Innate Immune Responses

Question 1

(T/F) Most infections are prevented by innate and adaptive immunity.

Answer 1

False!

Most infections are prevented by physical (anatomical & chemical) barriers.

If viruses bypass these barriers, a series of immune responses (intrinsic, innate, adaptive) are engaged.

Question 2

Match the following responses to their definitions:

1) Physical barriers
2) Intrinsic
3) Innate
4) Adaptive

A) interferons, apoptosis, etc. cell-autonomous responses (can be achieved by a single cell in isolation). immediate.

B) t/b cells. tailored to pathogen (specific). hours/days.

C) mucus, saliva, tears, acid, etc. continuous. block majority of infections.

D) natural killer cells, complement, antigen-presenting cells, etc. induced by infection (non-specific). minutes/hours.

Answer 2

Physical barriers: mucus, saliva, tears, acid, etc. continuous. block majority of infections.

Intrinsic: interferons, apoptosis, etc. cell-autonomous responses (can be achieved by a single cell in isolation). immediate.

Innate: natural killer cells, complement, antigen-presenting cells, etc. induced by infection (non-specific). minutes/hours.

Adaptive: t/b cells. tailored to pathogen (specific). hours/days.

*intrinsic and innate can be grouped together

Question 3

How do individual cells detect a virus infection?

Answer 3

Receptor-mediated recognition of MAMPs (microbe-associated molecular patterns).

Then, cell signalling is induced by MAMP-receptor engagement, causing cellular changes to occur (gene expression).

Question 4

(T/F) Receptors recognize MAMPs only on the cell surface.

Answer 4

False!

They can recognize MAMPs within the cell also. MAMPs can be viral proteins or nucleic acids.

Question 5

Match the following terms to their definitions:

1) Microbe-associated molecular patterns (MAMPs)

2) Pattern recognition receptors (PRRs)

A) host cell receptors that recognize MAMPs. can be located on host cell surface, endosomal membranes, cytoplasmic, or secreted. examples: Toll-like receptors (TLRs), Rig-1-like receptors (RLRs), and protein kinase R (PKR).

B) MACROMOLECULES shared among groups of microorganisms and are recognized as FOREIGN to host. examples: dsRNA, peptidoglycan, LPS, proteins, flagellin, etc.

Answer 5

Microbe-associated molecular patterns: MACROMOLECULES shared among groups of microorganisms and are recognized as FOREIGN to host. examples: dsRNA, peptidoglycan, LPS, proteins, flagellin, etc.

Pattern recognition receptors (PRRs): host cell receptors that recognize MAMPs. can be located on host cell surface, endosomal membranes, cytoplasmic, or secreted. examples: Toll-like receptors (TLRs), Rig-1-like receptors (RLRs), and protein kinase R (PKR).

Question 6

(T/F) Toll-like receptors (TLRs) are membrane-bound, while Protein Kinase R receptors are found in the cytoplasm.

Answer 6

True!

Question 7

MAMP-PRR engagement leads to signalling events that ultimately activate transcription factors such as ______ and the _________.

The major outcome is the expression of ______ genes such as:

Answer 7

NFkB; Interferon Regulatory Factors (IRF3/7)

Cytokine; Inflammatory Cytokines; Type I Interferons (IFNs)

Question 8

(T/F) PRRs are expressed in equal amounts in all cells.

Answer 8

False!

Expression of PRRs depends on the cell type!

Question 9

Match the PRRs to their brief description:

1) RIG-1 & MDA5

2) Protein Kinase R (PKR)

3) cGAS

A) Cyclic GMP-AMP synthase; binds to viral dsDNA in the cytoplasm

B) cytoplasmic RNA helicases that function as RNA sensors

C) sensor for viral dsRNA, inhibits cap-dependent translation by eIF2α

Answer 9

RIG-1 & MDA5: cytoplasmic RNA helicases that function as RNA sensors

Protein Kinase R (PKR): sensor for viral dsRNA, inhibits cap-dependent translation by eIF2α

cGAS: Cyclic GMP-AMP synthase; binds to viral dsDNA in the cytoplasm

Question 10

RIG-1 and MDA5 are part of RIG-1-like receptors (RLRs) that detect slightly different things.

What do each detect?

Answer 10

RIG-1: detects 5’ triphosphate RNA without 5’ cap

MDA5: detects dsRNA

Question 11

Both RIG-1 and MDA5 contain tandem ___ ________ _______ domains, which are phosphorylated in __________ cells (inactive conformation).

