Virology term 1 - positive strand viruses. Flashcards Preview

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1

Positive sense ssRNA virus families.

Picorna, Calici, Astro, corona, flavi, toga and more!

2

+ssRNA Rdrp present on entry?

No. Translation must occur first to synthesise this.

3

+ssRNA Rdrp

Synthesises new RNA, the template moving 3' to 5', producing RNA 5' to 3'.
Error prone. Generation of mutants and quasi-species.

4

5' cap structure

m7GpppNpN
2'O methylation identified as self/non-self marker.

5

5' region cellular mRNAs

3-1000 nt in length. RNA secondary structures unwound for ribosome to pass.
Affects translation efficiency.

6

3' region cellular mRNAs

Regulates translation efficiency, mRNA stability.
PolyA tail required for efficient translation.

7

Circularisation of cellular mRNAs

eIF4E binds cap, recruits eIF4G. Recruits eIF4A, eIF3 and 40S subunit. Also recruits PabIp, which binds polyA, causing ciruclarisation.

8

Making multiple proteins (4 mechanisms).

Make a polyprotein and cleave it.
Use a segmented genome
Produce functionally monocistronic sgRNAs.
Access multiple ORFs in mRNAs.

9

Accessing multiple ORFs in mRNAs

Polyprotein synthesis and cleavage
Altering termination.
Altering initiation.

10

Viruses that make and cleave polyproteins

Picornaviruses, flaviviruses.

11

Viruses that use segmented genomes.

Reoviruses, orthomyxoviruses.

12

Viruses that produce functionally monocistronic sgRNAs

Coronaviruses and closteroviruses.

13

IRES basics

No cap needed. Translation in host-shut off. If shut-off via eIF2 phosphorylation, uses ligatin.
Can direct to more than one ORF as scanning required.

14

Picornavirus replication cycle control and coordination

Controlling translation
Controlling replication.

15

Picornavirus replication cycle control and coordination - controlling translation.

Controlling initiation (types of IRES comparison, VPg comparison).
Controlling available RNAs in cell - host shut off.
Control of protein proportions.

16

Picornavirus replication cycle control and coordination - control of protein proportions.

Not at transcription level (unlike alphaviruses)
Polyprotein cleavage is main technique.

17

Picornavirus replication cycle control and coordination - control of replication.

o Initiation – co-ordinating priming
o Control of position
o Control of template selection. - both for replication and packaging.

18

Major groups of viral IRESes.

Type I. Poliovirus
Type II. EMCV and FMDV.
Type III. Hep A.
Type IV. Porcine teschovirus.

19

Type II IRES discovery

1988. 5' non-translated region of encephalomyocarditis directs interan entry of ribosomes.

20

Common conformational change in Type II IRESs - binding of eIF4G/eIF4A.

eIF4G/eIF4A binds J-K domains and restructures region of ribosomal attachment via HEAT-1 domain of eIF4G. This promotes restructuring of 3’ border and facilitates binding of pre-initiation 43S complex.

21

ITAFs required by EMCV and TMEV IRESs.

Stimulated by pyrimidine tract binding protein.

22

ITAFs required by FMDV.

PTB and ITAF45

23

Positioning of PTB on Type II picornavirus IRESs.

PTB has 4 RNA binding domains (RBDs). RBDs 1 and 2 bind K, while 3 and 4 bind H and base of I and L. Constrains and stabilizes structure.

24

How to discover what bases are exposed when an IRES is bound by an ITAF.

Hydroxyl radical cleavage occurs where the genome is exposed.

25

Type II IRES conformational change on binding of cognate ITAFs.

Binding of ITAFs promotes relative reorientation of I and JK domains, to make them in closer proximity.

26

Type II IRES - binding of 40S

40S subunit directly interacts with H and I domains of IRES. Role of eIF4G/4A is to stabilize and promote this conformational change and therefore acts like an ITAF.

27

Picornavirus Type I IRES. ITAFs - 4.

PTB (poly pyrimidine tract binding proein) stimulates the IRES by modulating eIF4G.
PCBP - binds poly C tracts
UNR
La

28

Picornavirus Type I IRES. PTB binding

Binding of PTB occurs in localized manner at base of Domain V, and short flanking regions. Binding sites of PTB and central domain of eIF4G overlap, so they reciprocally modify each others binding.

