Lecture 5 - Attachment and Entry

Attachment and Entry
Lecture 5
Biology 4310
Virology
Spring 2021
Who hath deceived thee so

often as thyself?
--BENJAMIN FRANKLIN
Viruses are obligate intracellular parasites
Virus particles are too large to diffuse across the plasma membrane
Virology Lectures 2021 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
Finding the ‘right’ cell receptor
• Step 1: adhere to cell surface (random collisions & electrostatics)

- No specificity
• Step 2: Attach to specific receptor molecules on cell surface
- More than one receptor may be involved
• Step 3: Transfer genome inside the cell
Cellular receptors for viruses
• Essential for all viruses except those of fungi (no extracellular

phases) and plants (enter cells by mechanical damage)
• 1985: one receptor known, sialic acid for influenza virus
Enabling technologies
• Recombinant DNA
• Monoclonal antibodies
• Flow cytometry
• Nucleotide sequencing
• siRNA
• CRISPR/Cas9
Criteria for identifying cell receptors for viruses
• Receptor binds virus particle

• Antibody to receptor blocks infection
• Receptor gene confers susceptibility
- More than one receptor may be involved
• Disruption of receptor gene blocks infection
ACE2 is receptor for SARS-CoV-2
DAPI ACE2-FITC NP-Cy3 MERGE
HeLa-hACE2
HeLa cells do not

produce ACE2
HeLa-bACE2
Plasmid DNA
encoding ACE2
HeLa-mACE2
HeLa that produce
ACE2
HeLa
Virology Lectures 2021 • Prof. Vincent Racaniello • Columbia University https://www.nature.com/articles/s41586-020-2012-7

Hydrophobic
core
Polar head groups
Phosphatidyl- Phosphatidyl- Phosphatidyl-

serine inositol ethanolamine
Cytoplasm
Outside of cell
Indirectly anchored
protein Carbohydrate
Integral
membrane Lipid-anchored
proteins protein
Indirectly anchored protein

Cytoplasm
Cell functions!
ScEYEnce Studios
Principles of Virology, 4e
Volume 01
Fig. 02.06
3-25-14
Different viruses can bind the same receptor
• Adenovirus and Coxsackievirus B3 have common primary receptor

• The swine herpesvirus, pseudorabies virus, binds same receptor as
human poliovirus
Viruses of the same family may bind different receptors
• Rhinoviruses (3), retroviruses (16)

• One virus may bind multiple receptors
How do virus particles attach to receptors?
Poliovirus Rhinovirus 2
CAR
Knob
Adenovirus
http://pdb101.rcsb.org/motm/132
Influenza virus attachment to cells
Hemagglutinin (HA)
Neuraminidase (NA)
Virology Lectures 2021 • Prof. Vincent Racaniello • Columbia University Monomer of HA protein ©Principles of Virology, ASM Press
Sialic acid: receptor for influenza viruses
Transmembrane glycoprotein
• Sialic acids: N-acetylneuraminic acid (A,B); 9-O-acetyl-N-neuraminic acid (C)
• α(2,6) preferentially bound by human strains, α(2,3) by avian

HIV-1 attachment
SU
TM
Envelope
Virology Lectures 2021 • Prof. Vincent Racaniello • Columbia University http://pdb101.rcsb.org/motm/169 ©Principles of Virology, ASM Press
SARS-CoV-2 attachment
Receptor binding domain

(ACE2)
Coronavirus cell receptors
HCoV-229E Human amino peptidase N

HCoV-NL63 Angiotensin converting enzyme 2
HCoV-OC43 N-acetyl-9-O-acetylneuraminic acid
SARS-CoV Angiotensin converting enzyme 2
MERS-CoV Dipeptidyl peptidase 4
SARS-CoV-2 Angiotensin converting enzyme 2
Go to:
b.socrative.com/login/student
room number: virus
Viral receptors on the cell surface:
A. Can bind directly to icosahedral virus capsid proteins

B. Interact with glycoproteins of enveloped viruses
C. Can be carbohydrate or protein molecules
D. Have cellular functions
E. All of the above
Virology Lectures 2021 • Prof. Vincent Racaniello • Columbia University

1
Entry into cells
https://ccsb.scripps.edu/goodsell/
The cytoplasm is crowded!
Movement of large protein

complexes will not occur by
diffusion!
Fusion of viral and host cell
membranes mediated by
viral fusion proteins
Movement of endosomes
XVIVO Scientific Animation http://www.xvivo.net/

Entry at plasma
membrane
HIV-1
HIV-1 Env mediated fusion
Virology Lectures 2021 • Prof. Vincent Racaniello • Columbia University https://youtu.be/51z2dwdOLUE