Answer 11

N-terminal CARD; uninfected

Question 12

What happens when MDA5 and RIG-1 bind to viral RNA?

Answer 12

Binding of viral RNA, CARDs are dephosphorylated by specific phosphatases, and now are in ACTIVE conformations.

In their active conformations, RIG-1 and MDA5 are targeted to the mitochondrial antiviral signalling protein (MAVS).

Then, there is activation of IRF3/7 and NFkB which promotes expression of IFNs and pro-inflammatory cytokines to induce antiviral state.

Question 13

What is the difference between RIG-1 during inactivation and activation compared to MDA5?

Answer 13

Phosphorylation leads to the inactivation of both. However, RIG-1 is also held in the inactive conformation via INTRAMOLECULAR interactions.

Dephosphorylation leads to the activation of both. However, RIG-1 is also activated by the UBIQUITYLATION of its CARD & C-terminal domains (as signalling molecules not degradation).

Question 14

How can viruses evade RIG-1 and MDA5 responses (list 4 ways)?

Answer 14

1) Sequestration or modification of viral RNA ligands (prevents recognition by PRR)

2) Manipulation of post-translational modifications on RIG-1, MDA5, MAVS (phosphatase blocked)

3) Cleavage of RIG-1, MDA5, MAVS

4) Sequestration or relocalization of RIG-1, MDA5 (restricting their access)

Question 15

What is the role of eIF2?

Answer 15

eIF2 promotes the recruitment of tRNA^met to initiate cap-dependent translation when bound to GTP.

Once bound to the ribosome, translation is initiated; GTP –> GDP.

Question 16

What does PKR do? What is its mechanism of action?

Answer 16

dsRNA-activated protein kinase PKR causes translation arrest.

When PKR binds to viral dsRNA, it undergoes DIMERIZATION and AUTOPHOSPHORYLATION which leads to its activation.

Activated PKR phosphorylates the α subunit of eIF-2 which causes it to remain in an INACTIVE GDP-bound form.

Inactive GDP-bound eIF-2 is not able to recruit Met-tRNA, which leads to an arrest of translation and can cause APOPTOSIS.

Question 17

How can viruses evade PKR responses?

Answer 17

1) VIral phosphatase can be activated to remove the phosphate added by PKR

2) Viruses can use eIF2 INDEPENDENT translation mechanism

3) Viral protein PKR antagonists can block PKR action

Question 18

Fill in the blanks regarding cGAS mechanism of action.

1) cGAS binds to viral _______ in the _______.

2) Following binding, cGAS generates ________.

3) This binds and activates _______ which is located on the ____. It dimerizes and is further activated by __________.

4) Activated STING translocated to ________ structures where it promotes expression of Type I IFNs and proinflammatory cytokines.

Answer 18

1) dsDNA; cytoplasm

2) Cyclic GMP-AMP (cGAMP)

3) STING; ER; ubiquitylation (signalling not degradation)

4) Perinuclear

Question 19

How can viruses evade cGAS responses?

Answer 19

1) Viral manipulation of STING post-translational modifications (no more ubiquitylation)

2) Cleavage of STING

3) Prevent/limit cGAS sensing of nucleic acid ligand (hide dsDNA by viral factories)

Question 20

1) What are cytokines?

2) What are interferons (IFNs)?

Answer 20

1) Cytokines are small SIGNALLING MOLECULES that are secreted by specific immune cells. They mediate CELL-CELL COMMUNICATION to regulate a range of immune responses.

2) IFNs are a group of cytokines that are generated in response to several pathogens (‘interfere’ with viral infection). There are 3 types: Type I, II, and III.

Question 21

What are Type I interferons (IFNs)?

Answer 21

Viruses (or viral components) bound by PRRs trigger downstream signalling events that lead to the production of Type I IFNs (α/β).

These cytokines are released from the cell and then bind to IFNAR receptors on the surface of ADJACENT cells to stimulate expression of ‘IFN RESPONSIVE GENES’.

*protects uninfected cells from being infected
*establishes antiviral state

Question 22

What does ISRE stand for?

Answer 22

IFN-sensitive response element

Question 23

What is a key molecular/cellular outcome of Type I IFN signalling?

Answer 23

Induces resistance to viral replication in all cells by upregulating antiviral proteins such as Mx proteins, 2’-5’ linked adenosine oligomers, and the kinase PKR.

