Antiviral system of the host / Innate immunity / Viral antagonists of the innate immune system

Question 1

Virus defense

Answer 1

• Innate immunity / resistence („innate“, „inborn“, „natural“ immunity
  „natural“ resistance)
• Adaptive immunity („acquired“ immunity, „specific“, „adaptive“ Immune response)

Question 2

Virus defense more specific

Answer 2

Innate immunity
Immediate response
- Prevents infection
- Blocks infection / stops virus spread
- Decelerates virus infection: gain of time
- physical /chemical barrier
- solvent factors
Interferons, cytokines, defensins
- cellular
Macrophages / Monocytes
NK-cells (natural killer cells)
Granulocytes

Adaptive immunity
Needs time to evolve
1.Full recovery: overcoming of first infection (immunosuppressed, newborn) 2. Protection against re-infection (active vaccination!)
3. Immunopathology (excessive or misdirected immune answer)
4. Auto-immunity
5. Co-existence (virus persistence, reactivation from latency)
-> Humoral or Cellular

Question 3

Immune response against viruses

Answer 3

1. Induction of interferon (innate, unspecific)
   - time period within hours after infection (p.I.)
2. Proliferation of T-killer cells (CD8) (acquired, specific)
   - specific lysis of virus-infected cells
   - T-cell receptor, viral antigen on the target cell/MHC class I - time period within days p.I.
3. Proliferation of B-cells (acquired, specific)
   - Secretion of specific antibodies against viral antigens - time period within weeks p.I.

Question 4

Active components of the innate immune response

Answer 4

1. Preset components
   - Physical barrier
   - Cells of defense: NK-cells, Macrophages, etc. - Chemical substances
2. Inducible components
   - humoral signaling molecules
   * Interferons (IFN) of typ I: a-IFN b-IFN w-IFN, k-IFN
   -> induce „antiviral “ state
   * Chemokines
   * Other cytokines

Question 5

interferon

Answer 5

• Interferon Type I
• Interferon-a („leukocytes-interferon“) is produced by virus-infected leucozytes
• Interferon-b („fibroblasts-interferon“) is produced by virus-infected fibroblasts or virus-
  infected epithelial cells
• IFN-ω, IFN-k, IFN-t
• Signalling trough IFN-α receptor (IFNAR 1 or 2)
• Inteferon type II
• Interferon-g („immune-interferon“) is produced by specific activated T-cells and NK-cells
• Interferon-g is generated as response to antigens (including viral antigens) or mitogenic stimulation of lymphocytes
• Activates macrophages
• Signalling through IFN-g receptor (IFNGR)
• Inteferon type III (IFN-l? still under debate)
• Role in clearance of HCV persistence upon interferon-a treatment?

Question 6

Interferon typ I (alpha und beta)

Answer 6

• In normal cells no expression
• Expression inducible in all cell types via:
  -> Bacterial elements (Lipopolysaccharides)
  -> Infection with viruses (DNA and RNA), especially dsRNA

Consequences of interferon induction:
- Induction of expression of up to 300 proteins (interferon-stimulated gene - ISG)
- Antiviral state of the cell

Question 7

Interferon induced gene expression

Answer 7

• Regulation of gene expression
• Genes in the genome (DNA within the nucleus)
• Regulation of expression of individual genes by selective transcription (TS) of specific mRNAs
• Regulation of transcription by proteins (transcription factors), which bind to regulatory DNA sequences 5‘ of the gene promoter and so stimulate TS via the cellular RNA Polymerase II
• In the INF-system:
  -> IRF: Interferon regulatory factors (TS factors)
  -> PRD: Positive regulatory domain (regulatory sequences)

Question 8

The Interferon alpha/Beta System

Answer 8

Consequence of Interferon stimulation: antiviral state

Question 9

Protective effect of IFN-alpha/Beta

Answer 9

Stain of cells by Cristal violet: no cells - no stain = plaque

Virus-proliferation (plaques) after IFN-treatment

Question 10

Viral PAMPs
-> Ds viral DNA as trigger

Answer 10

Binding of viral ds DNA to cGAS (nucleotidyl transferase) stimulates the synthesis of cGAMP (cyclic di-nucleotide). STING: (ER)-located stimulator of IFN genes. After stimulation by cGAMP STING ativates TANK-binding kinase 1 (TBK1) to phosphorylate IRF3.

