Virus Entry/Uncoating Delivery of genome to inside of a cell Genome must be available for first step of replication
The Problem--barriers to infection Virion Barriers: Non-enveloped viruses capsid Enveloped Viruses virus membrane capsid/nucleocapsid
Cell Barriers to Infection From Grove and Marsh, J Cell Biol. DOI; 10.1083/jcb201108131
Entry at Plasma Membrane nonenveloped (polio maybe in some cell types) enveloped paramyxoviruses (recently questioned for RSV) HIV and some retroviruses (recently questioned) Herpes virus Entry at Intracellular Membranes (most viruses) nonenveloped eg. Coxsackie virus, polyoma, SV40, poliovirus (some cell types) Enveloped eg. Flavi, Toga, influenza (recently questioned for Toga, alternate pathway for some paramyxoviruses)
Advantages of Entry at Intracellular membranes 1. Physiologically active cell 2. Get past cortical skeleton 3. Avoid immunodetection 4. Use of intracellular vesicle environment/enzymes for subsequent steps in infection (uncoating)
Entry at Internal Membranes Pathways for virus entry by endocytosis Most common Caveolin, lipid raft dependent Calveolin independent, lipid raft dependent Various caveolin, clathrin independent mechanisms Ligand induced, actin dependent
Cell endosomal pathway ER
Binding, endocytosis Caveolar endocytosis Virus in Clathrin coated vesicles Virus in early endosomes Virus in smooth ER
1. Clathrin mediated endocytosis (most common for enveloped viruses) Clathrin coated vesicles to endosomes to lysosomes acid environment of endosomes important 2. Caveolae mediated endocytosis (SV40, Coxsackie) Cholesterol/lipid rafts to caveosome to ER 3. Clathrin independent endocytosis---to endosomes 4. Caveolae independent but cholesterol dependent +/- dynamin 5. Macropinocytosis Adenovirus, pox virus, Ebola virus Some viruses use multiple pathways--eg influenza
Evidence used to discriminate pathways 1. Microscopic Colocalization of virus and host protein such as caveolin or clathrin (time courses) 2. Inhibitors Brefeldin A (clathrin endocytosis) Bafilomycin (inhibits acidification of endosomes) Ammonium chloride (raises endosome ph) Drugs that disrupt lipid rafts (nystatin, filipin, methyl beta cyclodextrin) caveolin scaffolding domain peptides 3. Dominant negative mutant proteins eg Eps 15, Rab5, Rab7-- Clathrin mediated endocytosis DN Caveolin. 4. sirna that targets key components of a pathway
Entry of Non-enveloped viruses Example--Picornaviruses polio Coxsackie virus Virus Attachment protein=vp1
Picornaviruses Structure of Virion Location of proteins Depth cued
Entry Non-enveloped viruses--picornaviruses Time course of infection: Size of capsid changes
Entry Non-enveloped viruses--picornaviruses 160S virion +soluble receptor 135S Change in antigenic sites and conformational shifts in capsid Sensitive to proteases Externalization of N term of VP1 inserts into membranes Exit of VP4 (binds membranes) 135 S binds to liposomes (hydrophobic) 135S (cell associated) 80S + RNA (80S contains no RNA) RNA exits capsid Trigger unknown for some Others acid ph
Cellular Site of Polio Uncoating varies with cell type Bergelson, J. Trends in Microbiology 16: 44
Coxsackie Virus Entry Coxsackie virus Attachment CAR =receptor DAF attachment (virus will bind to DAF but binding is not sufficient for infection) Coxsackie and polarized cells CAR at TJ DAF apical surfaces
5. Virus endocytosed and delivered to RER Site of 135 S to 80 S + RNA? trigger??? Coyne and Bergelson, 2006 Virus induced Abl and Fyn Kinase signals permit Coxackievirus entry through tight Junctions Cell 124; 119 Conclusions: 1. Binds DAF 2. Cross-linking of DAF by the virus results in migration of DAF (with bound virus) to tight junctions where CAR is located 3. Virus binds CAR --160 S converted to 135 S 4. 135 S Delivered to caveoli
Virus Initially binds to DAF Virus results in DAF clustering
Coxsackie Attachment Protein then associates with CAR Time Course of Infection With respect To CAR
Conversion to 135S requires CAR
Question: How does virus get inside cells?
VP1 co localizes with caveolin (Cav-1)
Cav-GFP 60-90 min post infection DN Caveolin--no internalization of CAV or the virus
caveolin virus virus Caveolin, VP1, VP4 co-localize in internal membranes That is, VP4 does not stay in TJ (implication--rna exit not in TJ)
Conclusions: 1. Binds DAF 2. Cross-linking of DAF results in migration to tight junctions where CAR is located 3. Virus binds CAR --160S converted to 135S 4. 135S Delivered to caveoli 5. 135S particle endocytosed and delivered to RER Likely site of 135S to 80S + RNA trigger for RNA release from particle???
Enveloped Virus Entry Membrane fusion at the Plasma membrane or at An intracellular membrane From Grove and Marsh, J Cell Biol. DOI; 10.1083/jcb201108131
Entry at PM (paramyxovirus) Virus Cell Plasma Membrane ATTACHMENT MEMBRANE FUSION INSERTION OF GENOME
Entry at Internal Membranes Pathways for virus entry by endocytosis Most common Caveolin, lipid raft dependent Calveolin independent, lipid raft dependent Various caveolin, clathrin independent mechanisms Ligand induced, actin dependent
Entry at Intracellular Membranes--influenza virus Endocytosis attachment Evidence for usage of several Different pathways
Influenza in Endosomes
How discriminate between PM and intracellular membrane fusion?? Virus binding and entry at 37 C What is localization of virion glycoproteins if fusion at PM or intracellular locations? How discriminate localization of virion associated glycoproteins in the two cell locations? What controls are required??
