World of Viruses

Question 1

Explain the dUTP problem

Answer 1

dUTP levels are high in non dividing cells and incorporation into DNA is potentially mutagenic. Cells have dUTPase which converts it into TTP with thymidylate synthase and thymidylate kinase.
This reduces levels of dUTP but also causes and increase in TTP favouring DNA synthesis.

Question 2

Which viruses have dUTPase

Answer 2

Poxviruses
Herpesviruses
Some retroviruses

Question 3

Which viruses also encode thymidylate kinase

Answer 3

Herpesvirus

Poxviruses

Question 4

Guanosine analogies as antivirals

Answer 4

Acyclovir and Gancyclovir.
Compete for guanosine and also acted on by viral TK enzyme (more promiscuous) which causes it to be incorporated into either the cell DNA or the viral DNA killing either them both.

Acyclovir –(VIRAL TK)–> Acylclovir monoP –(Cell TK)–> Acyclovir triP –> DNA

They are prodrugs as they have to be first acted on by the viral TK making them non-toxic in a normal cell.

Question 5

Viral uracil-DNA glycosylase (UNG)

Answer 5

Excises uracil if it has been incorporated into DNA leaving an abasic site which is repaired by cellular enzymes.

Question 6

Which viruses encode UNG

Answer 6

Herpes and poxviruses

HIV incorporates cellular UNG into its virus particles.

Question 7

Which viruses encode ribonucleotide reductase

Answer 7

Herpes and poxviruses

Question 8

Viral ribonucleotide reductase

Answer 8

Two subunit enzyme which converts NTPs into dNTPs by removing an OH group.
Increases pool of dNTPs for DNA synthesis - needed in non-dividing cells e.g. neurons where herpes reside as they have high NTPs and low dNTPs.

Question 9

Viral ribonucleotide reductase as an antiviral

Answer 9

Could have been a good antiviral drug but the cell also contain ribonucleotide reductases and its hard to specifically target the viral form.

Question 10

Explain cytosine deamination - an anti-retroviral response

Answer 10

Cellular encoded APOBEC3G cytosine deaminase - enzyme which deaminates cytosine to produce uracil.
First discovered in HIV.
Acts during reverse transcription where it converts C –> U in ~10% of the genome. RT means an A is incorporated into the complimentary strand (should have been a G).
These hypermutations cause misreplication and no virus produced or UNG pathway is activated which can lead to degradation.

Question 11

Explain HIVs response to APOBEC3G

Answer 11

HIV encodes a protein called Vif. Vif binds to APOBEG3G targeting it for degradation via the proteasome system.

Elongin B/C, CUL5 and RBX1 are the enzymes recruited by Vif.

Question 12

Explain ADAR

Answer 12

ADAR = Adenosine deaminase.
Acts on dsRNA and converts A –> I (Inosine)
Inosine base pairs with cytidine (A normally base pairs with U in dsRNA so generates a mutation).

Effects of this mutation include: RNA degradation, change in RNA structure (I:C less stable than A:U), mutations in mRNA replication and translation (loss of termination stop codon)

Question 13

Hep Delta Virus (HDV) - basics about the virus

Answer 13

Replication depends on presence of hep B.
Very small virus with unusual genome - circular RNA encoding 1 protein called the Delta antigen.
Infectious particles consist of nucleocapsid (D antigen and RNA) surrounded by envelope containing Hep B virus glycoprotein (share a receptor).
Coinfection leads to higher likelihood of severe acute or chronic disease.

Question 14

Explain - Hep Delta Virus (HDV) replication and RNA editting

Answer 14

HDV does not encode its own polymerases so requires the cells. RNAP1 generates the anti-genome and RNAP2 makes the genome from the anti-genome and makes the mRNA.

Contains 2 stop codons - antigenome can be editted by ADAR which causes the first stop codon to be changed to a Trp residue allowing the translation of an extra 19aa.
Therefore get 2 forms of the delta antigen - large and small.