Question 24

Match the following antiviral proteins upregulated by Type I IFN signalling to their definitions:

1) PKR
2) 2’,5’-oligo A synthetase
3) Mx GTPases

A) GTPase with diverse roles in the INHIBITION of virion ASSEMBLY.

B) dsRNA sensor that inhibits viral protein synthesis.

C) activated by dsRNA, promotes the production of oligo A which, in turn, activates RNase L. RNase L then promotes DEGRADATION of viral RNA.

Answer 24

PKR: dsRNA sensor that inhibits viral protein synthesis.

2’,5’-oligo A synthetase: activated by dsRNA, promotes the production of oligo A which, in turn, activates RNase L. RNase L then promotes DEGRADATION of viral RNA.

Mx GTPases: GTPase with diverse roles in the INHIBITION of virion ASSEMBLY.

Question 25

What are the four types of viral modulation for the interferon response?

Answer 25

1) Inhibition of IFN synthesis

2) IFN receptor decoys

3) Inhibition of IFN signalling

4) Block function of IFN-induced proteins (PKR, Mx, etc)

Question 26

What is the complement system of the innate response?

Answer 26

It consists of a series of SERUM proteins that form an ENZYMATIC CASCADE that ultimately serves to enhance the innate immune response.

These complement proteins circulate in the blood in inactive forms and are activated in a SEQUENTIAL manner upon infection to ultimately promote pathogen CYTOSIS and INFLAMMATION.

Question 27

What are the three major pathways of the complement system and what major outcomes do they lead to?

Answer 27

1) Classical pathway
2) Lectin pathway
3) Alternative pathway

They lead to:
1) Inflammation: production of cytokines and recruitment of immune cells to site of infection

2) Opsinin: substance that binds microbial surface that enhances uptake by phagocytic cells (opsonization)

3) Formation of membrane attract complex: pore within membranes which leads to the lysis of the cell

Question 28

(T/F) In each of the three major pathways of the complement system, there are lots of different complement proteins that activate each other in a sequential manner, ultimately leading to the major outcomes.

Answer 28

True!

Question 29

Match the following terms to their definitions:

1) Hematopoietic stem cells (HSC)
2) Myeloid progenitor lineage
3) Lymphoid progenitor lineage

A) cells of innate immunity such as macrophage, NK cell, dendritic cell, and mast cell.

B) precursor cells that form leukocytes in the bone marrow when they mature through a process called hematopoiesis.

C) cells of adaptive immunity such as B and T cells.

Answer 29

Hematopoietic stem cells (HSC): precursor cells that form leukocytes in the bone marrow when they mature through a process called hematopoiesis.

Myeloid progenitor lineage: cells of innate immunity such as macrophage, NK cell, dendritic cell, and mast cell.

Lymphoid progenitor lineage: cells of adaptive immunity such as B and T cells.

Question 30

What are natural killer (NK) cells?

Answer 30

Nk cells are FRONT LINE of innate defence that are ready to recognize and KILL INFECTED CELLS.

They are LARGE, GRANULAR cells that lack antigen receptors. They kill infected cells by releasing PERFORINS and GRANZYMES that generate pores in the target cell membrane, which leads to CASPASE-MEDIATED cell death.

NK cells may recognize infected cells by ‘missing self’ or ‘altered self’ signals.

Question 31

(T/F) NK cells are abundant.

Answer 31

True!

They make up ~2% of circulating lymphocytes.

Question 32

(T/F) MHC I is downregulated by viruses as they display viral proteins to immune cells.

Answer 32

True!

Question 33

(T/F) A target cell has MHC class I and activating receptor, which bind to inhibitory receptor and NK-cell activating ligand of NK cells, respectively.

Answer 33

False!

A target cell has MHC class I and NK-cell activating ligand, which binds to inhibitory receptor and activating receptor of NK cells, respectively.

Question 34

How do NKs function in normal cells vs in infected cells?

Answer 34

MHC class I on normal cells is recognized by NK inhibitory receptors; no cell death.

Lack of MHC class I on infected cell can’t stimulate an inhibitory signal; cell death

Question 35

How can viruses evade/modulate NK cell activity (4 ways)?

Answer 35

1) Expression of viral protein that has homology to cellular MHC I which engages with NK inhibitory receptor

2) Release of viral-encoded activating receptor antagonist; downregulation of activating ligand on infected cells

3) Inhibition of action of NK cell-stimulating cytokines by binding of viral factor to cytokine or functioning as a receptor agonist

4) Newly produced virus particles may engage the inhibitory receptor or infect NK cell