29

Picornavirus Type I IRES - structure, recruitment of 40S.

Domain 5 mimics tRNAGly anticodon to recruit glycyl-tRNA synthetase to apical part of domain V promoting accommodation of the initiation region of the IRES in the mRNA biding site of the ribosome. Interaction with GARS may be needed for correct positioning of 40S at PV IRES.

30

Picornavirus Type I IRES - basic structures.

Picornavirus Type1 IRES have 5 principle domains (dII-dVI)

31

Picornavirus Type I IRES - initiation.

Initiation starts with eIF4G/eIF4A, recruitment of 43S complexes with interaction between eIF3 and eIF4G. If the dVI is unstructured, scanning occurs, if structured initiation occurs without scanning.
Type I IRES initiation depends on PCBP2.

32

Dicistroviridae IRESes

Intergenic IRES unusual
Partly mimics E and P site tRNAs leads to binding of ribosome subunits. Precise placement prevents leaky scanning. Although an extra base-pairing in the P site leads to a different ORF.

33

Leaky scanning - general

Fail to initiate at first codon and initiate at alternative codon downstream. Can be long distance, even past a whole ORF and initiating next one.

34

Leaky scanning - context

G at +4 and purine at -3 is optimal. Doesn't have to be an AUG codon.
Close to 5’ end (<50 nt) not recognised efficiently.
Second AUG close to first.

35

Leaky scanning - failure to recognise due to proximity to 5' end.

Murine norovirus past 2 good AUG codons (for capsid).

36

Leaky scanning - second AUG causes initiation due to proximity.

Segment 6 of influenza virus.

37

Non-AUG initiation - general.

CUG, ACG can be recognised by Met-tRNAi when in strong context, and an RNA structure 14 nt 3’ delays it. Inefficient, leads to leaky scanning.

38

Non-AUG initiation - examples.

Sendai virus (-ssRNA)
o Extended C (ACG)
o P (AUG but poor context, no purine at -3)
o C (AUG).

39

Ribosome shunting.

Allows access to downstream ORFs; 5’ dependent, scanning independent. Bypassing RNA structures.

40

Ribosome shunting - causes.

Possibly due to retention of some initiation factors, as in reinitiation, while loss of others leads to discontinuous scanning.

41

Ribosome shunting - examples.

Caulimoviridae.
Suggested in Sendai Resp virus and gag gene of spumavirus.

42

Caulimoviridae wider family.

Pararetrovirus

43

Reinitiation

Sometimes 40S subunit doesn’t dissociate, but resumes scanning and reinitiates translation downstream. Especially after a short ORF: reacquires initiation factors.

44

Reinitiation upstream of termination.

Respiratory syncytial pneumovirus.

45

Reinitiation examples

Caliciviruses
o ORF1 from genome = non-structural
o ORF2 and 3 generally capsid proteins.
o ORF3 initiation very close to ORF2 termination: requires RNA sequence (TURBS) to retain the ribosome.

Caulimovirus TAV protein mediates.

46

Ribosomal frameshifting; -1 frameshifting. Examples.

RSV gag and pol.
HIV1, HIV2, HTLV1, HTLV2, coronaviruses.
Flavivirus to add 52 aa extension to make NS1'.

47

Ribosomal frameshifting - general mechanism.

Slippery sequence + downstream RNA structure (pseudoknot resistant to unwinding delays and tension in ribosome RNA binding). Tandem slippage allows perfect repairing except at the wobble position.

48

Ribosomal frameshifting - taxa with +1 or -2.

Closteroviridae, +ssRNA.

49

Stop codon read through.

Context important. AAstopcodon stimulates readthrough.
Also stimulated by conserved 3' adjacent nt, downstream sequences and secondary structures.

50

Types of stop-codon

UAA, UAG or UGA

51

Stop-carry on

Alternative to proteolytic cleavage in some picornaviruses. Amino acids in exit tunnel interact to prevent peptide bond forming between gly and pro.

52

Initiation of mRNA translation - techniques in +ssRNA viruses.

1) 5' cap dependent
2) IRES
3) Vpg dependent.