Question 11

How is intracellular dsRNA detected by the cell
1. Proteinkinase R (PKR)

Answer 11

-> Constitutive expression within many cell types
-> Two dsRNA-binding motives
-> Binding of dsRNA leads to autophosphorylation and so to the activation of PKR
-> Activation of PKR leads among other things to activation of NF-kB
-> Mechanism not yet fully understood
BUT:
-> In PKR 0/0- mice, the induction of IFN-b by dsRNA is not impaired, meaning PKR is not the only dsRNA-sensor

Later on:
Two more dsRNA sensors identified!

Question 12

Intracellular recognition of dsRNA

Answer 12

Pathogen associated Pattern Recognition Receptors (PRRs):
2. Retinoic Acid Inducible Gene I (RIG-I)
3. Melanoma Differentiation-Associated gene 5 (MDA-5)
bind dsRNA (DExD/H box RNA helicase-domain)
activates IFN-b promoter (via CARD domain dep. signaling)
Constitute the PRR family of RIG-like helicases

Substrates:
RIG-I:
- 5 ́-PPP RNA (also when ss)
- short blunt ds region (also without 5 ́PPP) e.g. panhandle of ss (-) RNA virus
- poly-Uridine stretches

MDA-5: longer dsRNA

Critical aspect:
Discrimination of cellular and foreign (“pathogenic“) RNA!

Question 13

Activity profiles

Largely non-overlapping pattern of virus susceptibility in mice deficient for either RLR

Answer 13

West Nile virus and reovirus are recognized by both RIG-I and MDA5

RIG-I is activated by blunt-ended double- stranded (ds)RNA with or without a 5’- triphosphate (ppp), by single-stranded RNA marked by a 5’-ppp and by polyuridine sequences.

ss (-) RNA:
- Orthomyxoviridae: Influenza A
- Paramyxoviridae: Measles, mumps, VSV and Sendai virus
ss (+) RNA:
Flaviviridae, HCV, Japanese encephalitis virus
-> RIG-I (dsRNA + 5’ PPP)

ss (+) RNA:
Picornaviridae: poliovirus and encephalomyocarditis virus
-> MDA5 (long dsRNA)

Question 14

The C-terminal regulatory domain is the RNA 5’ triphosphate sensor of RIG-I

Answer 14

The C-terminal regulatory domain (RD) of RIG-I binds viral RNA in a 5 ́-triphosphate-dependent manner and activates the RIG-I ATPase by RNA-dependent dimerization

Helicase domain and ATPase domain

Two caspase activation and recruitment domains (CARDs) transmit the signal

Question 15

How to discriminate pathogenic from self-RNA?

Answer 15

Cytosolic Viral Sensor RIG-I is a 5’ Triphosphate-Dependent Translocase on Double-Stranded RNA

Can recognize different PAMPS
- 5 ́-PPP-RNA
- short ds RNA
- ssRNA hairpins

• ATP-powered dsRNA translocation occurs pref. on dsRNA
• in the absence of 5’-triphosphate CARD domain suppresses translocation

Question 16

The link between RIG-I/MDA-5 and IRF-3 kinases:
IFN-b promoter stimulator (IPS-1) = mitochondial antiviral signaling (MAVS) (also called = VISA = CARDIF)

Answer 16

• binding partner for RIG-I and MDA-5
• activates NF-kB and IRF-3
• part of MAVS localizes to Peroxisomes; short term antiviral response
  no IFN induction but other ISGs
• mitochondrial MAVS essential for sustained antiviral effect

Question 17

MAVS/VISA/CARDIF/IPS-1

Answer 17

Peroxis. MAVS induces not IFN but other ISGs

• part of MAVS localizes to Peroxisomes; short term antiviral response no IFN induction but other ISGs
• mitochondrial MAVS essential for sustained antiviral effect

