[MCR 228]
[Chapter 4: Virus Entry]
Lecture Outline
I. Steps in Virus Entry
II. Penetration through Cellular Membrane
III. Intracellular Transport
Enveloped and non-enveloped viruses have
distinct penetration strategies
Enveloped viruses
• Use vesicle transport and coupled fusion (“coming
together”) and fission (“breaking apart”) of
membranes
• viral envelope: transport vessel in passage from
one cell to the next; nucleocapsid is the cargo
Budding
• viral envelope forms around nucleocapsid and
other internal components (matrix proteins) at cell
membrane
• takes place at external plasma membrane
• enveloped virion is then released through EC
space directly or after transport thru secretory
pathway
•
Fusion of envelope with cell membrane
• Delivery of nucleocapsid into cytosol of target cell
• takes place at plasma membrane or membranes
of early or late endosomes (membranous
organelles in which the virion is enclosed after
undergoing receptor-mediated endocytosis)
• advantage: viral macromolecules do not need to
pass directly across the hydrophobic interior of any
cellular membrane.
• Ex. Influenza viruses and togaviruses
Non-enveloped viruses
• no lipid membrane
• to exit from infected cell, they rely on cell
death/rupture of cell membranes to release virus
particles in nucleus or cytoplasm
• Examples:
o Adenoviruses – taken up in endosomes
where they proceed to rupture the
endosomal membrane, releasing the
virion and other contents in cytosol
o Picornaviruses – undergo conformational
changes during binding to surface
receptors or after entering endosomes
that allow viral proteins to form
membrane channels to direct genome to
cytosol
Some viruses can pass directly from cell to cell
Release of virions from infected cell is not always necessary
for transmission of infection
• Filopodia – use to infect neighboring cells through
contact
• Infected host cells play active role in transmission,
with minimal exposure of virus to extracellular
environment
• Also helps avoid recognition of virus by the host
immune system
[Bernadette Pedro]
• Ex. Retroviruses – virological synapse (special
region where viruses are preferentially assembled
and transmitted in the contact area between virus-
producing cell and neighbor)
Infection can also spread from one cell to another without
formation of a complete virus particle
• Measles and other enveloped viruses – induce
fusion of infected cells with neighboring uninfected
cells
• Results in formation of syncytia (multinucleated
cells)
o Responsible for cell-cell fusion when
incorporated into the plasma membrane
of infected cell
• Transmission is limited to cells located next to
each other
In plants, infection spreads directly through
plasmodesmata which connect the cytosolic
compartments of neighboring cells
• These channels traverse the cell wall and provide
a conduit of transport of viral genomes
• For some viruses, plasmodesmata are too narrow
for passage; virus-encoded “movement proteins”
are expressed to make plasmodesmata wider
A variety of cell surface proteins can serve as
specific virus receptors
• attachment factors and receptors in PM
o include proteins, carbohydrates and
lipids
o have their own cell functions
o highly conserved
o unlikely to undergo mutations
o presence or absence of receptors is one
of the most important factors to
determine the species and cell types that
can be infected by a given virus
• proteoglycans or glycoproteins
• immunoglobulin family (CD4, PVR, CAR, CD46)
• LDL (low density lipoprotein) receptor family
• Multimembrane-spanning transport proteins,
Integrins,Glycolipids
• Ex. Influenza and paramyxoviruses – bind to sialic
acid residues present on glycoproteins and
glycolipids
o To avoid from binding to fragments that
do not support infection, they carry
neuraminidases which release the
virions by cleaving the sialic acid
residues to which they are bound
1
 [MCR 228]
[Chapter 4: Virus Entry]
• Ex. HIV-1 and Adenovirus – require more than one
type of receptor
o Primary receptor – sufficient for
attachment and induce conformational
changes
o Secondary receptor (coreceptor) –
needed to trigger endocytosis or
penetration
o Association with the receptors can occur
sequentially because the binding sites for
the coreceptor are exposed only after
interaction of the virion with the primary
receptor
Receptors interact with viral glycoproteins surface
protrusions or “canyons” on the surface of the
virion
• In enveloped viruses: Specific surface spike
glycoproteins -serve as binding sites for receptors
o Same glycoproteins mediate both
attachment and membrane fusion; some
exclusively for attachment
o Ex. HA domain of trimeric influenza virus
HA protein
• In non-enveloped viruses: receptor-binding sites
are formed either by protrusions or cavities in
capsid surface
o Ex. Adenoviruses: prominent trimeric
fiber attached to each penton base, with
receptor-binding site located in a knob at
the tip; in Picornaviruses: indented
receptor binding site (“deep canyons”)
• Interaction between receptor and virus leads to
changes in both virus particle and cell
o Ex. Binding of HIV-1 to CD4 proteins
triggers a change in HIV-1 glycoprotein
trimers, allowing them to bind to
coreceptors in the cell membrane
• Attachment of adenovirus to integrins