53

Initiation of mRNA translation - techniques in +ssRNA viruses: 5' cap dependent.

Coronaviruses, flaviviruses (also IRES).
Capping occurs in cytoplasm.

54

Initiation of mRNA translation - techniques in +ssRNA viruses: Vpg dependent

Calici, astro

55

Coronaviruses: avoiding monocistronic nature of mRNAs

5' cap dependent, but...
-1 ribosomal frameshifting
produces functionally monocistronic sgRNAs.

56

Accessing multiple ORFs in mRNAs: altering termination

Change ORF - ribosomal frame-shifting.
Stop-codon read-through
stop-carry on.

57

Accessing multiple ORFs in mRNAs: altering initiation

Different ORFs: RNA stuttering and ribosomal frameshifting.
Leaky scanning.
Ribosomal shunting.
Non-AUG initiation.
Reinitiation.
True internal initiation

58

Flavivirus using IRES initiation

HCV, BVDV (which has cap, but uses IRES)

59

Flavivirus using cap dependent initiation

Dengue. Possibly. It has a cap at least.

60

Different Vpgs.

Picorna Vpg: 22aa, covalently attached.
Calici Vpg: 13-15 kDa, essential for replication. Also protective.

61

Role of Vpg in picorna

Protection endonucleases, avoidance of RLRs.

62

Roles of 5' caps

1) enhance translation
2) protect from 5' endonucleases
3) prevent detection by RIG-1.

63

Viral mRNAs: overcoming lack of polyA tail.

Overcomes natural decrease in stability and translation efficiency by 3' UTR structures which interact with cellular factors. E.g. PABP.

64

How IRES works - Hep C Virus

40S subunit binds IRES without proteins, since structure mimics E and P site. eIF3 needed for subunit joining. No scanning as functional AUG is internal to the virus.

65

How IRES works - Type I and II

Requires all eIFs to initiate translation, except eIF4E. Requires ITAFs.

66

Translation of Calicivirus dependent on Vpg

Required for calicivirus infectivity;
Burroughs et al 1978. Translation inhibited if RNA is treated with proteinase K prior to this.

67

Calicivirus translation.

VPg present. Has subgenomic RNA (ORFs 2 and 3). Genomic RNA has ORF1, which produces a polyprotein.

68

Calicivirus Vpg acts as proteinaceous cap-substitute how?

By binding eIF4E.

69

Calicivirus Vpg binds eIF4E in infected cells

Chaudhry et al. Recombinant norovirus Vpg binds eIF4E in ELISA. But is not required for translation in vitro. Vpg forms direct protein-protein interaction with eIF4G.

70

Types of initiation for Rdrp

De novo
Primer dependent

71

Where does replication of +ive ssRNA take place?

Either on or in membranous vesicles in cells.

72

Picornaviridae common viruses

Picornaviridae common viruses

73

Replication of +ive ssRNA - induction

by membrane bound replicase components, containing NTPase. 2B or 2BC thought to be inducing it.

74

Replication of +ive ssRNA - dependent on...

Host cell factors, which interact with the viral proteins and RNAs, and are recruited to the membranes.

75

Picornaviridae (coding region)

Single open reading frame. P1 is capsid proteins VP2, 3, 1. P2 and P3 are non-structural proteins.
Firth suggested another open reading frame.

76

Altering proportions when polyproteins are made.

1) terminate at different stages.
2) contain multiple copies of a protein - Apthovirus has multiple copies of 3B - rapid replication.
3) Use different proteases to give a cascade of functionally different proteins.

77

Poliovirus ends

Not capped but polyadenylated.

78

Cellular mRNA translation initiation.

5' cap recruits cap binding complex.
Recruits 40S subunit.
Scans until first AUG is found and 60S subunit is recruited.
Circularisation acts via PABP binindg to eIF4G.

79

Proteases in picornaviruses

2A and 3C

80

2A in picornaviruses

Protease; cleaves P1 from P2/P3

81

3C protease in picornavirus replication.

3Cpro or 3CD precursor cleaves P1, P2 and P3 into final proteins.

82

Picornavirus host cell manipulation

Host cell shut off by cleavage of eIF4G preventing translation
cleavage of TATA binding protein (by 3C) preventing transcription.
Alter localisation of proteins by cleaving nuclear pore complex proteins.

