The Replication of Viruses 1: PositiveStranded RNA Viruses

1
Lecture 2 The Replication of Viruses - 1
Positive-Stranded RNA Viruses
2
Positive-stranded RNA viruses?
M P . 7meG-5NCR-ATGCCC...TGA-3NC
R-poly(A) ? ve, mRNA sense
3NCR-TACGGG...ACT-5NCR ? -ve sense
5NCR-TCAGGGCAT-3NCR positive-stranded RNA
virus genomes have the same sense as mRNA they
can function directly as mRNA some virus genomes
possess a 5 mRNA cap structure (7meG), others do
not some virus genomes possess a poly(A) tail,
others do not
3
5000
10000
15000
20000
25000
30000
0
Picornavirus
PV1
Comovirus
CPMV
Key
Flavivirus
YFV
Encapsidation proteins
BDV
RNA replication proteins
HepC
Sub- genomic mRNA
Calicivirus
(Lagovirus)
RHDV
Leaky stop codon
(Sapporo-like viruses)
Sapporo
Ribosomal frame-shift
(Norwalk-like viruses)
Norwalk
(Vesivirus)
FCV
Alphavirus
SFV
Rubella
Arterivirus
AEV
ORF1a
4
2
6
ORF1b
3
5
7
2
3
4
5
6
7
Coronavirus
MHV
ORF1a
ORF1b
2a
2b
3
4a
4b
5a
5b
6
7
4

• Positive sense RNA virus mRNA sense
• Virus genome acts directly as an mRNA
• Naked RNA is infectious does not need any
  virus proteins associated
• 
  
  with the vRNA
• First step in replication is translation
  generates virus replication proteins
• 
  which can then copy the vRNA
• Needs to make RNA from an RNA template animals
  cells do not do this, they
• 
  make RNA from a DNA
  template
• RNA-dependent RNA polymerase
• Second step in replication is transcription of
  RNA
• 
  transcribes daughter RNA strands from input,
  parental, strand

5

• RNA-dependent RNA Polymerases
• - No proof-reading ability
• No form of post-replicative mis-match repair in
  virus replication
• High error-rate during RNA replication 1 x
  10-4
• (error rate
  in human (DNA) genome replication 1x 10-11)
• Can escape selection pressures (antibodies /
  antivirals etc.)
• High rates of recombination
• between daughter strands from
  the same parent
• possibly between strands from
  related viruses (co-infection of the same cell)
• - RNA-dependent RNA polymerases have the same
  overall structural architecture
• as DNA polymerases

6
-ve sense nascent RNA strands
RNA Recombination
ve sense template RNA
RNA-dependent RNA polymerase and incomplete
daughter RNA strand drops off template
re-associates with a different template
ve sense template RNA
RNA-dependent RNA polymerase
7
transcription continues
Copy-Choice mechanism of recombination
recombinant genome
8
The 3 (alternative) uses of daughter ve sense
RNA strands
ve sense input, parental, RNA strand
-ve sense template RNA strand
1. Replication used as template to make
more ve strands
ve sense daughter RNA strands
3. Encapsidation packaged into new
particles
2. Translation more virus proteins
9
3
5
An
5
3
An
(80x)
3
5
An
10
Eukaryotic mRNA
AUG
Stop
AAAAAAAAA
7meG
single open reading frame

• Virus must compete with cellular mRNAs for
  translational resources
• Must use cellular translational apparatus
• 
  initiation factors
• 
  elongation factors
• 
  termination (release) factors
• - Some viruses shut-off cellular mRNA
  translation
• - can
  sequester cells resources to increase the yield
  of virus particles

11

• RNA-dependent RNA polymerase
• Capsid protein(s)
• ??????

Viruses must be able to make multiple proteins
12

• RNA-dependent RNA polymerase
• Capsid protein(s)
• ??????