Question 18

Model for the RIG-I/MDA-5-mediated activation of IFN-beta promoters

Answer 18

• dsRNA binds to the helicase- domain of RIG-I; ATP hydrolysis
• This induces a conformational change, which allows the interaction of the CARD-domain with an adaptor molecule (e.g. IPS1=MAVS usw.)
• A TBK-1 mediated activation of IRF-3 leads to an activation of the IFN-b promotor
• Interaction of the CARD-domain of RIG-I with an adaptor molecule (e.g. IPS-1= MAVS usw.)
  -A TBK-1 mediated activation of IRF-3 leads to an activation of the IFN-b promotor

Additional regulatory step: Ubiquitination of RIG-I is essential for its signaling activity

Question 19

ZAPS electrifies RIG-I signaling

Answer 19

RD, repressor domain; PPP, 5′-triphosphate modification of the PAMP RNA ligand; Ub, polyubiquitin; ISGs, interferon-stimulated gene products; ZAPS, zinc-finger antiviral protein shorter isoform.

RESTING
Phosphorylation in the CARDs and RD maintains the autoinhibitory “closed” conformation where helicase and RD domains associate with the CARDS to obscure signaling

ACTIVATION
ATP hydrolyzation, dephospho rylation of CARDS and RD, and K63-linked ubiquitination of the RD allow change to “open” conformation upon ligand binding to facilitate oligomerization

SIGNALING
K63-linked ubiquitinatzion of the CARDs allows oligomerized RIG-I to associate with MAVS. Removal of acetylation licenses RIG-I to signal.

Anti-viral signalling requires RIG-I redistribution from the cytosol to membranes where it binds the adaptor protein, MAVS. Here we identify the mitochondrial targeting chaperone protein, 14-3-3ε, as a RIG-I- binding partner and essential component of a translocation complex or “translocon” containing RIG-I, 14-3-3ε, and the TRIM25 ubiquitin ligase.
14-3-3ε is essential for the stable interaction of RIG-I with TRIM25 and thus for targeting of RIG-I to mitochondrial membranes

Question 20

RIG-I-like receptor (RLR) family of pattern recognition receptors

Answer 20

The cytoplasmatic RLR family has three members:
- retinoic acid inducible gene I (RIG-I)
- melanoma differentiation-associated gene 5 (MDA5)
- laboratory of genetics and physiology 2 (LGP-2)

• DExD/H-box RNA helicase domain (positiv für RIG-I, MDA5, LGP-2)
• Two N-terminal caspase activation and recruitment domains (CARDs) (positiv für RIG-I, MDA5)
  -> LGP-2 does not interact with MAVS

Only regulatory function?

Question 21

LGP2 is a positive regulator of RIG-I- and MDA5-mediated antiviral responses

Answer 21

Generation of mice lacking LGP2:
LGP2 is essential for type I IFN production in response to infection by members of the picornaviridae.

Consequence of LGP2 knockout:
LGP2−/− mice highly susceptible especially to EMCV
Nevertheless, LGP2 and its ATPase activity were dispensable for the responses to synthetic RNA ligands for MDA5 and RIG-I.
Taken together, the present data suggest that LGP2 facilitates viral RNA recognition by RIG-I and MDA5 through its ATPase domain.

Question 22

Why are there two different RNA-Helicases for the detection of viral dsRNA

Answer 22

=> RIG-I binds ssRNA with at least short dsRNA with 5‘-triphosphates

The vRNAs of the NSVs have triphosphate groups at their 5’-ends

Question 23

Viral strategies against RNA induced IFN-induction

Answer 23

• hiding signals recognized by cell and/or cleavage/blockage of cellular sensors/signalling molecules by viral antagonists

Positive strand RNA viruses
- hiding of dsRNA (replication intermediates) in membrane vesicles - cleavage of signalling molecules by viral proteases

Negative strand RNA viruses
Processing of genome 5’ termini as a strategy of negative-strand RNA viruses to avoid RIG-I-dependent interferon induction.
Habjan M. et al., PLoS ONE. 2008 Apr 30;3(4):e2032.
- Genomic RNAs of Ebola virus, Nipah virus, Lassa virus, and Rift Valley fever virus strongly activated the interferon-beta promoter by a RIG-I and 5 ́-PPP dependent mechanism
- genomic RNAs of Hantaan virus, Crimean-Congo hemorrhagic fever virus and Borna disease virus do not trigger interferon induction
due to the absence of a 5 ́-PPP (viral enzyme?)

