Final Flashcards
Types of membane transport operation.
- uniport: moves a single substance at a time. Example is GLUT1 which is a passive uniporter.
- Symport: transports two different substances in the same direction across the membrane
- Antiport: Transports two different substances in opposite directions across the membrane.
Three methods of membrane transport
diffusion, passive transport, active transport
Diffusion in membrane transport
small non-polar substances.
Flux (diffusion rate) depends on the solubility of the molecule in lipid and the concentration gradient across the membrane.
Passive Transport and Facilitated diffusion
Facilitated diffusion: channels form a hole in the membrane through which molecules can pass down their concentration gradient. Can be selective. Can be saturated. .
Can have ungated or gated/regulated.
Ungated Channels
Passive Transport. always open. Selective or non-selective. Examples are porins and aquaporins.
Porins
Ungated passive transport.
also called beta-barrel Channels. Located on outer membranes of gram-negative bacteria and mitochondria..
Aquaporins.
Ungated Passive Transport.
lots of alpha-helices.
Only allow passage of water, it even excludes the flow of ions including H3O+.
There are two Asn residues at the center of the pore.
Gated or Regulated Channels in Passive Transport
highly selective, regulated by membrane potential, ligand binding, mechanical stress or other factors..
Examples include Voltage gated sodium and potassium channels, voltage dependent calcium channels, and two ligand gated GABAaR and nAchR
Voltage Gated Sodium and Potassium Channels.
Gated Passive Transport.
Resting state: neurons maintain a negative membrane potential with an excess of extracellular sodium ions and intracellular potassium ions.
In an action potential sodium channels open letting sodium into the cell causing depolarization.
Potassium channels open after sodium channels close and they let potassium out of the cell which restores the resting membrane potential.
Electrochemical Gradient
the combination of the concentration gradient and the membrane potential that influences the diffusion of ions across membranes.
Tetrodotoxin
found in certain pufferfish and it inhibits voltage-gated sodium channels and causes paralysis
Voltage Dependent Calcium Channels (VDCCs)
Gated Passive Transport.
found in muscle cells and neurons.
Activated by depolarization of the membrane and allow Calcium into the cell. They are open for much longer than voltage-gated Na channels and cause muscle contraction and neuron excitation.
Calcium Channel Blockers
amlodipine. used to treat hypertension. Inhibit VDCCs in the heart muscle, decreasing cardiac contractility. Also inhibit VDCCs in the arterial smooth muscle causing vasodilation.
Name the two ligand gated channels in passive transport.
GABAaR and nAChR
GABAa Receptor (GABAaR)
Ligand gated channel in passive transport.
Cl- channel found in CNS. Activation allows Cl- into the cell and causes membrane hyperpolarization causing neural inhibition.
Benzodiazepines and barbiturates enhance GABAaR activity.
Nicotinic Achetocholine Receptor (NAChR)
gated channel (passive transport) found at neuromuscular junctions.
Binding opens channel allowing Na+ to enter the cell and K+ to exit. Result is local depolarization initiating muscle contraction.
Acetylcholinesterase rapidly degrades ACh in the synaptic cleft.
Non-depolarizing nAChR inhibitors
bind to and competitively inhibit ACh binding.
Examples are d-tubocurarine (poison arrow dart) and muscle relaxants like atracurium.
Depolarizing nAChR inhibitors
bind to nAChR and open the channel resulting in membrane depolarization causing muscle contraction first then paralysis.
Example is succinylcholine which is a short-acting skeletal muscle relaxant used in IV with anesthesia.
The two types of active transport
Primary involves ATP consumption
and Secondary is coupled to an existing electrochemical gradient.
Na/K-ATPase
Primary Active Transport.
maintains the concentration gradients of sodium and potassium.
In each cycle it hydrolyzes 1 ATP, pumps 3 Na+ ions out of the cell and pumps 2 K+ ions into the cell.
P-glycoprotein (P-gp)
Primary Active Transport
Efflux transporter in the gut, liver, kidney and blood-brain barrier.
Potential source for drug-drug interactions. inhibitors include quinidine which increases exposure to substrate drugs like digoxin.
Na-glucose transporter
Secondary Active Transport.
located in renal epithelial cells. Symport so glucose import is powered by the sodium gradient set up by the Na/K-ATPase.