• Viruses must be able to make multiple proteins
• Proteolytic processing of a polyprotein
• 
  host-cell proteinases
• 
  virus-encoded
  proteinases

13
Picornaviruses
Capsid proteins (P1)
An
P2
P3
Replicative proteins
Enteroviruses Poliovirus serotypes 1, 2, 3.
Coxsackie A and Coxsackie B
viruses ECHO viruses
various Ovine, Bovine and
Equine enteroviruses Rhinoviruses common cold
virus Cardioviruses EMC, Theilers Murine
Encephalitis Virus Aphthoviruses Foot-and-Mouth
Disease Viruses (serotypes O, A, C, Asia, SAT
1-3) Equine rhinitis A
virus (ERAV) Hepatoviruses Human and simian
hepatitis A viruses Other Genera Erbovirus
Equine rhinitis B virus
(ERBV) Parechoviruses Human Parechovirus
1 2 Teschovirus Porcine
teschoviruses Kobuvirus
Aichi virus
14
Picornaviruses
60 copies of 4 proteins
VP1 (1D) (blue)
VP2 (1B) External (green)
VP3 (1C) (red)
VP4 (1A) - internal to capsid (myrstoylated)
Combination of linear and conformational epitopes
Icosahedral symmetry
Non-enveloped, 25nM
Genome Structure
Infected-cell Proteins
5NCR
3NCR
(IRES)
3B
Time

1C
1D
2B
2B
2C
3A
3C
3D
A
1A
1B
n
P1
P2
P3
P3
P1
Capsid
proteins
Replicative proteins
P2
7,500 bases positive (mRNA) polarity
15
Picornavirus Polyproteins
CAPSID PROTEINS
REPLICATIVE PROTEINS
P2 REGION
P3 REGION
Entero-/Rhinoviruses
3C
2A
Cardioviruses
3C
2A
Aphthoviruses
L
3C
2A
Hepato-, Parechoviruses
3C
2A
16
Polyprotein Primary cleavages intramolecular,
in cis
P1
P2
P3
An
80 ribosomes
17
Virus-encoded proteinases are molecular mimics
of host-cell proteinases
His18
Cys106
Asp35
Human Rhinovirus 2 2A Proteinase
Cys112
Cys52
Cys54
His114
18
Virus-encoded proteinases are molecular mimics
of host-cell proteinases
Cys147
His40
Glu71
Human Rhinovirus 2 3C Proteinase
19
Enveloped virus, Icosahedron - 240 copies of 1
protein
Flaviviruses
capsid protein
blue surface glycoprotein green lipid
bilayer orange capsid protein
surface glycoproteins
matrix protein
40-60nM
20
5000
10000
15000
20000
25000
30000
0
Picornavirus
PV1
Comovirus
CPMV
Key
Encapsidation proteins
Flavivirus
YFV
RNA replication proteins
BDV
Sub- genomic mRNA
HepC
Leaky stop codon
Calicivirus
RHDV
Ribosomal frame-shift
FCV
Alphavirus
SFV
Rubella
Arterivirus
AEV
ORF1a
4
2
6
ORF1b
3
5
7
2
3
4
5
6
7
Coronavirus
MHV
ORF1a
ORF1b
2a
2b
3
4a
4b
5a
5b
6
7
21
Medically Significant Flaviviruses
22
Flavivirus replication
23
Host-cell signalase cleaves the flavivirus
glycoproteins
COP I - coated vesicles
COP I
COP I I
secretory protein
COPII - coated
vesicles
ER
Trans Golgi
Network (TGN)
vesicular tubule
clusters (VTCs) / ER-
ribosome free
Golgi intermediate
Golgi
transitional elements -
compartments (ERGIC)
transitional ER (tER)
24
Polyprotein Processing Flavi, Pesti- and
Hepaciviruses
Hepatitis C
5A
5B
4B
3
2
E2
E1
C
4A
Flavivirus
NS1
NS5
NS4B
4A
NS3
2A
E
C
2B
prM
Pestivirus
E2
E1
C
Npro
NS5A
NS5B
NS4B
NS3
NS2
4A
- cleavage by virus-encoded proteinase
- cleavage by host-cell signalases
25
E1
NS5A
NS5B
NS4B
NS3
2
E2
C
4A
Hepatitis C
NH2
Dengue 2
4A
prM
NS5
NS4B
NS3
2A
1
E
C
2B
26

• Surface glycoproteins in virus membrane
• Glycoproteins need to enter the cells exocytic
  pathway
• Virus glycoproteins processed from a polyprotein
  precursor by host-cell signalases
• Flavivirus polyproteins also processed by a
  virus-encoded proteinase
• 
  - the
  proteolytic domain of NS3
• Flavivirus polyproteins are proteolytically
  processed by a combination of
• 
  host-cell and virus-encoded
  proteinases

27

• RNA-dependent RNA polymerase
• Capsid protein(s)
• ??????