Question 24

Role of defective viral genomes in natural Influenza infections of humans

Reduced accumulation of defective viral genomes (DVGs) contributes to severe outcome in influenza virus infected patients.

Answer 24

• Viruses collected from cohort of previously healthy individuals who suffered highly severe IAV infection requiring admission to Intensive Care Unit and patients with fatal outcome who additionally showed underlying medical conditions.
• These viruses were compared with those isolated from a cohort of mild IAV patients.
• Viruses with fewer DVGs accumulation were observed in patients with highly severe/fatal outcome than in those with mild disease, suggesting that low DVGs abundance constitutes a new virulence pathogenic marker in humans.
• Identified polymerase PA D529N mutation detected in a fatal IAV case is associated in two different viral backbones with reduced defective viral genomes (DVGs) production.

Polymerase complex shields 5 ́PPP from RIG-I detection; this does not work in DVGs; therefore, DVGs lead to
the induction of IFN and immune protection.

A Conserved Histidine in the RNA Sensor RIG-I Controls Immune Tolerance to N1-2’O-Methylated Self RNA.

Question 25

Extracellular dsRNA: Toll-like receptor 3 (TLR3)

Answer 25

TLRs: Third Family of (PRRs)

IL-1R/TLR superfamily: IL-1R: Interleukin-1 receptor TLR: Toll-like receptor

-> Common element:
cytosolic domain, termedToll-IL-1R (TIR) domain

-> Sequence-similarities of IL-1R1 receptors and the Drosophila melanogaster protein Toll

TLR7/8 and 9 detect viral nucleic acids as well

Question 26

Mouse hepatitis virus (Coronavirus) ns2 Protein is an RNase L antagonist

Answer 26

• MHV ns2 protein inhibits the IFN-induced OAS-RNase L pathway
• ns2 reduces the intracellular level of 2′,5′-oligoadenylate (2-5A)
• ns2 has a 2′,5′-phosphodiesterase activity to directly cleave 2-5A
• ns2 mutant virus recovers wild-type virulence in RNase L-deficient mice

Question 27

Interferon-induced antiviral pathways

Answer 27

• ADAR 1/2 RNA-editing enzyme, adenosine deamination
• ISG20: Exonuclease with specificity for ssRNA
• ISG56 (p56): binds translation factor eIF3, blocks protein synthesis
• PML: promyelocytic leukemia protein, antiviral (HSV-1)
• p200 family: inhibition of gene transcription, RNA synthesis
• hGBP-1: large GTPase, antiviral
• MHC: presentation of antigenic peptides
• IRF-7: amplification of IFN response, „priming“
• STAT-1: amplification of IFN response, „priming“
• and many, many more (300?) …

Question 28

The IFN-induced protein Mx

Answer 28

• Broad antiviral activity (esp. negativ-strand RNA viruses, but also HBV, African swine fever virus…)
• Discovery with help of „influenza-hypersensitive“ mouse strains
• GTPase
• Related to dynamin
• Different forms: Localized in the nucleus and/or the cytoplasm
• Assembles into aggregates (antiviral form); interaction with other host proteins or with viral proteins?

Question 29

Mode of action Mx -> Thogotovirus (Orthomyxovirus)

Answer 29

• Viral RNA is replicated in the nucleus
• Transport of viral nucleoprotein complex into the nucleus is essential for viral replication
• Mx binds the nucleoprotein complex in the cytoplasm and therby inhibits its transport into the nucleus
• Block of viral replication

Control: Infection, IF staining
MxA: Infection, Mx expression, IF staining
MxA + 2C12: Infection, Mx expression, Neutralisation of Mx by injection of anti-Mx Ab, IF staining

2C12 is mAk against MxA

Question 30

Mode of action Mx -> La Cross Virus (Bunyavirus)

Answer 30

• Viral RNA is replicated in the Golgi apparatus
• Proteins are translated in the cytoplasm
• Mx binds to the viral N-protein and inhibitis its transport into the Golgi apparatus
• Block of viral replication

