1. Entry and Uncoating of Viruses Virology Unit 4, 2015 Version

2. Main Points and Plan  Introduction-some general background  Generic information  Specific examples-animal viruses: Influenza A (also evolution) Paramyxoviruses Plant viruses

3. Viral Entry Hard to Study  Cellular processes obscure  Technology weak  Diverse mechanisms  Entry mechanisms tough to conceptualize  Not always as expected-more sophisticated?  Are there any generalizations?

4. Plasma membrane  Common to all cells-a barrier to viruses  They must penetrate to infect- (a) trick the cell into bringing virus in? (b) or penetrate on their own?  (a) requires subversion of cellular mechanisms  (b) requires mechanical “puncture”

5. Some Generic Mechanisms Used by Cells to Internalize Large Particles-Can Viruses Subvert These?  Receptor-Mediated Endocytosis involving ligand and receptor (RME)  Endocytosis-generic term for non-specific pinocytosis or phagocytosis  Caveolae-separate pathway, no clathrin

6. Entry of Influenza A is the Paradigm  Best-studied (important pathogen)  Integrates many types of studies  Reveals unsuspected sophistication of viruses  Complex, multi-step process

7. ORTHOMYVIRIDAE Orthomyxoviridae comes from the Greek word “Ortho” meaning correct and “Myxo” meaning mucus. Orthomyxoviruses replicate in the nucleus. The Orthomyxoviridae contains five genera (Influenza A, B, C and D or thogotovirus and isavirus). Genera can be distinguished based on antigenic differences in their Nucleocapsid and Matrix proteins and on the number of genome segments. Influenza A contains eight segments and has two glycoproteins in the envelope Hemagglutinin (HA) and Neuraminidase (NA) that can be used to distinguish virus species. Infects birds, humans and a large number of other mammals. Most common cause of flu in humans. Influenza B has eight segments and is known only in humans. Influenza B strains evolve slowly and normally cause a mild disease. Influenza C has seven segments and particles have a single glycoprotein. The virus causes a relatively mild disease in humans and occasionally in swine. Thogotovirus is tick-borne and has only a single glycoprotein. Little is known about the molecular biology of this virus.

8. Influenza Disease Hippocrates recorded influenza (probably) in 400 BC. Probable epidemics noted throughout middle ages. “Grippe”, “epidemic catarhh”, “sweating sickness” old names for influenza. “influenza” come from the idea that sick people were under the influence of something bad.

9. Influenza Clinical Symptoms Rapid Onset Fever Shivering Prostration Myalgia Coughing, sneezing, runny nose (coryza) Usually no GI involvement Usually not life threatening for adults Secondary infections problematic

10. -Pleomorphic virions are 80-100 nm in diameter -Filamentous forms -may occur -Prominent 16 nm surface spikes INFLUENZA VIRUS STRUCTURE

11. Virion Components

12. Virus Binding to Cells Influenza virus attaches via HA trimer Cellular receptor molecule Is a carbohydrate Sialic acid (N-acetylneuraminic)

13. Receptor Distribution

14. Influenza Virus HA Protein 1) The HA 0 protein subunits form a homotrimer 2) N-terminal heads contain variable antigenic determinants & sialic acid binding pockets 3) C-termini are membrane anchors 4) HA 0 converted to HA 1 and HA 2 by proteolysis.

15. Proteolysis of HA0 “Late” event Host protease Absolute requirement for infection

17. Influenza A particles internalized via receptor-mediated endocytosis

18. Physiological pH form is metastable Lower pH induces massive conformation change

19. Conformation Change of HA Protein

20. Fusion peptide penetrates endosomal membrane pH 7.0 pH 5.0

21. Cytoplasm Virus Snapback of HA trimer forms fusion pore Contents of virus exit to cytoplasm

22. Ion Channel is Closed at neutral pH Ion Channel is open at low pH The M2 Ion Channel Protein also has a Major Role in Entry 1) M2 tetramers form four to six ion channels in virions that allow H + ions to flow into the virus particle as the endosome moves into the interior of the cell. 2) The ion channel is closed at neutral pH but opens at low pH 3) As the pH is reduced inside the virus particle, the matrix protein (M1) dissociates from the RNPs 4) The "free" RNP is primed and ready to enter the nucleus after fusion of the viral envelope with endosomes 5) RNPs containing M protein are unable to enter the nucleus. THE RNP GETS AN ACID BATH

23. Summary for Influenza A  Conformation changes  pH dependent  In cytoplasm  Irreversible  Checkpoints passed in response to signals

24. Functions of the Neuraminidase (NA) Spike  NA also functions to bind host plasminogen, converted to the protease plasmin.  Plasmin then cleaves HA0 to produce HA1 and HA2.  NA functions after virus release to remove sialic acid residues from the viral G protein & cellular glycoproteins.  This prevents virus aggregation upon release to promote even delivery of progeny virus to cells.  The NA glycoprotein forms a tetramer of box- like heads on a slender stalk.  The neuraminidase active site is on the top surface of each box.  The tetramer is not evenly distributed & occurs in patches on the surface of the virion. Model for HA0 Cleavage by Plasmin