Viruses must be able to make multiple
proteins 1. Proteolytic processing of a
polyprotein
host-cell
proteinases

virus-encoded proteinases 2. Partite genomes
28
5000
10000
15000
20000
25000
30000
0
Picornavirus
PV1
Comovirus
CPMV
Key
Encapsidation proteins
Flavivirus
YFV
RNA replication proteins
BDV
Sub- genomic mRNA
HepC
Leaky stop codon
Calicivirus
RHDV
Ribosomal frame-shift
FCV
Alphavirus
SFV
Rubella
Arterivirus
AEV
ORF1a
4
2
6
ORF1b
3
5
7
2
3
4
5
6
7
Coronavirus
MHV
ORF1a
ORF1b
2a
2b
3
4a
4b
5a
5b
6
7
29
Picornaviruses
Capsid proteins
An
Replication proteins
Comoviruses - bipartite genome
Capsid proteins
An
An
Replication proteins
Only one RNA segment per particle needs two
(different) particles to cause infection Plant
viruses Can now control (a) transcription of
different RNA strands
(b) how much protein is translated from the
different strands
30

• RNA-dependent RNA polymerase
• Capsid protein(s)
• ??????

Viruses must be able to make multiple
proteins 1. Proteolytic processing of a
polyprotein
host-cell
proteinases

virus-encoded proteinases 2. Partite genomes 3.
Synthesis of sub-genomic mRNAs
31
5000
10000
15000
20000
25000
30000
0
Picornavirus
PV1
Comovirus
CPMV
Key
Encapsidation proteins
Flavivirus
YFV
RNA replication proteins
BDV
Sub- genomic mRNA
HepC
Leaky stop codon
Calicivirus
RHDV
Ribosomal frame-shift
FCV
Alphavirus
SFV
Rubella
Arterivirus
AEV
ORF1a
4
2
6
ORF1b
3
5
7
2
3
4
5
6
7
Coronavirus
MHV
ORF1a
ORF1b
2a
2b
3
4a
4b
5a
5b
6
7
32
Coronaviruses
The envelope carries two glycoproteins
S - Spike glycoprotein (
) receptor binding, cell fusion,
major antigen M - Membrane
glycoprotein ( )
transmembrane - budding envelope
formation In a few types, there is a third
glycoprotein HE -
Haemagglutinin-esterase ( ) The
genome is associated with a basic phosphoprotein
( N )
33

• Pathogenesis These viruses infect a variety of
  mammals birds. The exact number of human
  isolates are not known as many cannot be grown in
  culture. They cause
• respiratory infections (common)
• enteric infections (occasional - mostly in
  infants gt12 mo.)
• neurological syndromes (rare)
• Transmitted by aerosols of respiratory
  secretions, growth appears to be localized in
  epithelium of the upper respiratory tract, but
  there is no adequate animal model for the human
  respiratory coronaviruses.
• Clinically, most infections cause a mild,
  self-limited disease (classical 'cold' or upset
  stomach), but there may be rare neurological
  complications. Greatest incidence in children in
  winter, less common in adults.
• Number of serotypes/extent of antigenic variation
  unknown. Re-infections appear to occur throughout
  life (implying multiple serotypes (at least four
  are known) and/or antigenic variation) hence
  prospects for immunization appear bleak.

34
SARS
The most common reported symptom is fever (94),
with 5172 of patients reporting general
influenza-like symptoms, chills, malaise, loss of
appetite, and myalgia. Gastrointestinal
symptoms are less common at presentation,
including diarrhoea (27), vomiting (14), and
abdominal pain (13). The mean incubation
period of SARS is estimated to be 64 days. The
estimated case fatality rate is
132
for patients younger than 60 years and