Question 31

IFIT1 is an antiviral protein that recognizes 5’-triphosphate RNA

Answer 31

Antiviral protein IFIT1 (interferon-induced protein with tetratricopeptide repeats mediates binding of a larger protein complex containing other IFIT family members to PPP-RNA (IFIT5 is functional ortholog)

IFIT proteins form an interferon-dependent multiprotein complex on viral PPP-RNA and thereby interfere with its translation (sequestration)

Efficient antiviral activity requires all three family members (IFIT1, IFIT2 and IFIT3) and the PPP-RNA-binding ability of IFIT1

Knockout mouse with Ifit1 deficiency:
- no general defect in IFN response (cytokines, IRF3 phosphorylation…)
but
- higher replication level of VSV (has 5 ́PPP)
- no enhanced replication of EMCV (has no 5 ́PPP)

Conclusion:
IFIT1 specifically binds and sequesters RNAs with 5 ́PPP

Question 32

Interferon induced genes: ZAP

Answer 32

Zinc-Finger Antiviral Protein (ZAP) = poly(ADP-ribose) polymerase 13 (PARP13)
- Part of the innate immune system; induced via interferon

Inhibits:
Genus Alphavirus (Togaviridae):
Sindbis Virus, Semliki Forest Virus, Ross River Virus,
Venezuelan equine encephalitis virus
Filoviridae: Ebola Virus, Marburg Virus Retroviridae: HIV
poly(ADP-ribose) polymerase 13
Hepadnaviridae: hepatitis B virus (PARP13)

Not inhibited:
Vesicular Stomatitis Virus, Poliovirus, Yellow Fever Virus, Herpes Simplex Virus 1

Mechanism
- binding of ZAP to CpG dinucleotides in RNA via N-term. RNA binding domain
- ZAP binds also polyADP-ribose (PAR) as cofactor
- PAR fascilitates formation of non-membraneous sub-cellular compartments
- degradation of viral RNA in those cytosolic RNA stress granules

Question 33

Viral strategies against interferon

Answer 33

• Avoidance of interferon induction
• Blocking of the activation of interferon- specific TS-factors (IRF3, IRF9)
• Blocking of interferon receptors of not infected cells
• Blocking of effectors
• Interception of Mx protein e.g. by Bunjaviruses
• Blocking of kinases like PKR

Question 34

Influenza A virus NS1 targets the ubiquitin ligase TRIM25 to evade recognition by the host viral RNA sensor RIG-I

Answer 34

Influenza A virus nonstructural protein 1 (NS1) specifically inhibits TRIM25-mediated RIG-I CARD ubiquitination, thereby suppressing RIG-I signal transduction

Question 35

Dengue Virus NS3 Binds to 14-3-3epsilon Thereby Blocking the Transfer of RIG-I to MAVS at Mitochondria

Answer 35

Influenza A virus nonstructural protein 1 (NS1) specifically inhibits TRIM25-mediated RIG-I CARD ubiquitination, thereby suppressing RIG-I signal transduction

Question 36

Dengue Virus NS3 Binds to 14-3-3epsilon Thereby Blocking the Transfer of RIG-I to MAVS at Mitochondria

Answer 36

Binding of 14-3-3ε to Dengue NS3 interferes with RIG-I signalling.
Replication efficiency of wildtype Dengue virus DV2(WT) and a virus mutant deficient in14-3-3ε-binding DV2(KIKP) in cells with functional RIG-I (Huh7) or non-functional RIG-I (Huh7.5)

Result:
In cells with functional RIG-I only wildtype DV can replicate efficiently. Thus, binding of 14-3-3ε to NS3 is of high importance and is
not compensated by other mechanisms.

Question 37

Effect of viral interferon antagonists on the virulence of viruses

Answer 37

Example, the Thogotovirus (Orthomyxovirus)
- Genom: - ss negativ-strand RNA - 6 segments
- Long version of matrixprotein = ML
- is an interferon antagonist

Question:
- How important is this protein for the virulence of the virus?

