RNA polymerase to membranes.
• 2C anchors viral RNA to membranes during replication.
• 3AB anchors the viral primer, VPg to membranes.
• 3Dpol is brought to the replication complex by binding to
Poliovirus replication:
Polioviral RNA is linked to VPg via a tyrosine.
This bond is cleaved by a cellular enzyme to
produce viral mRNA containing a 5’ terminal
Up.
Minus strand synthesis:
• 5’ end of polioviral RNA contains a cloverleaf structure
and the 3’ end contains a pseudoknot
• Precursor of VPg, 3AB, acts as a VPg donor
• A ribonucleoprotein complex is formed when PCbp and
3CDpro bind to the cloverleaf structure
• This complex interacts with PAbp1, producing circular
genome
• Protease 3CDpro
cleaves membrane
bound 3AB to produce
VPg
• VPg is uridylated by
3Dpol and transferred
to the 3’ end of the
genome
• 3D pol uses
uridylated VPg as a
primer for (–) strand
RNA synthesis
Plus strand synthesis:
• The strands of the RF
are separated by 2C,
which binds to cloverleaf
in the (-) strand
• Membrane bound 3AB is
cleaved to produce VPg
• Uridylated VPg is
synthesized by 3D pol,
using (–) strand RNA as a
template
• Uridylated VPg is then
elongated by 3D pol to
Synthesize (+) strand RNA
Host factors required for poliovirus replication
 Poly(rC) binding protein 2- Binds to
cloverleaf structure at the 5’ end.
Formation of the 5’ cloverleaf, 3CD pro and
poly(rC) binding protein is necessary for
initiation of viral RNA synthesis.
 Poly(A) binding protein 1-interacts with
poly(rC) binding protein 3CDpro and 3’
poly(A) tail, circularizing the genome.
Imbalance of (-) and (+) strand synthesis:
 In poliovirus infected cells, genomic RNA
is produced at 100-fold higher concentration
than the (-) strand RNA
 Ribosomes must be cleared from RNA
before it can serve as a template for (-)
strand synthesis