Question 15

Overview of host cell protein synthesis

Answer 15

See a diagram.

eIF4F complex: eIF4A (ATP dep. RNA helicase), eIF4E (binds m7G cap) and eIF4G (scaffold)
43S complex: eIF1, eIF1A, eIF2 (GTP and met-tRNA), eIF3, eIF5, 40S ribosome subunit.
PABP x2 and MNK1 kinase which P’s eIF4E.

Question 16

Explain how eIF4E is regulated

Answer 16

4EBP1 binds eIF4E and sequesters it from the eIF4F complex and get no translation.
When 4EBP1 is P it can no longer bind eIF4E, which can be part of the eIF4F complex allowing translation to occur.

Question 17

Regulation of 4EBP1

Answer 17

Complex regulation of 4EBP1 - see a diagram.

Receptor tyrosine kinase activates P13K which activates Akt which blocks TSC. TSC converts RHEB-GTP into RHEB-GDP. The GTP form activates MTORC1 which phosphorylates eIF4E and allows transcription initiation.
Nutrient deprivation activates AMPK activating TSC and Hypoxia also activates TSC which ultimately through the same pathway as above blocks P of eIF4E and therefore translation.

Question 18

Regulation of 4EBP1 - Viruses which activate P13K therefore inhibit trasnlation

Answer 18

HCV
HVP-16
EBV
HIV-1
KSHV

Question 19

Regulation of 4EBP1 - Viruses which block TSC therefore inhibit translation

Answer 19

HSV-1
HCMV
HPV-16

Question 20

Regulation of 4EBP1 - Virus which activates mTORC1 therefore actives translation

Answer 20

West Nile virus

Question 21

Regulation of 4EBP1 - Viruses which increase 4EBP1 binding to eIF4E therefore inhibit translation

Answer 21

VSV and Polyomavirus

Question 22

Regulation of 4EBP1 - Virus which increases P of 4EBP1 therefore activates translation

Answer 22

EMCV

Question 23

Viruses which cleave eIF4G

Answer 23

Picornaviruses (Polio and rhino) - 2A protease
FMDV leader protease
Calicivirus 3C protease
Retrovirus (HIV, HTLV-1) protease

Question 24

Virus proteins which interact with eIF4G

Answer 24

Influenza NS1 - physical binding prevents translation
Adenovirus 100K - Binds and excludes MNK1 preventing P of eIF4E
Rotavirus nsp3 - Binds and prevents interactions with PABP so only get one round of translation and then it falls off

Question 25

Viral proteases which cleave PABP

Answer 25

Picorna virus 3C and 2A proteases Calicivirus 3C protease Lentivirus e.g. HIV protease

Question 26

Herpes simplex virus protein vhs and eIF4F dysregulation

Answer 26

vhs protein is a component of the virus particle (tegument) and is an mRNA-specific endoribonuclease which interacts with eIF4A and degrades the mRNA

Question 27

Explain cap snatching and give examples of viruses which do this

Answer 27

Viruses needs to have a cap on its own mRNA to allow it to be translated therefore these viruses snatch the cap from cellular mRNAs using a protease/nuclease. Vaccinia virus Influenza virus Hantavirus This both blocks host cell translation and allows virus proteins to be translated.

Question 28

Explain regulation of translation by eIF2

Answer 28

eIF2 has 3 subunits: alpha, beta and gamma eIF2 is a critical part of the initiation complex and needs to be recycled by eIF2B (guanine nucleotide exchange, GDP --> GTP in gamma subunit) so it can reinitiate translation. This is regulated by the alpha subunit of eIF2 - If Serine 51 is P it switches off this whole process by preventing the interaction with eIF2B therefore get no GDP switch and no recycling. Delayed block of translation as doesn't prevent the first round of translation.

Question 29

Name the eIF2 kinases and what they respond too

Answer 29

GCN1 - responds to aa starvation to UV HR1 - responds to haem deprivation PERK - responds to ER stress PKR - responds to dsRNA (part of innate immune response).

Question 30

Explain protein kinase R (PKR)

Answer 30

Responds to dsRNA which is a pathogen-associated molecular pattern. It autophosphorylates and then phosphorylates eIF2alpha which inhibits translation. PKR consist of a regulatory domain (binds to dsRNA) and a catalytic domain (dimerisation and phosphorylates substrates). Expression is stimulated by IFN but activation requires binding to dsRNA. Activation of PKR also stimulates IFN genes.