Measure association of labeled virion proteins with cells 100% 37 C 50% Time Glycoproteins inside or on PM??
100% Entry at PM 37 C 50% +protease Time Entry at Intracellular Membranes 100% - or +protease 37 C 50% Time
Alternative Approach Virus + Cells incubate at 37 C Add complement and antibody Result if entry at PM?? Result if entry at intracellular membranes??
Uncoating---- nonenveloped virus Genome/Core delivered into the cytoplasm Accessible for next stage of the replication cycle
General Considerations for Nonenveloped Viruses Virion structure must be destabilized or reorganized In order to release the genome
Different Host Factors Involved in Entry/uncoating of Nonenveloped Viruses Virus receptor interactions Proteases, Thioreductases, Acid ph Motor proteins Different ways to destabilize virions to release the genome
Picornavirus entry different models 135S 80S +RNA Exit of VP1 N terminus Receptor binding VP4 B-D Various models for Entry (virion/membrane interactions) polio virus From Bubeck et al JVI 79: 7745 VP1
Uncoating Enveloped Virus Fusion between virus envelope and vesicle membrane Genome/Core delivered into the cytoplasm Accessible for next stage of the replication cycle
Influenza Entry Membrane proteins--ha, NA, M1, M2 Attachment sialic acid receptors Membrane fusion (type 1 fusion)
Entry at Intracellular Membranes--influenza attachment virus Endocytosis Fusion of viral and cell membrane
Hemagglutinin--HA Attachment to cell receptors sialic acid Membrane Fusion--entry Target of neutralizing antibody Determinant of Organ Tropism--cleavage site HAo cleaved to HA1 +HA2
Domains of HA protein linear representation of sequence Cleavage site HR TM CT FP HA1 HA2 Typical of many fusion proteins (type 1 fusion proteins) FP fusion peptide HR heptad repeat TM transmembrane domain CT cytoplasmic domain
What Activates Fusion after Attachment? 1. Endosome-virus fusion (influenza) acid ph of endosome evidence-- a. ammonium chloride or chloroquine b. acid treat virus stimulates conformational change 2. PM fusion (eg paramyxoviruses) VAP-receptor interactions stimulates conformational change in fusion protein
Structure of HA Trimer H1--gray H2--colors membrane Cytoplasmic tail
Effect of acid ph on HA conformation HA at ph 7 HA at ph 4.5 From Fields Virology
Intermediate Conformations upon Acid treatment FP irreversible From Fontana, et al J. Virol. 86:2919 (2012)
How do conformational changes translate to membrane fusion? Prefusion Post fusion HA2 HA2
Over view--membrane fusion FP ph 7 Acid FP insert Into target membrane TM HA refolds FP and TM in Proximity Drawing Two membranes together
Target membrane Fusion peptide Close approach TM Virion membrane hemifusion Steps in Membrane Fusion Pore formation
From Fields Virology
From Fields Virology
Flavivirus Fusion type 2 fusion protein Virion Prefusion E (dimer) Post Fusion E (trimer) Pierson and Kielian Current Opinion in Virology 3: 3 (2013)
Flavivirus membrane fusion Pierson and Kielian Current Opinion in Virology 3: 3 (2013)
Rhabdovirus Fusion type 3 fusion proteins
General Steps in Viral Protein Mediated Membrane Fusion Synthesis and folding of fusion protein into metastable conformation Activation of fusion protein (acid ph or receptor binding) conformational changes Insertion fusion peptide into the target (cell) membrane anchoring of virion membrane to cell membrane Refolding of the fusion protein drawing target and virion membranes together Hemifusion, then pore formation, then pore expansion
After membrane Fusion: Final uncoating--the problem How is core of virus released from virus membranes (NP-M interactions required for assembly must be disrupted)?
Key to mechanism for influenza: Amantadine effects on infection Inhibits infection Effect is very early in infection (consistent with entry Blocks release of RNPs into cytoplasm How determine target of amantadine?
Envelope M1 M2 HA Lipid Bilayer--membrane Four Proteins HA--hemagglutinin NA--neuraminidase M1--matrix M2--membrane Component ion channel NA
Acid ph and the M2 protein Acid opens M2, an ion channel Acidification of virion interior M2 allows hydrogen ions into virion interior loosening M1-NP interactions
Endocytosis virus Influenza Penetration and Uncoating Acidification of vesicle Acidification of virion Interior and Activation of HA Fusion of viral and cell membrane Release Of NP-M interactions
After entering the cytoplasm, some viral genomes must get cross the nuclear membrane through the nuclear pore (35-40 nm) Genome segments Enter nuclear pore separately Genome exits core at nuclear membrane Virus disassembles At nuclear port Capsids are small Enough to directly Pass through the pore
Entry of HBV cores into nucleus HSV at nuclear pore Core line up inside pore channel. Cores disassemble In basket of NPC DNA exits core, transits Through the pore, leaving An empty core behind In the cytoplasm
Reading Fields Virology Chapter 4 Sections of Chapters 24, 47, and 48 that relate to virus entry Coyne and Bergelson, 2006 Virus induced Abl and Fyn kinase signals permit Coxsackievirus entry through tight junctions Cell 124; 119