Na/Ca exchanger (NCX)
Secondary Active Transport
antiport in cardiac cells that is powered by the sodium gradient. It exchanges 3 Na+ per Ca+ exported.
Cardiac Glycosides
natural products that increase the intensity of heart muscle contraction and are used to treat congestive heart failure.
Example is digoxin. Digoxin has a narrow therapuetic index.
Inhibit Na/K-ATPase decreasing the Na+ gradient which in turn inhibits NCX. This increases intracellular Ca++ causing an increase in contractility of the cardiac muscle and increasing intensity of cardiac contraction.
The two types of receptors in signal transduction
cell surface receptors and intracellular/nuclear receptors.
Mechanisms of intercellular signal transduction
Diffusion based:
- endocrine: specialized sender cells synthesize and secrete molecules (hormones) into the blood. All cells are exposed but only those containing the appropriate receptor are affected.
- paracrine: sender cell secretes molecules into the local environment only.
- autocrine: sender cell secretes molecules into the local environment and receives them itself.
Non-Diffusion based:
- juxtacrine: cell-to-cell contact.
- matricrine: cell to extracellular matrix.
Intracellular Receptors
also called nuclear receptors
Acted on by small hydrophobic molecules including steroids, lipid-soluble vitamins etc
Most common are transcription factors that are activated by hormone binding.
Two categories: Type I nuclear receptors and Type II nuclear receptors
Type I Nuclear Receptors
bind to ligand in cytoplasm, homodimerize and are translocated into the nucleus via the receptor pore complex where they can bind to the transcription element and initiate transcription.
Examples include sex hormone receptors, glucocorticoid receptors, mineralcorticoid receptors.
Type II Nuclear Receptors
exist in the nucleus of DNA and bound to a corepressor protein.
Ligand binding triggers dissociation of the corepressor and association of a coactivator allowing the recruitment of RNAP and initiation of transcription.
Examples include thyroid hormone (TR), retinoic acid receptor (RAR), aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), constitutive androstane receptor (CAR).
AhR, PXR and CAR all bind to xenobiotic response elements which is important for CYP induction.
The two types of cell surface receptors
Receptor tyrosine kinase and G-protein coupled receptors.
Receptor Tyrosine Kinase (RTK)
bind growth factors and trigger intracellular phosphorylation cascades
Binding of the hormone to the extracellular domain activates autophosphorylation of specific Tyr residues and activates the receptor.
The activated receptor binds to and phosphorylates Tyr residues on specific target proteins which are often kinases.
Examples: insulin receptor, EGFR (epidermal growth factor receptor) and VEGFR (vascular endothelial growth factor receptor).
EGF
EGF binds to EGFR (cell surface receptor) and stimulates cell proliferation, which is an active pathway in cancer.
EGFR-targeting drugs include therapeutic monoclonal antibodies (mAbs) such as cetuximab (Erbitux) which inhibits EGF binding and also small molecules like erlotinib (Tarceva) that inhibit autophosphorylation and downstream signaling.
structure is erlotinib.
VEGF
VEGF binding to VEGFR (cell surface receptor) stimulates blood vessel growth (angiogenesis) which is important in tumor growth and “wet” age related macular degeneration (AMD).
Bevacizumab (Avastin) is approved for cancer and off label to treat AMD. Ranibizumab (Lucentis) is a fragment of same mAb approved specifically for AMD.
GPCRs
generate intracellular second messengers.
1. resting state: GPCRs are associated with heterotrimeric G-proteins (alpha, beta and gamma subunits) with the Galpha bound to ADP.
- Ligand binding causes GPCR to undergo a conformational change that causes Galpha to release GDP and bind GTP.
- Galpha dissociates and activates both subunits.
4 Galpha and Gbeta,gamma interact with downstream effectors like adenylate cyclase and phospholipase C to change intracellular second messenger levels.
cAMP and PKA
- adenylate cyclase catalyzes the synthesis of cAMP. Activated Galpha subunits regulate activity of adenylate cyclase (stimulating and inhibiting).
- PKA: In turn, cAMP regulates the activity of protein kinase A (PKA). PKA is an inactive heterotetramer consisting of two regulatory and two catalytic subunits. cAMP binds to a regulatory subunit which releases a catalytic subunit. Activated PKA phosphorylates Ser and Thr on target proteins.
Example is the insulin receptor, glycogen synthase, phosphorylase kinase (in glycogen breakdown).