Question 31

Viruses which inhibit PKR

Answer 31

Polioviruses activates a cellular pathway to degrade PKR (d not fully understand yet) Vaccinia E3L, Reovirus sigma3, Rotavirus NSP3 and influenza NS1 - are dsRNA binding proteins which sequester the dsRNA so it cannot active PKR. Adovirus VAI RNA, EBV EBER RNA and HIV Tar RNA - produce lots of short dsRNA which is able to bind to PKR but fails to activate it as it is too short to allow dimerisation. Hep C NS5A - binds to PKR and blocks dimerisation Vaccinia K3L protein, HSV US11 protein, HIV Tat protein - act as a substrate for PKR and therefore inhibit (psuedosubstrate). Herpes simplex virus (HSV) gamma1 - 34.5 recruits a phosphatase called PPIalpha which removes the P on eIF2alpha added by PKR.

Question 32

Mechanism of Vaccinia virus K3L inhibition of PKR

Answer 32

PKR is a kinase so transfer P group. K3L protein have homology to eIF2alpha but Ala not Ser (no OH group) which locks PKR + ATP onto the K3L protein in an inactive form.

Question 33

Explain internal ribosome entry sites (IRES) and give examples of viruses which have them

Answer 33

IRES are an RNA sequence which fold up with complex secondary structures and allow cap-independent initiation of translation. 43S ribosome can bind directly onto the IRES with a reduced requirement for initiation factors. Exmaples of viruses: Picornaviruses, Flaviviruses (e.g. hep C) and lentiviruses 4 classes of IRES - (1-4) which have different requirements for initiation factors Can use CryoEM to visualise IRES interactions

Question 34

IRES type 1 example

Answer 34

Poliovirus IRES binds to the C-terminal domain of eIF4G (poliovirus cleave eIF4G) Requires eIF5, eIF2, Met-tRNA, eIF4A, MNK1 Stimulated by eIF1 and eIF4A

Question 35

IRES type 3 example

Answer 35

HCV IRES binds to eIF3 and 40S ribosome Requires eIF5, eIF2 and Met-tRNA No requirement for eIF4F

Question 36

Calicivirus protein synthesis (Vpg protein)

Answer 36

+ sense ssRNA e.g. Norovirus, winter vomiting disease Genome has a 5' Vpg (protein) but no IREA Subgenomic RNA also has 5' Vpg - translated onto structural proteins Vpg binds to eIF4G and eIF3 (may be others as has homology to eIF4A) Recruits the 43S complex Ofr1 only 10x nucleotides from the 5' end so no ribosome scanning Vpg is a protein cap analogue

Question 37

Benefits of a single translation initiation event

Answer 37

+ sense RNA viruses have a single open reading frame which is translated into a polyprotein and then cleaved by proteases e.g. Hep C has 10x proteins from 1 polyprotein Advantageous as initiation is the rate-limiting step in translation so only need to recruit the factors and machinery once to get 10x proteins. Much for efficient for the virus.

Question 38

Benefits of RNA virus replication platforms

Answer 38

Increase local concentration of enzymes and substrates Increase local reaction rates Correct orientation of components Protection from cytoplasmic innate immunity Cytoplasm is a complex environment for a virus - high protein conc ~200mg/ml, lots of membrane structures, cytoskeleton and trafficking.

Question 39

Picornavirus life cycle and polyprotein

Answer 39

E.g. Polio or FMDV ssRNA + sense, non-enveloped Genome is translocated across the plasma membrane and viral proteins are translated, RNA is replicated and the virus particles are assembled before cell lysis and virus release. Genome produces a polyprotein which is cleaved into P1, P2 and P3 P1 - structural proteins: VP4, VP2, VP4 and VP1 P2 - 2A (protease), 2B, 2C and 2BC P3 - 3A (tail mem anchored protein 10kDa), 3B (Vpg), 3C protease) and 3D (polymerase). Replicates and assembless on cytoplasmic double-membraned vesicles - electron micrographs.