433 for patients aged 60 years or older. Case
clusters have played an important part in the
course of the epidemic.
35
SARS Genome
0
5,000
10,000
15,000
20,000
25,000
29,713
E
Leader protein
S
NSP7
NSP1
NSP10
NSP3
NSP12
NSP9
NSP2
NSP4
N
NSP11
NSP13
MHV p65 counterpart
NSP5
M
NSP6
36
Picorna-, Flavivirus
Progeny virus particles
Encapsidation
mRNA, translation
ve sense
Replication
Replication
- ve sense
37
Calici-, Alpha-, Arteri-, Coronavirus
Progeny virus particles
mRNA, translation of replication proteins
Encapsidation
ve sense
Replication
Replication
- ve sense
ve sense
Sub-genomic mRNA, translation of capsid proteins
38
The synthesis of sub-genomic mRNAs
ve sense input, parental, RNA strand
-ve sense template RNA strand
1. Replication used as template to make
more ve strands
Sub-genomic mRNAs
ve sense daughter full-length RNA strands
Translation virus proteins
3. Encapsidation packaged into new
particles
2. Translation more virus proteins
39
Translation from Sub-Genomic mRNAs
Mouse Hepatitis Virus Genome (Kb)
28
0
2
4
6
32
8
30
10
26
12
24
14
20
16
22
18
ORF 1a
ORF 1b
translated (dark blue)
untranslated (light blue)
A(n)
7meG
can only translate the first open reading frame
of any mRNA
A(n)
7meG
Sub-genomic mRNAs
A(n)
7meG
A(n)
7meG
7meG
A(n)
7meG
A(n)
3 co-terminal, nested set of subgenomic mRNAs
40
The Replication of Coronaviruses
41
Sub-genomic mRNAs can control when (during the
infectious cycle) proteins are made can control
(via transcription) how much protein is made
42

• RNA-dependent RNA polymerase
• Capsid protein(s)
• ??????

Viruses must be able to make multiple
proteins 1. Proteolytic processing of a
polyprotein
host-cell
proteinases

virus-encoded proteinases 2. Partite genomes 3.
Synthesis of sub-genomic mRNAs 4. Translational
tricks
43

• RNA-dependent RNA polymerase
• Capsid protein(s)
• ??????

Viruses must be able to make multiple
proteins 1. Proteolytic processing of a
polyprotein
host-cell
proteinases

virus-encoded proteinases 2. Partite genomes 3.
Synthesis of sub-genomic mRNAs 4. Translational
tricks (a) Leaky stop codons
44
5000
10000
15000
20000
25000
30000
0
Picornavirus
PV1
Comovirus
CPMV
Key
Encapsidation proteins
Flavivirus
YFV
RNA replication proteins
BDV
Sub- genomic mRNA
HepC
Leaky stop codon
Calicivirus
RHDV
Ribosomal frame-shift
FCV
Alphavirus
SFV
Rubella
Arterivirus
AEV
ORF1a
4
2
6
ORF1b
3
5
7
2
3
4
5
6
7
Coronavirus
MHV
ORF1a
ORF1b
2a
2b
3
4a
4b
5a
5b
6
7
45
Alphavirus Non-Structural Proteins
Read-through of leaky stop codon
nsP4
nsP1
nsP2
nsP3
For many alphaviruses, translation of the first,
long, open reading frame (ORF) terminates at an
opal stop codon (UGA) between NS3 and NS4
producing the translation product
NS123. Read-through of this stop codon,
however, results in the incorporation of an
arginine residue to produce the translation
product NS1234 Read-through occurs at 10-20
of translation events, but is poorly
understood. Some alphaviruses (Semliki forest
virus SFV Onyong-nyong - ONN) do not have a
stop codon in this position, but encode an
arginine residue translation producing only
NS1234.
46

• RNA-dependent RNA polymerase
• Capsid protein(s)
• ??????

Viruses must be able to make multiple
proteins 1. Proteolytic processing of a
polyprotein
host-cell
proteinases

virus-encoded proteinases 2. Partite genomes 3.
Synthesis of sub-genomic mRNAs 4. Translational
tricks (a) Leaky stop codons (b)
Programmed ribosomal frame-shifting
47

• Programmed Ribosomal
  Frame-Shifting
•  
• First described for the retrovirus Rous Sarcoma
  Virus (1985) - also described in
•  
• Retroviruses,
• Coronaviruses, Toroviruses, Arteriviruses,
  Astroviruses, Plant viruses (RNA DNA)
• giardiaviruses
• Drosophila retrotransposons,
• a virus-like element in Saccharomyces
  cerevisiae,
• eukaryotic genes,
• bacterial genes,
• bacteriophage genes,
• bacterial insertion elements.

• M A N L W
• ATG GCA AAU UUA UGG -
• I Y G

48
Programmed Ribosomal Frame-shifting
49
Retrovirus Genomes
50
RNA Pseudoknots
Structure first proposed (with experimental
data!) to explain the tRNA-like properties
of turnip yellow mosaic virus (TYMV) 3
non-coding region (1982).
51
Base-pairing within an RNA pseudoknot
52
Slippery Heptamer Sequence   A AAU UUA
RETROVIRUS Rous sarcoma virus U UUA AAC
CORONAVIRUS Infectious bronchitis virus X XXY YYN
CONSENSUS  
Frame-shifting efficiency G GGU UUA -
16.1 U UUA AAC - 41.7   spacer
region of ? 6nts, then a pseudoknot.
Slippery sequence
pseudoknot
53

• RNA-dependent RNA polymerase
• Capsid protein(s)
• ??????