Phosphoinositide Cascade
activated by GPCR.
mediated by phospholipase C (PLC) a membrane protein. PLC hydrolyzes phosphoatidylinositol (PIP2) to diacyl glycerol (DAG) and inositol triphosphate (IP3).
IP3 diffuses into the cytosol and binds to ligand-gated calcium channels in the ER releasing Calcium which acts as another second messenger.
DAG and calcium activate protein kinase C which is a Ser/Thr protein kinase involved in cell growth and differentiation.
GPCR drug targets
- loratidine: antihistamine that blocks the histamine H1 receptor.
- ranitidine: heartburn medication that is a histamine H2 antagonist
- perphenazine: antipsychotics which block the dopamine D2 receptor
- methylxanthines: caffeine, theobromine. inhibit phosphodiesterases and prolong the effects of cAMP. phosphodiesterasesgradually degrading cAMP.
5 Phases of Coagulation
- vasoconstriction
- Formation of Hemostatic Plug
- Formation of a blood clot
- Control of Coagulation
- Fibrinolysis
Platelets
circulating anuclear cells that are fragments of megakaryocytes which are produced in the bone marrow.
They contain mitochondria and glycogen granules but have no DNA or protein synthetic capacity.
They are proloaded with signaling molecules including ADP, serotonin (5-HT) and platelet factors.
Phase 2: Formation of a Hemostatic Plug
- Adhesion: damage to endothelial cells exposes collagen to which platelets bind and release von Willebrand factor (vWF) which enhances collagen:platelet binding.
- Activation: binding stimulates platelets to release ADP, serotonin (5-HT), thromboxane A2 (TxA2) and platelet factors. These factors mediate further vasoconstriction and the transition to the “sticky” platelet.
TxA2: promotes aggregation but also promotes vasoconstriction
serotonin: promotes vasoconstriction only - Aggregation: sticky platelets form a hemostatic plug that serves as a scaffold for subsequent clotting cascade.
Prostacyclin
(PGI2): produced by undamaged endothelial cells. It causes vasodilation and inhibits platelet aggregation.
Phase 3: Formation of a Blood Clot, Fibrinogen
proteolysis of fibrinogen to fibrin which polymerize to form a mesh network called a fibrin clot.
Fibrin clot is strengthened by cross-links between Lys/Glu residues catalyzed by a transglutaminase enzyme.
Vitamin K dependent clotting factors
IX, VII, X, II and all require Ca++.
Thrombin Sensitive Clotting Factors
V, VIII, XIII, I
Clotting Factor I
Fibrinogen and Ia fibrin
Clotting Factor II
Prothrombin and IIa thrombin
Clotting Factor III
tissue factor
Clotting Factor XIIIa
transglutaminase
Xa and Va Clotting Factor complex
Prothrombinase
Tenase Activity
VIIa and VIIIa/IXa
Phase 3: Formation of a clot, Intrinsic Factor
- triggered by exposure to collagen and von Willebrand Factor (vWF)
- Factor XII binds the site of injury and autoactivates to XIIa which is promoted by vWF.
- XIIa proteolytically activates XI to XIa.
- XIa activates factor IX to IXa which is vitamin K dependent.
- together with factor VIIIa, IXa activates factor X to Xa to start the common pathway.
Phase 3: Formation of a clot, Extrinsic Pathway
- triggered by exposure to tissue factor (III).
- TF binds to Factor VII and activates to VIIa. Factor VIIa has tenase activity and is vitamin K dependent.
- Factor VIIa converts X to Xa to start the common factory.
- more important of the two in vivo and produces a “thrombin burst”
Phase 3: Formation of a Blood Clot, Common Pathway
- Factor Xa is a vitamin K dependent factor. When complexed with Va it has prothrombinase activity to cleave factor II (prothrombin) to factor IIa (thrombin).
- Thrombin is a vitamin K dependent factor that is a protease that cleaves fibrinogen to fibrin (factor I to factor Ia). fibrin polymerizes to form a soft fibrin clot.
- Thrombin also activates factor V and VIII.
- Thrombin activates factor XIII to factor XIIIa which is the transglutaminase that cross-links fibrin polymers to form a hard fibrin clot.