Question 40

Explain the role of phosphatidylinositol-4-kinase (PI4K) in cholesterol trafficking

Answer 40

Lipid kinase which converts PI into PI4P on the membrane OSBP protein moves PI4K to the ER or other sites in the cell while also trafficking cholesterol in the opposite direction - cholesterol accumulates on the Golgi/TGN which drives curvature due to the asymmetry in the lipid bilayer.

Question 41

Which virus and viral protein recruits (and stimulates) PI4K(3beta isoform) to the Golgi/TGN?

Answer 41

Picornavirus protein 3A - 10kDa tail-anchored membrane protein. It is associated with the Golgi/TGN membranes and recruits PI4K3beta which causes a 6x fold increase in PI4P lipids at the Golgi/TGN. The Golgi gets disassembled and membranes become 'replication organelles'

Question 42

Anti-picornavirus compounds targeting OSBP

Answer 42

Enviroxime family - major target is PI4K however some target OSBP. The anti-fungal drug itraconazole also inhibits OSBP and some picornaviruses

Question 43

PIK93 is an inhibitor of what protein and what viruses?

Answer 43

Inhibits PI4K and therefore inhibits some picornaviruses like Polio, coxsackie virus B and human rhinovirus Does NOT inhibit FMDV so this must have an alternative mechanism for membrane remodeling.

Question 44

Describe the Hep C virus polyprotein

Answer 44

+ sense RNA, enveloped virus which makes a polyprotein. Polyprotein consists of 10x proteins with the structural at the 5' and the non-structural at the 3'. Structure proteins: Core, E1, E2, p7(?) Non-structural proteins: NS2, NS3, NS4A, NS4B, NS5A, NS5B.

Question 45

Describe the HCV lifecycle

Answer 45

Binding --> Receptor-mediated endocytosis --> Fusion and uncoating --> Translation and poly protein prcessing --> RNA replication --> Virus budding into intracellular vesicles --> Transport --> Fusion and release

Question 46

Explain the 'membraneous web'

Answer 46

Double membraned vesciles (DMV) which concentrate the recplication compentents and protect against innate immune response (dsRNA). 1000:1 NS5B protein to - RNA The membranes are distint from the ER - still closely associated though.

Question 47

Which HCV protein recruits PI4K

Answer 47

NS5A binds to PI4K(3alpha) and stimulates it's activity increasing PI4P levels. P315A mutant causes a 10x reduction in replication and loss of PI4K activation and colocalization. Also disrupts Golgi structure as a consequence of PI4K activation.

Question 48

Explain autophagy

Answer 48

'Self-eating' - degradation of cytoplasmic proteins and organelles which can provide energy under metabolic stress. Perturbation of autophagy can be involved in diseases e.g. degenerative conditions, cancer and virus infection. Stages of autophagy: Form a double membrane vesicle - autophagosome containing the components to break down. This autophagosome fuses with lysosomes to form an autolysosome where the double membrane is important in keeping the contents separate from the cell. Lysosome has a permease which allows nucleotides, lipids etc back into the cell. It is a very efficient way of breaking down cellular compartments. Early stages of autophagy use Beclin, LC3, PE lipid - see diagram. Can fuse LC3 and GFP to visualize

Question 49

Poliovirus and autophagy

Answer 49

Polio switches on autophagy and stimulated production of lipids in general for the production of membranes, more specifically to dervives more autophagosomes from the ER - viral proteins which do this are 3D, 3A and 2BC. Think the virus induces autophagy as a double-membraned vesicle as a platform for viral replication - don't know if it is inside or outside. The virus is blocking some stages of late autophagy as doesn't make sense for degradation to happen

Question 50

Can autophagy be anti-viral

Answer 50

Yes - Autophagic vesicles containing the virus can fuse with endosomes and with lysosomes and degrade them.

Question 51

Which viruses inhibit autophagy?