83

Cleavage of eIF4G in picornaviruses

By protease 2A or L. Cleaves 4G and so separates eIF4E and eIF4A. But the 4G-4E product readily acts at IRES.

84

Picornaviruses - the nuclear pore complex

Cleavage of Nup153, part of nuclear ring, and p62. Allows redistribution of nuclear proteins for use of virus in cytoplasm.

85

Synthesis of VPg-pUpU primer in picornaviruses.

The cre(2C) structure acts as a recruiter for proteins, and A6 and A7 act as template for pUpU.

86

The Vpg-pUpU action in picornaviruses

Acts as primer for 3Dpol.

87

Coronavirus genome replication.

Genome is translated to give replicase. This replicates the RNA to give an antisenseRNA, which is replicated to give a +ive sense RNA.
A subgenomic antisense RNA is then made by pausing at certain sites, allowing the RNA to hybridise to near the 5' end and so finish.

88

Rdrp specificity for polymerases

4 common motifs –ABCD. C; Gly-Asp-Asp is what gives specificity for RNA.

89

Results of Rdrp being error prone.

Cloud of viruses round consensus sequence.
Upper limit on genome size.

90

Quasi-species key-points.

• Sub-populations can modify expression of phenotypic traits
• Contribute to pathogenesis
• Mutant spectra are the target on which drift and selection act.

91

Rdrp upper limit on genome size - key points.

multifunctional proteins.
Potential for viral intervention?

92

Efficient utilisation of the template in picornaviridae

Oligomerisation of Rdrp.
Circularisation of RNA due to 3CD and PCBP2. PolyA tail vital.

93

Initiation of replication picornaviruses.

Vpg and pUpU required for +ssRNA – priming. 3CD binding cre, recruit Rdrp (3D?) and vpg; uridylates vpg, used as primer.

94

Initiation of replication togaviridae

conserved sequence elements, but possibly not involving polyA tail. Probably host factors.

95

Initiation of replication - caliciviridae.

Vpg much larger, essential for translation. Interaction with cellular factors.

96

+ssRNA viruses: choosing between translation and replication.

Location.
Inhibiting translation initiation.
Cleavage of IRES bound PCBP.

97

+ssRNA viruses: choosing between translation and replication. Location.

On membranous structures – induced by proteins 2B or 2BC; induced by membrane bound replicase components. 3AB anchors replication to structure (DIAGRAM), possibly stimulates 3D pol.

98

+ssRNA viruses: choosing between translation and replication. Inhibiting translation initiation and stimulating replication complex.

Binding of 3CD to cloverleaf formation recruits host factors that cause replication and decrease affinity of those that cause translation.

99

+ssRNA viruses: controlling packaging.

Massive bias towards +ssRNA production.

100

Picornavirus: 2B or 2BC

Accumulates on Golgi, many host protein effects including formation of vesicles.

101

Picornavirus role of 3A

Membranous vesicle formation. Disrupts ER-to-Golgi trafficking.

102

Picornavirus role of 3AB in replication.

multifunctional. Anchoring replication complex to membranous vesicle? Stimulates 3CD protease, anchors 3D pol, possibly stimulates 3D pol

103

Picornavirus role of 3CD protease

Processes P1 precursor. Contributes to circularisation.

104

Picornavirus role of 3D.

Polymerase. Oligomerization for template utilisation.

105

Cis elements RNA secondary structures in picornavirus.

5' UTR stem loop I.
PolyA tail on 3'.
Synthesis of Vpg-pUpU.
IRES.

106

Picornavirus: 5' UTR stem loop.

bound by 3CD and PCBP2 for circulisation

107

Picornavirus: polyA tail

1) bound by 3CD and PCBP2 for circulisation
2) initiation for intermediate ssRNA synthesis if enterovirus. Binding of 3CD and 3AB for replication.

108

Picornavirus: switch from translation to replication.

Binding of 3CD to cloverleaf promotes replication (maybe). Recruits host factors that cause replication, decrease affinity of these for sites causing translation.

109

Basic replication cycle for +ssRNA

o Entry
o Translation first for synthesis of Rdrp.
o Replication of genome
o Capsid assembly
o Aggregation and egress

110

Poliovirus unanswered question.