- feedback activation because thrombin activates factors that promote more clotting: VIIIa, Va
Phase 4: Control of Coagulation and Clot Retraction
- Tissue Factor Pathway Inhibitor (TFPI): plasma protein that inhibits thrombin and Xa. The Xa/TFPI complex further inhibits VIIa.
- Anti thrombin (AT): inactivates thrombin and Xa. Stimulated by heparan sulfate which is a glycosaminoglycan
- Protein C and Protein S: Protein C is activated by a complex of thrombin and thrombomodulin and then binds to Protein S. The complex inhibits Va and VIIIa. PC/PS is vitamin K dependent.
Phase 5: Fibrinolysis
- fibrinolytic pathway: restore normal blood flow through the vessel.
- thrombin/thrombomodulin complex induces the release of tissue plasminogen activator (tPA).
- tPA activates plasminogen to plasmin.
- plasmin is a protease that degrades fibrin to soluble degradation products.
- Urokinase (produced in the kidney) and streptokinase (produced by certain bacteria) can also activate plasmin.
- Inhibition of fibrinolysis: direct inhibition by alpha2-antiplasmin or inhibition of plasminogen activation by plasminogen activator inhibitor (PAI).
Aspirin
- covalently inactivates cyclooxygenase (COX) in both platelets and endothelial cells. Inactivation of platelet COX shuts down thromboxane synthesis and inhibits platelet aggregation. Inactivation of endothelial COX shuts down prostacyclin synthesis.
- at lower concentrations of aspirin, platelet COX is inactivated but endothelial can still synthesize more COX. Rationale for low-dose aspirin therapy post MI and to prevent stroke
- at higher concentrations of aspirin, both platelet and endothelial COX are inactivated
Plavix
Clopidogrel. inhibits a platelet ADP receptor thus inhibiting aggregation.
Heparin
1.glycosaminoglycan similar to heparan sulfate. Promotes inhibition of thrombin IIa and Xa by anti-thrombin. Given by injection and sometimes induces thrombocytopenia which is decreased platelet count.
- unfractionated heparin: 12-15 kDa
- low molecular weight heparins: less than 8 kDa. More predictable activity, shorter half-life and lower incidence of side effects.
Fondaparinux
pentasaccharide that binds to anti-thrombin and inhibits factor Xa but not thrombin. Used to treat DVTs and pulmonary embolisms
Recombinant tPA, streptokinase or urokinase
used as thrombolytics to disrupt blood clots in the first few hours following a stroke or MI.
Warfarin
- inhibits vitamin K epoxide reductase.
- four factors requiring vitamin K: II, VII, IX, X. Each must be post-translationally modified to form gamma-carboxyglutamate residues by gamma-glutamyl carboxylase. These side chains bind to calcium enabling tight binding to the platelet surface.
- Vitamin K is an essential cofactor for gamma-glutamyl carboxylase.
- metabolized by CYP2C9 and has a very narrow therapeutic index.
Hemophilia
hereditary genetic diseases that impair coagulation process.
von Willebrand Disease: deficiency of vWF.
hemophilia A: Factor VIII deficiency
hemophilia B: Factor IX deficiency. Recombinant factors VIII and IX are commercially available for treatment.
Hypercoagulability Disorders
also called congenital thrombophilias. Involves defects in anticoagulation
Factor V Leiden thrombophilia: mutation in Factor V that prevents inactivation by PC/PS complex.
Atherosclerosis
accumulation of cholesterol rich deposits (plaques) in the walls of the vessels. Thickened walls can narrow the diameter of the vessel but blood flow is actually only blocked if a clot forms. This can happen when the plaque ruptures or if the inflammation from the plaque causes tissue factor exposure.
Myocardial Infarction
produced by a clot in a coronary artery
Stroke
produced by a clot in a cerebral artery
Deep Venous Thrombosis (DVT)
clot that forms during a period of stasis or immobilization.
Pulmonary Embolism
when a clot dislodges and travels through the circulation to the lung.
The Complement System: Outcmes
- Inflammation: complement stimulates basophil and mast cell degranulation.
- Opsonisation: 3b coats foreign organisms which stimulates phagocytosis by white blood cells. The phagocytes have receptors that recognize complement proteins bound to pathogen.
- Pathogen lysis: C5b ultimately leads to a transmembrane pore called the Membrane Attack Complex (MAC) in the pathogen cell and causes cell lysis.