Answer 51

Herpesviruses: - HSV-1 gamma1 34.5 protein binds to Beclin - Kaposis sarcoma herpesvirus (KSHV) M11 binds to Beclin - Human cytomegavirus (HCMV) TRS1 binds to Beclin Adenovirus E1B-19K HIV - Nef protein binds to Beclin and inhibits the late stages of autophagy (lysosome fusion)

Question 52

The 2 ways apoptosis can be induced

Answer 52

Intrinsic signals e.g. inappropriate DNA synthesis, shut off of protein synthesis Extrinsic signals e.g. CTL, NK cells, cytokines

Question 53

Name the viruses which can inhibit apoptosis

Answer 53

Adenovirus - linear dsDNA genome, early/late gene expression and causes respiratory, GI tract and eye infections Human papilloma virus (HPV16) - circular dsDNA genome, early/late gene expression, causes cervical carcinoma Simian virus 40 (SV40) - circular dsDNA genome, not associated with disease but has transforming proteins which can induce tumours.

Question 54

Describe the p53 pathway - Intrinsic apoptosis

Answer 54

See diagram mdm2 Ub's p53 for degradation - in normal cells p53 is turned over rapidly. However under low rNTPs, hypoxia, DNA damage from gamma/UV radiation p53 gets phosphorylates which stabilizes it can it is no longer a substrate for mdm2. This P allows it to act as a TF to switch on genes responding to stress or activate the mitochondria apoptosis pathway with Bax (pro-apoptotic factor)

Question 55

Describe pRb and the cell cycle

Answer 55

Rb is associated with E2F through G1 phase which keeps E2F inactive. When the cell is ready to enter S phase, Rb becomes P by CDK2/4/6 (which is blocked by p21) and this released E2F which can switch on genes involved in DNA replication and cell cycle progression

Question 56

Describe the coordination of p53 and pRb

Answer 56

P-p53 can block DNA replication and cell cycle progression if Rb function is lost. So if you mutate either p53 or Rb the other will step in a block the processes.

Question 57

Which (best-characterised example) virus and it's proteins modifies pRb and p53?

Answer 57

HPV16 is the best-characterised example. E6 targets P-p53 for degradation E7 targets Rb for degradation This allow DNA replication and cell cycle progression to continue allowing replication of the DNA genome, virus is not trying to create a tumour just trying to replicate its genome efficently

Question 58

Viral proteins which target p53

Answer 58

``` Adenovirus E1B 55K, E4-Orf6 HPV E6 protein SV40 large T antigen HBV pX protein HCV NS3 and NS5A ```

Question 59

Viral proteins which target pRb

Answer 59

Adenovirus E1A HPV E7 protein SV40 large T antigen HCV virus NS5B

Question 60

Explain the role of mitochondria in apoptosis

Answer 60

See diagram. BCL-2 is anti-apoptotic and keeps the mitochondrial permeability transition pore closed and Bax is pro-apoptotic and keeps the MPTP open. When the pore is open this causes the release of cytochrome C and the loss of the membrane potential. Cytochrome-C, Apaf-1 and dATP form the apoptosome which induces apoptosis through the activation of caspase 9, which in turn actives caspase 3 leading to cell death. The ratio of pro-apoptotic to anti-apoptotic factors is important in determining whether a cell will apoptose

Question 61

Which viral proteins target mitochondrial apoptosis as they are BCL-2 homologues

Answer 61

Adenovirus E1B/19K Herpes virus BCL-2 like proteins (EBV BHRF1) African swine fever virus A179L Vaccinia virus N1 and F1L All keep the pore closed and inhibit the loss of membrane potential and cytochromeC

Question 62

Which virus has a novel interaction with complex 1

Answer 62

``` Human cytomegalovirus (HCMV - betaherpes virus) Has an abundant 20% untranslated RNA produced early in infection called beta2.7 which is mitochondrially located and binds to complex 1 of the ETC and stabilises it protecting it from metabolic stress ```

Question 63

Describe the ER stress response

Answer 63

ER stress is caused by the accumulation of unfolded proteins by Ca2+ imbalance, hypoxia or viral infections. This activates the unfolded stress response (UPR) which tries to restore the balance by reducing protein translation and inducing ER chaperones however if it is unsuccessful it induces mitochondrial apoptosis by Ca2+ release and caspase 12 activation. 3 proteins located in the nucleus which are activated by Bip disassociation: 1) . IRE1 which moves into the nucleus where it recognises a single mRNA for the transcription factor Xbp1. IT is an endonuclease which splices out 18bp which allows the translation of an active transcription factor which can bind and active genes involved in the UPR. 2) . ATF6 which is cleaved from the membrane which allows it to enter then nucleus and bind to the promoter or genes to address the balance. The gene involved are for proteins involved in protein folding and/or degradation. 3) . PERK which P's eIF2alpha and blocks translation

Question 64

Does HCV block the UPR?

