Entry and Uncoating of Entry and Uncoating of VirusesViruses

Virology Unit 4, 2015 VersionVirology Unit 4, 2015 Version

Main Points and PlanMain Points and Plan Introduction-some general backgroundIntroduction-some general background Generic informationGeneric information Specific examples-animal viruses:Specific examples-animal viruses:

Influenza A (also evolution)Influenza A (also evolution)

ParamyxovirusesParamyxoviruses

Plant virusesPlant viruses

Viral Entry Hard to StudyViral Entry Hard to Study

Cellular processes obscureCellular processes obscure Technology weakTechnology weak Diverse mechanismsDiverse mechanisms Entry mechanisms tough to conceptualizeEntry mechanisms tough to conceptualize Not always as expected-more Not always as expected-more

sophisticated?sophisticated? Are there any generalizations?Are there any generalizations?

Plasma membranePlasma membrane

Common to all cells-a barrier to viruses Common to all cells-a barrier to viruses They must penetrate to infect-They must penetrate to infect-

(a) trick the cell into bringing virus in?(a) trick the cell into bringing virus in?

(b) or penetrate on their own?(b) or penetrate on their own? (a) requires subversion of cellular (a) requires subversion of cellular

mechanismsmechanisms (b) requires mechanical “puncture”(b) requires mechanical “puncture”

Some Generic Mechanisms Used by Some Generic Mechanisms Used by Cells to Internalize Large Particles-Can Cells to Internalize Large Particles-Can

Viruses Subvert These?Viruses Subvert These?

Receptor-Mediated Endocytosis involving Receptor-Mediated Endocytosis involving ligand and receptor (RME)ligand and receptor (RME)

Endocytosis-generic term for non-specific Endocytosis-generic term for non-specific pinocytosis or phagocytosispinocytosis or phagocytosis

Caveolae-separate pathway, no clathrinCaveolae-separate pathway, no clathrin

Entry of Influenza A is the ParadigmEntry of Influenza A is the Paradigm

Best-studied (important pathogen) Best-studied (important pathogen) Integrates many types of studiesIntegrates many types of studies Reveals unsuspected sophistication of Reveals unsuspected sophistication of

virusesviruses Complex, multi-step processComplex, multi-step process

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.

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.

Influenza Clinical Influenza Clinical SymptomsSymptoms

Rapid OnsetFeverShiveringProstrationMyalgiaCoughing, sneezing, runny nose (coryza)Usually no GI involvementUsually not life threatening

for adultsSecondary infections

problematic

-Pleomorphic virions are 80-100 nm in diameter

-Filamentous forms -may occur

-Prominent 16 nm surface spikes

INFLUENZA VIRUS STRUCTURE

Virion ComponentsVirion Components

Virus Binding to Cells Influenza virus attaches via HA trimer

Cellular receptor moleculeIs a carbohydrate

Sialic acid(N-acetylneuraminic)

Receptor Distribution

Influenza Virus HA Protein

1)The HA0 protein subunits

form a homotrimer 2) N-terminal heads contain

variable antigenic determinants & sialic acid binding pockets

3) C-termini are membrane anchors

4) HA0 converted to HA1 and

HA2 by proteolysis.

Proteolysis of HA0

“Late” eventHost proteaseAbsolute requirement for infection

Influenza A particles internalized via receptor-mediated endocytosis

Physiological pH form is metastableLower pH induces massive conformation change

Conformation Change of HA ProteinConformation Change of HA Protein

Fusion peptide penetrates endosomal membrane

pH 7.0 pH 5.0

Cytoplasm

Virus

Snapback of HA trimerforms fusion poreContents of virusexit to cytoplasm

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

Summary for Influenza ASummary for Influenza A

Conformation changesConformation changes pH dependentpH dependent In cytoplasmIn cytoplasm IrreversibleIrreversible Checkpoints passed in response to signalsCheckpoints passed in response to signals

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