Antibody Structure
IgG antibodies consist of 2 heavy chains and 2 light chains each with a variable (V) and constant (C) domain.
antigen binding sites (complementarity determining regions) are located on the variable regions of both heavy and light chains.
Fc region is composed of constant domains from both heavy chains and is a binding site for receptors on immune cells.
Therapeutic Antibodies
- Consist of entire IgG molecule: cetuximab
- Consist of isolated Fab fragments: ranibizumab
- Consist of single-chain variable fragments (scFv): pexelizumab. complement inhibitor (C5). picture
The Classical Complement Pathway
(Antibody-Dependent Complement Pathway)
C1 binds to the Fc region of an antibody bound to the surface of a pathogen which activates C1.
Activated C1 cleaves C2 and C4
C4a stimulates an inflammation response and is an anaphylatoxin.
C4b and C2b form a complex called C4b2b which is also called C3 convertase.
The Lectin Pathway of the Complement System
- Mannan-binding lectin (MBL) is a pattern recognition receptor that recognizes pathogen-derived carbohydrates and binds to them.
- Binding of MBL recruits MBL-associated serine proteases (MASPs)
- MASPs cleave C2 and C4
- C4a acts as an anaphylatoxin
- C4b and C2b form C4b2b complex.
The Central Step of the Complement System
Activation of C3
1. The C4b2b complex (C3 convertase) binds to C3 and cleaves it.
- C3a acts as a chemokine to stimulate chemotaxis of phagocytes to the site of infection. It also acts as an anaphylatoxin.
- C3b remains associated with the pathogen surface where it acts as an opsonin and enhances phagocytosis of the pathogen.
The Alternative Complement Pathway
also called the intrinsic complement pathway. Constitutively active.
When C3b binds to pathogen surfaces it recruits factor B and factor P
formation of a C3bBb complex which is the alternative C3 convertase
Feedback Activation (positive feedback). Enables rapid immune response even if the adaptive immune system has not mounted an antibody response and it also amplifies the effects of the classic complement pathway and the lectin pathway.
Formation of the MAC complex in the Complement System
- C3b is also a protease that cleaves C5.
- C5a is a chemokine and an anaphylatoxin.
- C5b recruits other complement factors to form a pore in the bacterial membrane called a membrane attack complex (MAC). This causes bacterial cell lysis and death in an antibody-independent mechanism.
- Formation of the MAC in host cells is inhibited by CD59
Disorders of the Complement System
Factor P Deficiency: linked to recurrent bacterial infections
Systemic Lupus erythematosus (SLE): linked to deficiencies in the classic pathway. Thought to result from defects in the clearance of immune complexes from the blood.
What is a virus?
an infectious, obligate parasite containing RNA or DNA genetic material in complex with or surrounded by protein and/or lipid membrane.
They have an extracellular and intracellular stage in their replication cycle.
They are small and do not by themselves carry out any metabolic processes.
Variolation
practice in 11th century where material from a smallpox infected pustule would be introduced into the scratched skin of a healthy individual to confer resistance.
Hershey Chase Experiment
on bacteriophage to determine that nucleic acid carries the genetic information. radiolabeled protein or DNA and detected whether the progeny virus particles have radioactivity or not.
Replication Cycle of Viruses: two major stages
- Extracellular stage: viruses are essentially inert until they encounter a host cell
- Intracellular Stage: virus hijacks the host cell and repurposes it in order to make more copies of the virus.
Stages of the Infectious Cycle - Virus
- host cell recognition (attachment)
- entry: endocytosis or diffusion
- genome delivery (uncoating): release genetic material
- replicate components
- assembly
- maturation
- egress: release of infectious virus from cell
Eclipse vs. Latent Period - Virus
The eclipse period is the time from virus attachment to production of the first virion (intracellular). The latent phase is the time to first extracellular virus. A virion is a complete, infectious particle
Susceptible vs. Permissive vs. Resistant - Virus
susceptible: The cell has the right receptor on its surface so that the virus can bind to that cell.
Resistant: A cell that lacks the necessary receptor and is thus resistant to infection by that virus.
Permissive: A cell that can support the replication of virus once it is inside the cell.
Classifying Viruses
- nucleic acid type: RNA/DNA, +/- sense, single/double stranded and its sequence
- structure, symmetry, dimensions of the the protein capsid.