Viruses must be able to make multiple
proteins 1. Proteolytic processing of a
polyprotein
host-cell
proteinases

virus-encoded proteinases 2. Partite genomes 3.
Synthesis of sub-genomic mRNAs 4. Translational
tricks (a) Leaky stop codons (b)
Programmed ribosomal frame-shifting (c)
Internal ribosome entry sequences (IRESes)
54
Eukaryotic Initiation Factor 4G (eIF4G)
m7GpppN
eIF4E
PABP
eIF3
eIF4A
eIF4A
Entero-, Rhinovirus 2A Proteinase
635 / 636
Aphthovirus L Proteinase 642 / 643
shut-off of host-cell cap-dependent mRNA
transation
eIF4E - cap binding eIF4A -
bidirectional RNA helicase eIF3 - ribosome
binding PABP poly(A) binding protein
55
Foot-and-mouth Disease Virus Internal Ribosome
Entry Sequence (IRES) - RNA secondary structure
in the 5 non-translated region - cap independent
translation of virus RNA
Open Reading Frame
VPg
poly(C) tract
5 Non-coding region
56
m7GpppN
eIF4E
PABP
eIF3
eIF4A
eIF4A
IRES can bind this portion of the (cleaved)
initiation complex directly to initiate virus
translation
57

• Picornaviruses
• cap structure is an oligopeptide (not 7meG as
  for cellular mRNAs)
• proteinases can cleave host-cell translation
  initiation factor 4F
• this shuts-off host-cell, cap-dependent, mRNA
  translation
• virus has an IRES in the 5 non-translated
  region
• IRES is able to bind the C-terminal, cleaved
  portion directly
• cap-independent translation of picornavirus
  mRNAs
• can use the cells translational resources to
  make more viruses !
• IRESes also used by many other types of
  virus.........
• e.g. Hepatitis C virus

58

• RNA-dependent RNA polymerase
• Capsid protein(s)
• ??????

Viruses must be able to make multiple
proteins 1. Proteolytic processing of a
polyprotein
host-cell
proteinases

virus-encoded proteinases 2. Partite genomes 3.
Synthesis of sub-genomic mRNAs 4. Translational
tricks (a) Leaky stop codons (b)
Programmed ribosomal frame-shifting (c)
Internal ribosome entry sequences (IRESes) (d)
Ribosome skipping
59
Capsid Proteins
Replication Proteins
P1 Region
P2 Region
P3 Region
Entero-, Rhinoviruses
3C
2A
2B
Cardioviruses
3C
2A
2B
Aphthoviruses
L
3C
2B
2A
Cardiovirus 2A
(119aa)GIFNAHYAGYFADLLIHDIETNPG
P- (109aa)KAVRGYHADYYKQRLIHDVEMNPG P-
-Q LLNFDLLKALGDVESNPG P-

Cardiovirus (EMCV) Cardiovirus (TMEV) Aphthovirus
(FMDV)
2B
Aphthovirus 2A
60
GFP-GUS Reporter System
single ORF
GFP
GUS
pGFP2AGUS
pGUSGFP
2A
GFP
GUS
GFPGUS
GUS2AGFP
--
--
--
GUS

• Analysed using translation systems in vitro
• (coupled transcription / translation systems)
• translation products labelled with 35S-met

GFP2A
--

• cleavage mediated by just 18aa (2A)
• N-terminal
  proline of 2B
• cleavage efficient (90)
• cleavage co-, not post-, translational
• cleavage at C-terminus of 2A as in FMDV

61
Translational model of cleavage occurs within
the ribosome
Exit tunnel (100Å)
exit tunnel accommodates 30-34aa 2A cleavage
activity maps entirely within this length of
peptide known that other nascent peptide
sequences can interact with exit tunnel 2A
interacts with ribosome exit tunnel glycly-prolyl
peptide bond not formed cleavage (hydrolysis)
not of a peptide bond, but the ester bond between
the nascent peptide and its tRNA a
psudeo-reinitiation at the A site - to
translate the sequences downstream of 2A
Cleavage (eRF1 / eRF3)
COOH
OH
OH
A
P
E