Go to:
room number: virus
Which of the following does not play a role in virus entry:
A. Clathrin-mediated endocytosis
B. Fusion of viral and plasma membranes
C. Diffusion of virus particles in the cytoplasm
D. Microtubule-mediated transport
E. Sialic acids
Virology Lectures 2021 • Prof. Vincent Racaniello • Columbia University 2

Influenza virus membrane fusion
Three states of influenza virus HA
Post-fusion
Class I fusion proteins
• Perpendicular to
membrane - spikes
• Mostly α-helical
• Form trimers
Influenza virus entry

SARS-CoV-2 entry
TMPRSS2
RBD
Protease cleavage of spike

needed for fusion: this
process must be regulated Furin cleavage site
TMPRSS2
Class II fusion proteins
• Mostly β-sheet
• Form dimers
• Parallel to the membrane
Dengue virus entry

Ebolavirus entry
Fusion is regulated
• Must not occur in the wrong location

• Neutral pH (plasma membrane)
• Second protein receptor interaction
• Low pH fusion
•Proteolytic cleavage activates the fusion protein for cleavage (class I)
•Endosome fusion receptor
Go to:
room number: virus
Viral fusion peptides are exposed for insertion into the host cell membrane when:
A. The virus particle is near a cell

B. The virus particle is in the cytoplasm
C. Trimers of the fusion peptides form
D. The endosome becomes acidified
E. The virus is docked on the nuclear pore

3
Adenovirus
How are genomes released

from non-enveloped
viruses?
Uncoating of adenovirus at nuclear pore complex
Release of poliovirus RNA
“Umbilicus” connector
Calicivirus portal on receptor engagement
t=3, 180 copies of VP1
For poliovirus and calicivirus, only one portal is formed

Reovirus entry
Four modes of nuclear entry
Receptor polymorphism and susceptibility
Rhinovirus receptors
• HRV A, B: 90 serotypes, ICAM-1; 10 serotypes LDLR
• HRV C: 49 genotypes, CDHR3
ICAM-1 LDLR CDHR3
HRV-C
• 529Y in CDHR3 linked to increased surface production, CDHR3

increased risk of wheezing illnesses and hospitalization
for childhood asthma
529C
• Cells with CCHR3 529Y have 10x increased HRV-C
binding and yields
• Risk factor for HRV-C wheezing illnesses
• 2013 outbreak of HRV-C in chimpanzees, Uganda: all
homozygous 529Y
• Only humans have 529C, recently selected as
Neanderthal and Denisovans have 529Y
Betty
Next time: RNA directed RNA synthesis

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Who hath deceived thee so

• Step 1: adhere to cell surface (random collisions & electrostatics)

• Essential for all viruses except those of fungi (no extracellular

• 1985: one receptor known, sialic acid for influenza virus

• Receptor binds virus particle

HeLa cells do not

Virology Lectures 2021 • Prof. Vincent Racaniello • Columbia University https://www.nature.com/articles/s41586-020-2012-7

Polar head groups

Phosphatidyl- Phosphatidyl- Phosphatidyl-

Indirectly anchored protein

• Adenovirus and Coxsackievirus B3 have common primary receptor

• Rhinoviruses (3), retroviruses (16)

• Sialic acids: N-acetylneuraminic acid (A,B); 9-O-acetyl-N-neuraminic acid (C)

• α(2,6) preferentially bound by human strains, α(2,3) by avian

Receptor binding domain

Coronavirus cell receptors

HCoV-229E Human amino peptidase N

Viral receptors on the cell surface:

A. Can bind directly to icosahedral virus capsid proteins

Virology Lectures 2021 • Prof. Vincent Racaniello • Columbia University

The cytoplasm is crowded!

Movement of large protein

XVIVO Scientific Animation http://www.xvivo.net/

Virology Lectures 2021 • Prof. Vincent Racaniello • Columbia University https://youtu.be/51z2dwdOLUE

Which of the following does not play a role in virus entry:

Virology Lectures 2021 • Prof. Vincent Racaniello • Columbia University 2

Virology Lectures 2021 • Prof. Vincent Racaniello • Columbia University

Protease cleavage of spike

Virology Lectures 2021 • Prof. Vincent Racaniello • Columbia University

• Must not occur in the wrong location

A. The virus particle is near a cell

Virology Lectures 2021 • Prof. Vincent Racaniello • Columbia University

How are genomes released

t=3, 180 copies of VP1

For poliovirus and calicivirus, only one portal is formed

ICAM-1 LDLR CDHR3

• 529Y in CDHR3 linked to increased surface production, CDHR3

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