- enveloped vs. naked/non-enveloped
Particle/PFU in virus
can be greater than 1. Example is if the ratio is 500, then 1 out of every 500 particles is infectious.
7 Classes of Viral Genomes
- +RNA retrovirus: converted to - RNA by viral nuclei polymerase then to dsDNA by vnp then to + mRNA by hnp
- +RNA: Poliovirus, Rhinovirus. converted to -RNA by vnp then to +mRNA by vnp.
- ssDNA: Parvovirus. converted to dsDNA by vnp and hnp then to +mRNA by hnp
- fragmented dsDNA: Hepatitis B virus. converted to dsDNA by vnp then to +mRNA by hnp.
- dsDNA: Herpesvirus, Adenovirus. converted to +mRNA by hnp.
- dsRNA: Rotavirus, Reovirus. converted to +mRNA by vnp.
- RNA: Influenza, Ebola. converted to +mRNA by vnp.
RNA polymerases
- nucleic acid replication carried out by an RNA-dependent RNA polymerase, RdRp
- no proofreading, so high error rate which may limit size and complexity of virus.
- structurally homologous between different viruses.
usually localized in replication “factories”
Polycistronic Genome
single transcript encodes information for multiple proteins.Three options to create multiple proteins
- RNA translated as a single long polyprotein that is chopped up by a virally encoded protease
- segmented genomes with multiple “monocistronic” mRNAs
- long mRNA that is processed by host splicing machinery
+ RNA
Poliovirus
Plyiomyelitis, small, non-enveloped, capsid. Exclusive to humans and infects the GI tract but can migrate to CNS. Highly contagious even when asymptomatic.
+ssRNA converted to -RNA through RdRp then to +mRNA through RdRp
covalently attached VPg protein used in place of a 5’ end cap. And also must have an Internal Ribosome Entry Site (IRES) where the ribosome can initiate translation.
- RNA influenza
-RNA converted to mRNA through RdRp
Has a segmented genome with 8 segments each encoding for a protein and each converted to + mRNA strand for translation.
RdRp “steals” mRNA caps from host mRNAs and uses them as the starting point for producing the + sense complementary viral mRNA strand.
Reassortment is common due to the segmented genome and occurs when a cell is infected with two strains of the virus. These can give rise to pandemics and antigenic shifts.
+ RNA Retrovirus
HIV
- Virally encoded reverse transcriptase (RT) converts +RNA to -DNA. Then RT produces dsDNA. HIV integrase enzyme integrates dsDNA into the host DNA.
- Finally, RT has RNA-digestion (ribonuclease) activity and digests the original +RNA
- HIV virion encapsulates 2 copies of +RNA associated with nucleocapsid protein (NP) and is contained within cone-shaped capsid (CA).
- Virally encoded proteins contained within capsid: RT, integrase, protease and miscellaneous proteins used to modulate host cell response.
- HIV produces two polyproteins that are cleaved by virally encoded protease.
dsRNA
rotavirus.
- One of the most common causes of gastroenteritis. Symptoms of severe watery diarrhea, vomiting, fever and abdominal pain. Common in infants and children. Most serious side effect is dehydration.
- RNA strand not available for translation because bound up in the dsRNA.
- Viral polymerase opens dsRNA and transcribes new - and + RNA.
- 12 individual monocistronic strands encapsulated in viral particles
VP1: polymerase function (RdRp)
VP3: adds 5’ cap onto mRNA being produced
VP6: shell that contains channels that newly synthesized viral mRNA exits from
Vaccines for Rotavirus dsDNA
Rotarix: monovalent live attenuated strain
RotaTeq: pentavalent vaccine produced by reassortment of human and bovine strains. Reassortants all have VP7 and VP4
+ RNA: Dengue
- Transmitted to humans by infected mosquitos. 4 main serotypes.
- Virus has a highly dynamic structure from the mosquito body temperature to the human body temperature. At human temp the membrane is exposed. Enveloped virus
already capped and can be directly translated to proteins. - RdRp makes the complementary - RNA to make more + RNA copies.
- Virally encoded protease, NS3, and host proteases process polyprotein into functional units.
- Virus attachment through E proteins on membrane.
+ RNA: Hep C
- sever liver disease, fibrosis, cirrhosis, liver cancer.
- Transmission through blood. Currently no vaccine.
- Rapidly evolving and very diverse virus.