How does it select the correct AUG? Many in 5' UTR, but does not translate multiple ORFs. Not primary sequence that we can see.

111

Picornavirus description of genomic replication.

• Sense as template
• Formation of dsRNA replicative form
• Antisense = replicative intermediate. Many more initiations.

112

Initiation of replication in +ssRNA not polioviruses.

de novo priming, in which polymerase binds template and 2 NTPs, synthesises first phosphodiester bond, then elongates.

113

Origin of membranous vesicle for replication of +ssRNA viruses.

Origin of vesicle uncertain: ER or autophagosomes suggested. Appears to require Arf.

114

Anchoring to membranous vesicle in picornavirus replication.

3AB has a loop inserted into the membrane of the membranous vesicle. Also binds PCbp, which binds the cloverleaf structure.

115

RNA structures that can act as cis-acting factors.

Stem loops, hairpin loops, bulge loops, interior loops and multibranched loops, pseudoknots.

116

Coronaviruses translation of genomic RNA

replicase-transcriptase protein genes.
ORF1a and ORF1b (via frameshift) result in 2 polyproteins, pp1a and pp1ab.

117

Coronavirus replication - proteins.

Non-structural proteins recruited to replication-transcription complexes. Includes replicase-transcriptase gene products from ORF1. Also include N protein and some cellular proteins.

118

Discontinuous replication model (coronaviridae). Detailed.

Synthesise -ssRNA subgenomic RNA from full length genomic RNA. Copy this to make +ssRNA subgenomic RNA.
 Initiation of synthesis at 3' end of genomic RNA, to make 5' end of subgenomic -ssRNA
 Elongation until first functional body TRS motif encountered.
 Some RTCs with stop synthesis here, and relocate, guided by complementarity between 5' end of the subgenomic RNA and leader TRS motif.
 Translocated strand extended by copying the 5’ end of the genome. This is then copied to give the positive strand subgenomic RNA.

 N protein appears important in maintaining production of genome-length templates.

119

Unanswered questions about discontinuous replication.

Which signal in the TRS stops transcription?
How is relocation mediated?
Is there a role for splicing of the -ive strand which is discontinuously copied? Splicing is ruled out in the mRNAs.

120

 Synthesis of VPg-pUpU primer

• 3CD dimers bind Cre
• 3D binds, uses adds pUpU to VPg using cre as a template.
• To allow this sort of priming, active site must be more accessible than for viruses using de novo initiation.

121

MLV stop-codon read-through

Pseudoknot downstream from stop codon.
If open ,then translation terminates.
If tight - due to protonation of a residue - then translation progresses.

122

Poliovirus genome

7.5kb long genome
No cap but VPg instead at 5’ end
Then has 5’UTR which is not linear but has secondary structure which is very important for protein binding in replication —> must be unwound for passage of genome —> length and secondary structure effects translation efficiency
3’ UTR can regulate translation initiation and efficiency, and mRNA stability, with the poly(A) tail being necessary for efficient translation

123

Similarities between IRESes

Few, if any, convincing similarities between IRES elements in terms of sequence, size, or structure except for those from families of related viruses —> implication is that there is no universal mechanism of internal ribosomal entry.

124

Differences with Type II IRESes

Binds 40S directly
Different family - EMCV and FMDV.
Different factors (e.g. different PTB binding).

125

Control of translation replication switch polio.

CLOVER LEAF : critical conc of 3CD reached binds the clover leaf → switch
mutate the clover leaf: more translation less negative strand synth
Build up of 3CD and 3D
mutations of 3CD or the cloverleaf binding site shift balance to translation
PCBP2 in association with stem-loop B of clover leaf enhances translation ten fold
3CD cleaves PCBP2 and competes for binding site
Cleavage of PCBP2 (Kirkegaard 2014)
cleaved PCBP2 still has a role in replication IS THIS THE SWITCH
localisation
nuclear recruitment

126

Type I IRES recruitment of 43S

eIF4G/A binding recruits 43S.
eIF4G binding modified by PTB binding.
43S positioning modified by GARS.

127

Corona: subgenomic RNAs code for...

NS, Spike, E, M and N proteins.