Answer 64

No - RT-PCR shows the short form of Xbp-1 to be present but it is still able to replicate in the presence of ER stress so don't fully understand this yet

Question 65

Which viruses block the activity of PERK?

Answer 65

Dengue and HSV

Question 66

Which virus blocks the activation of ATF6?

Answer 66

HCMV

Question 67

Do you need all 3 protein of the UPR to be active to induce a stress response or just one?

Answer 67

Just one and this is why viruses just target one protein in this pathway

Question 68

Explain extrinsic apoptosis

Answer 68

See diagram. FAS ligand binds to the FAS receptor and the death domain of FAS interacts with other death domain proteins e.g. FADD which activate caspase 8 and this activates caspase 3 from procapase 3 leading to cell death. TNF is a soluble factor released from cells which can bind to the TNFR which also contains a death domain and can activate as above. However also activates cPLA2 which is a phospholipase enzyme and translocates to the membrane acting on arachidonic acid which activates inflammation and apoptosis.

Question 69

Adenoviruses and apoptosis

Answer 69

dsDNA genome which produces different RNAs which can be divided into early and late. 5 proteins are produced from the E3 transcript and these regulate extrinsic apoptosis. One is involved in MHC1 downregulation and the 4 others where 2 of these form a complex called RID. RID targets the Fas and TNF receptors and internalises them into an endosome then lysosome so they get degraded therefore they cannot respond to pro-apoptotic factors coming from outside the cell. 14.7K protein targets caspase 8 and inhibits its function. RID complex and 14.7K associate with cPLA2 and prevent it from going to the surface and therefore preventing apoptosis. 6.7K co-operates with RID to endocytose other TNF receptor family proteins called TRAIL which respond to a different cytokine, they also have death domains and interact with FADD to active caspase 8 and adenovirus also blocks these. The E3 region of adenovirus has specifically evolved to prevent extrinsic apoptosis

Question 70

Which viral proteins have homology to death domains

Answer 70

vFLIPs (viral FLICE-like inhibitory proteins (FLICE-FADD-like)). Bind to the FADD proteins. - Molluscum contagiosum (poxvirus - benign cutaneous lesions) - MC159 protein - gamma-herpes viruses like Kaposi's sarcoma herpesvirus

Question 71

Which viruses inhibit caspases?

Answer 71

Cowpox CrmA, Rabbitpox SPI-1, Vaccinia SPI-2 - Serpins (SERine Protease inhibitors) - CrmA inhibits caspases and gramzyme B Vaccinia F1L (BCL-2 inhibitor) - Specific to caspase 9 Mousepox p28 and rabbitpox N1R -Specific to inhibit caspase 3 activation and induced by UV Other DNA viruses like adenovirus E3 - 14.7K protein Blocking caspase inhibits both extrinsic and intrinsic apoptosis

Question 72

What is the hallmark of late stages of apoptosis?

Answer 72

Phosphotidyserine (PS) on the cell surface. Normally Flippase (ATP11C) flips it back but caspase cleavage of this means PS on surface and can be recognised by specific receptors on other cells e.g. phagocytes

Question 73

Is PS recognised directly or indirectly

Answer 73

Both Direct - e.g. T-cell immunoglobulin and mucin receptors (TIM) Indirect - e.g. bridging molecule GAS6 is a soluble molecule which binds to PS and interacts with receptors

Question 74

Does apoptosis induce an inflammatory response?

Answer 74

No - hence this is useful for viruses

Question 75

What happens when apoptotic bodies/PS binds the receptor of other cells?

Answer 75

Activates a cellular signalling pathway which actives TFs to switch on genes which will block the inflammatory response and the innate immune response. They will produce cytokines e.g. IL-10 which is anti-inflammatory and produce SOCs proteins which inhibit the innate immune response.