- Irregular shapes and structures and have some association with lipoprotein.
- IRES for translation therefore no 5’ cap.
- Polyprotein that is cleaved into subunits by protease NS3-NS4A partly by viral protease but mostly by host protease.
Two Types of Viral Structure
- non-enveloped, icosahedral viruses: repeating units assembled symmetrically that produce a closed shell that encapsidate and protect the nucleic acid genome
- enveloped virus: proteins are embedded in the membrane and the membrane is used as the vessel.
Cellular Barriers to Cell Entry
virus needs to bypass the membrane of the cell and the actin cortex. Once inside the virus has to uncoat to release genetic material.
- non-enveloped virus: employ lytic peptides or hydrophobic conformations of their capsid.
- enveloped virus: employ virally encoded fusion proteins and surface glycoproteins.
Poliovirus Cell Entry
- uses clarithrin-dependent endocytosis.
- Initially attaches with low affinity and then shifts toward high avidity with receptor.
- Canyon and Pocket binding.
receptor binds in the canyon of the virus. Binding triggers a structural reorganization of the capsid proteins causing a release of the RNA genome.
The pocket usually contains a protein that supports the structure when not bound to the receptor and is released when it becomes bound. Site of drug targeting. Create a pocket protein with stronger affinity so that when the virus binds to the receptor the virus cannot undergo the necessary conformational change. Pleconaril
Binding proteins in cell entry
recognize receptors on cell surface
affinity: high strength of attachment of binding protein to receptor
avidity: multivalent attachments which together form a strong binding of binding protein to receptor.
Attachment Proteins
- viral attachment proteins may bind initially to the cell surface with low affinity and selectivity to increase the concentration of particles to the cell surface.
- Now viral particle only has to diffuse along the cell surface to find specific receptors for viral receptor binding proteins.
- Examples of viral attachment proteins: glycans binding to lectin proteins and binding to heparan sulfate on the host cell surface.
Influenza Membrane
- virions form by budding from host cell plasma membrane.
- Two glycoproteins on viral surface
HA: hemagglutinin. 18 types. Function is to bind to the virus’ receptor, sialic acids on glycoproteins, on the cell surface
NA: neuraminidase. 11 types. Function is as a sialidase, clipping sialic off cell surface so particle can float away to infect new host cell. - multivalent, high avidity binding leads to endocytosis
- Fusion protein: structural change in response to change in pH. At pH 7 the protein is in the normal form. At pH 5 in the endosome the fusion protein flips upward as well as the fusion peptide which causes membrane fusion.
- Fusion proteins between virus’ have a common structure.
Influenza Treatments
- have a drug that blocks uncoating. However, influenza is 100% resistance to these compounds so they are effectively useless
- Oseltamivir, Zanamivir: block release by binding to NA and preventing NA from cleaving saliac acid.
- Favipiravir: initially developed to target RdRp of influenza but has been used experimentally for Ebola.
HIV Genome
- single stranded RNA retrovirase. Viral coded RT. Transcribed DNA is integrated into host chromosome by viral integrase.
- High mutation rate combined with high HIV virions produced per day in an individual leads to a huge pool of raw genetic material capable of developing resistance within months of starting single therapies.
- Within an individual infected with HIV, the diversity of the genome is equal in size to the diversity of the global human influenza genome.
Receptor Binding and Cell Entry for HIV drug targets
HIV has three stages of attachment.
- Heparan sulfate for initial adhesion. CD-4 receptor binding for initial triggering and co-receptor binding for fusion initiation.
- Some northern Europeans do not have a functional co-receptor CCR5 and are resistant to infection.
- Maraviroc: entry inhibitor which binds to co-receptor CCR5 preventing attachment by virus.
Fusion for HIV drug target
Target the HIV envelope protein and prevent virus from carrying out fusion with the host membrane.
Enfuvirtide (Fuzeon, Roche) forms “prehairpin” intermediate conformation inhibiting fusion.
Reverse Transcriptase for HIV drug targeting
nucleosidic RT inhibitors: target drug to dNTP active site
non-nucleosidic RT inhibitors: bind to an allosteric site on RT and decrease RT activity
Integration for HIV drug targets
target integrase. Raltegravir (Isentress).
Polyprotein proteolysis for HIV drug targeting
Protease is the target to target gag and pol which are initially cleaved and responsible to produce the rest of the proteins.
