Entry into host cells
Chapter 5, pp. 151 -- 180
Lecture 11 BSCI437
Overview
Once attached to a cell, viruses must

Enter the cell o Active uptake o Passive uptake

Target themselves to the appropriate compartment o Cytosol o Nucleus

Make their genomes accessible for the next step o Disassembly.
Uptake of Macromolecules by cells (Fig. 5.15)
Some small molecules can freely traverse lipid bilayer of cell membrane
Water, gas, small hydrophobic molecules
Most small metabolites are actively transported in and out by pores or specific receptors
Glucose, ATP, nucleosides, amino acids, proteins, cations
Larger molecules and particles require active transport by
Phagocytosis
Endocytosis
Pinocytosis – non-specific
Receptor-mediated endocytosis – specific
Receptor-mediated endocytosis (Fig. 5.16)

Ligands bind to membrane receptor proteins

Migrate to clathrin coated pit

Pit invaginates, then pinches off

Clathrin coat falls off, now a vesicle

Vesicle fuses with endosome – early endosome

Contents transported to late endosome – acidification

Late endosomes fuse with lysosomes – degradation
Cell membrane fusion
 Cell membranes do not fuse all by themselves…otherwise our bodies would be just one gigantic syncytium.

Proceeds by specialized mechanisms mediated by proteins
1.
Help bring into close proximity
2.
Targeting and docking proteins must interact
3.
Water molecules must be removed (energetically unfavorable)
Virus uncoating/disassembly (Fig. 5.18)


Viruses use capsids (and sometimes envelopes) to protect their genomes from the environment

On entry into cells, genetic material must be released

Effected by uncoating/disassembly

Three general pathways
1.
Uncoating at plasma membrane
2.
Uncoating within endosomes
3.
Uncoating at nuclear membrane
Uncoating at plasma membrane (Fig. 5.19)

Generally used by enveloped viruses.

Receptor-ligand interaction brings envelope and cell membranes into close juxtaposition.

Fusion generally mediated by a second, viral fusion protein
 vSNARES evolved from cellular SNARES, proteins that effect fusion of intracellular vesicles.
Acid-catalyzed uncoating within the endosome (Fig. 5.20)

Many enveloped viruses undergo fusion within the endosome.
Example: Influenza

At cell surface, virus attaches to sialic acid via viral HA glycoprotein

Virus-receptor complex internalized by clathrin-dep. Endocytosis.

In late endosome, pH = 5.0

HA undergoes acid-catalyzed conformational change

Exposes fusion peptpide/

Viral and endosomal membranes fuse

Viral ribonucleoprotein released into cytoplasm.
Entry of nonenveloped viruses
General points

Entry into cells cannot be mediated by membrane fusion.

Much more complex

Many mechanisms
Disruption of the endosomal membrane (Fig. 5.26)
Example: Adenoviruses. dsRNA genome in icosahedral capsid.

Internalized by receptor-mediated endocytosis

In endosome, low pH exposes penton base, which lyses endosome

What remains of viron enters cytoplasm

Docks with nuclear pore complex

Capsid interacts with histone H1 and importins

Capsid disassembles at nuclear pore

DNA imported into nucleus

Formation of pore in the cell membrane (Fig. 5.27)
Example: picornaviruses, ssRNA genomes in icosahedral capsids.

Virus interacts with cell surface receptor (pvr for poliovirus)

Internalized by endocytosis

Interaction with pvr results in conformational change

VP1 and VP4 move from inside virion to outside

Exposes hydrophobic domains

Result in pore through endosome

Viral genome exits through pore into cytoplasm
Lysosomal uncoating (Fig. 5.28)
Example: Reoviruses: dsRNA genomes, double shelled icosahedral capids

Enter cell via receptor-mediated endocytosis

VERY low pH of lysosome induces conformational change

Outer capsids shell uncoated

Inner capsids revealed

This penetrates lysosomal vessicle
Transport into the Nucleus
General

Many viruses begin replication in the nucleus

They need to get from cytoplasm to nucleus

Examples: adenoviruses, retroviruses, influenza, Epstein-Barr virus

Require nuclear localization signals o Short amino acid sequences: 2 general motifs (Fig. 5.31)

Hydrophobic – basic
3-7
– hydrophobic

Basic
2/3
– X
10
– basic
3/5
Transport through nuclear pore: see Fig. 5.32A, C, and 5.26
Nuclear membranes contain pores
Allow passage of molecules into and out of nucleus
Very complex structures
Viruses have developed strategies to hijack nuclear pore complexes.

Example: Adenoviruses.