Experimental approach:
- Creation of recombinant Thogotoviruses, which cannot express this protein any longer

Question 38

Comparison of virulence of THOV/ML- (Without ML) and THOV/ML+ (with ML)

Answer 38

Strong attenuation by deletion of the interferon antagonist!

Question 39

Susceptibility to severe COVID-19

Answer 39

… On the other end of the spectrum is severe disease, with an overall estimated fatality rate near 1%. Zhang et al. and Bastard et al., respectively, report analyses of >1600 patients infected with SARS-CoV-2 from >15 countries to identify endogenous factors that determine susceptibility to severe COVID-19.
… The authors focused on the type I IFN pathway and analyzed 13 candidate genes that have previously been linked with susceptibility to other viral infections. Deleterious variants that can impair gene function were identified in 3.5% (23/659) of cases….
… Autoantibodies recognize and thereby may inhibit host proteins; they are a hallmark of many autoimmune diseases and are thought to be a contributor to autoimmune pathophysiology. Neutralizing autoantibodies against type I IFNs, mostly IFN-α2 and IFN-ω, were found in up to 13.7% (135/987) of patients with life-threatening COVID-19 and were shown to neutralize activation of the pathway in vitro…

This study shows that about 17% of the severe covid cases are connected to a misfunction in the innate immune system

Question 40

Global absence and targeting pf protective immune states in severe COVID-19

Answer 40

While SARS-CoV-2 infection has pleiotropic and systemic effects in some patients, many others experience milder symptoms. We sought a holistic understanding of the severe/mild distinction in COVID-19 pathology, and its origins. We performed a whole- blood preserving single-cell analysis protocol to integrate contributions from all major cell types including neutrophils, monocytes, platelets, lymphocytes and the contents of serum. Patients with mild COVID-19 disease display a coordinated pattern of interferon- stimulated gene (ISG) expression across every cell population and these cells are systemically absent in patients with severe disease. Severe COVID-19 patients also paradoxically produce very high anti-SARS-CoV-2 antibody titers and have lower viral load as compared to mild disease. Examination of the serum from severe patients demonstrates that they uniquely produce antibodies that functionally block the production of the mild disease-associated ISG-expressing cells, by engaging conserved signaling circuits that dampen cellular responses to interferons. Overzealous antibody responses pit the immune system against itself in many COVID- 19 patients and perhaps in other viral infections and this study defines targets for immunotherapies in severe patients to re-engage viral defense.
Central role for innate immune cells/immunity in clearance of SARS CoV-2 without severe disease!

Question 41

BVDV: A model for virus persistence
-> Strategy of suppression of the innate immune system

Answer 41

Npro: N-terminal protease
- releases itself in a autocatalytic way from the polyprotein
- binds to interferon-regulating factor 3 (IRF3)
- leads to degradation of IRF3 in the proteasome in an ubiquitin dependent fashion - consequence: no interferon synthesis in BVDV inf. cells

Erns: Envelope protein ribonuclease secreted
- essential envelope protein of the virion
- is secreted by infected cells (significant amounts in the plasm of p.I. animals) - has RNase-activity
- binds dsRNA and blocks IFN induction via dsRNA in the supernatant of cell cultures; consequence: no IFN synthesis induction by free dsRNA

Question 42

Comparing Pestiviruses and HCV

Answer 42

PESTIVIRUS
Nero und Erns Block innate immune system

HEPACIVIRUS
NS3/4A serinprotease blocks innate immune system

Question 43

HCV: Strategies of virus persistence
-> Strategies of suppression of the innate immune system

Answer 43

• NS3 needs NS4A as a cofactor for its full protease activity; it is essential for viral replication
• NS3/4A complex cleaves TRIF:
  -> no signal via TLR 3 (dsRNA)
  -> no signal via TLR 4 (viral glycolipids)
• NS3/4A complex cleaves Cardif (= MAVS; VISA; IPS-1)
  -> no signal from RIG-I or Mda-5
  thus no detection of cytoplasmatic dsRNA / RNA with 5 ́triphosphat

Question 44

Immune system / Interferon -> Summary

Answer 44

1. The innate (or „natural“) immune response is a crucial part of the host defense. It keeps the viruses at bay, until the adaptive immune response is active.
2. The interferon-system is an important component of the innate immune system.
3. Many viruses have evolved unique strategies to undermine the interferon defense system.

Question 45

Kinetics of a antiviral immune response

Answer 45

ordered by time:

1. IFN-alpha/beta
2. NK-cells
   3- cytotoxic T-cells
3. Antibodies

Question 46

Viral immune evasion strategies

Answer 46

Lytic infection:
- Blocking IFN-system
Latent infection:
- Providing no target
Persistent infection:
- Immune tolerance (BVDV)
-> No immune response
- Immune evasion (HCV, HIV, HCMV)
-> Permanent adaption of B- and T-cell response
-> Block of immune response

Question 47

Immune evasion of the human cytomegalie virus (HCMV)

Answer 47

• Virus coded MHC-dummies prevent NK attack
• Binds to MHC blocks T-cell-binding
• Translocation of MHC into cytoplasm
• Blocks MHC-transport in cis Golgi
• Blocks MHC-export out of ER
• Blocks proteasom
• Blocks peptide transport into ER

Question 48

Viroceptor

Answer 48

• soluble or surface moleclues
• decoy rec. bind cyto-/ chemokines

Question 49

Virokines

Answer 49

• secreted agonists or antagonists for cell. rec

Question 50

Resistance through host factors
-> Resistance to virus through cellular restriction factors
-> How where they identified

Answer 50

• Different strains of mice differ in their permissiveness
• Different cells in cell culture differ in their permissiveness
• Restriction by crossbreeding / cell fusion transferable to a permissive system

Question 51

Intrinsic antiviral factors -> SAMHD1

Answer 51

Target Virus: HIV-1

SAMHD1 restricted infection by hydrolyzing intracellular deoxynucleoside triphosphates (dNTPs), lowering their concentrations to below those required for the synthesis of the viral DNA by reverse transcriptase (RT). Antagonist is Vpx.

Question 52

Resistance through host factors
-> Résistance to Retroviruses through capsid specific restriction factors

Answer 52

• cellular binding partners for viral capsids?
• block replication! Mechanism?

Fv1: Friend virus susceptibility factor 1 gene defect in mouse strain leads to sensitivity to Friend Virus strain of MLV; thus the gene product blocks replication

Lv1 (Lentivirus susc.) blocks HIV-1 in rhesus monkey cells = TRIM5a

TRIM5a:
Tripartide interaction motif 5 Interaction with retroviral capsids

TRIM-Cyp
Insertion mutant: TRIM5-Cyclophilin A fusion protein; interaction with HIV capsid via Cyclophilin domain

Question 53

Tetherin as inhibitor for budding of HIV/Filoviruses

Answer 53

• HIV without Vpu: viruses get stucked to the cell surface when budding
• IFNa induces CD317 (tetherin, Bst-2); which has membrane anchor on both ends - Tetherin is integrated during the budding event into the virus membrane
• Tetherin in the virus membrane interacts with tetherin in the cell membrane:

Consequence for HI virus: Re-uptake, degradation?

Kaposi’s sarcoma- associated herpesvirus:
- viral protein K5 has ubiquitin-ligase activity
- Ubiquitinylated CD317
- Degradation of CD317 in proteasome
Vpu?
- Reduces amount of CD317
- Cell. Ubi.-ligase?
- direct interaction with Vpu is not yet shown

Question 54

Resistance caused by host factors
-> Resistence aginast retroviruses via cytidine-deaminases

Answer 54

Cellular APOBEC1 (apolipoprotein editing complex)
Apo. B mRNA editing; AID (activation-induced cytidine desaminase) antibody diversification

Antiviral APOBEC3G Leads to hypermutation in retroviral cDNAs
APOBEC3G is packaged into the capsids;
ssDNA is substrate

HIV-1 Vif (viral infectivity factor) Recruits APOBEC3G as well as APOBEC3F to the proteasome
Degradation!

Binding of viral ds DNA to cGAS stimulates the synthesis of cGAMP to STING: (ER)-located stimulator of IFN genes. STING stimulates TANK-binding kinase 1 (TBK1) activity to phosphorylate IRF3.