activates a signaling cascade inside the cell that
temporarily increases endocytic activity and
prepares the cell for the incoming virus
o Activation of signaling pathways –
essential process for entry/replication
o Signaling pathways – promote changes
in cell architecture and induce
micropinocytosis and caveolar/lipid raft-
dependent endocytosis
Many viruses enter the cell via receptor-mediated
endocytosis
• Majority of viruses depend on endocytosis to be
first carried into cytoplasmic vacuoles, from which
they can then enter the cytosol
[Bernadette Pedro]
• Endocytosis occurs via clathrin-coated pits and
vesicles
o Ferry virus particles through network of
cytoskeletal fibers and transport them
away from surface and toward the cell
nucleus
o after leaving the cell surface, virions are
delivered to early endosomes as other
substances are taken up by clathrin-
coated vesicles
o after internalization, virus particle is still
separated from the cytosol by barrier of
endosomal membrane
• Early endosomes – complex organelles
composed of tubular and bulbous membrane
elements in peripheral cytoplasm
o Sorting and recycling station for
incoming fluid, ligands, receptors, and
membrane proteins
o Responsible for dissociation of incoming
ligand-receptor complexes, recycling of
empty receptors to cell surface, and
dispatching non-recycled material to late
endosomes and lysosomes to be
degraded
o Mildly acidic (6.3-5.5), maintained by
membrane-associated vacuolar-type
ATPases that pump protons from
cytosol into lumen (interior of the
vesicle)
• Incoming molecules sorted for degradation are
exposed to successively acidic pH from early to
late endosomes, and ultimately to lysosomes
• Late endosomes – have characteristics of
multivesicular bodies
o Contain intralumenal vesicles
o Move by microtubule-mediated transport
into perinuclear region of cell and fuse
with lysosomes
Entry via caveolae and lipid rafts
• Ex. Simian virus 40 (polyomavirus)
• Small, flask-shaped invaginations in the plasma
membrane are enriched in cholesterol and
sphingolipids
o Invaginations are formed by induction of
membrane curvature caused by tight
multivalent interaction of capsid proteins
on the virion with receptor gangliosides
on cell surface
• Caveolae – contain cellular proteins called
caveolins and cavins
o Has a role in signaling and uptake of
cholesterol and selected ligands
2
 [MCR 228]
[Chapter 4: Virus Entry]
Macropinocytosis
• Ex. Vaccinia virus – triggers complex signaling
cascade that induces transient ruffling of PM
followed by internalization of fluid and virus
particles in large vacuoles called
macropinosomes
Passage from endosomes to cytosol is often
triggered by low pH
• Endosomes – major site of virus penetration into
cytosol
• Trigger for penetration: low pH, which induces
conformation change in spike of glycoproteins
(enveloped viruses) or in capsids (non
enveloped)
• Low pH signals virus that it has reached
intracellular location and penetration should begin
• Virus particles attached to PM can be artificially
activated to penetrate prematurely simply by
lowering pH in the medium of bathing cells
• Most viruses adjust timing of penetration by
titrating activation threshold to a pH 6.5-5.3 range
• Conversion occurs 5-30 mins after uptake into
early and late endosomes, before virus can be
delivered to lysosomes
• Many viruses can penetrate directly thru PM,
where they first encounter their receptors
o Adenoviruses – need both receptor-
induced and low pH-induced
conformation change to penetrate
Membrane fusion is mediated by specific viral
“fusion proteins”
Penetration of enveloped viruses involves membrane
fusion event mediated by viral glycoproteins
Viral fusion proteins
• type I transmembrane proteins
o Mass lies external to viral membrane
o Organized as oligomers
• Synthesized, folded, and assembled into
oligomers in ER of infected cells
• To become fusion-active, they are cleaved by
cellular proteases in transit from ER through golgi
complex to PM
[Bernadette Pedro]
o Ex. Influenza virus HA – cleaved into
peripheral N-terminal fragment HA1 and
C-terminal transmembrane fragment
called HA2
• In HA and some viral spike glycoproteins,
activating cleavage occurs to N-terminal side of
hydrophobic sequence of 10-12 amino acid
residues
o fusion peptide- sequence in the
cleaved protein which contains the N-
terminus of the transmembrane subunit
and important in the fusion reaction
o energy to induce fusion is derived from
conformational change in fusion protein
(independent of metabolic energy in the
form of ATP)
Fusion proteins undergo major conformational
changes that lead to membrane fusion
• activated by low pH or receptor binding
• studied in most detail for trimeric influenza virus
HA
o Step 1: exposure to low pH in late
endosomes
o Step 2: fusion peptides in HA2 subunits
are exposed and insert into outer bilayer
leaflet of target membrane
o HA1 subunits remain attached but no
longer form a trimeric domain at the top
of oligomer
o Viral HA2 protein is now attached to
both the viral membrane (via
transmembrane sequence) and to target
membrane (fusion peptide)
o This attaches the membrane to each
other but does not cause them to fuse
due to the great distance between them
o Step 3: Several HA molecules form
cluster
o Step 4: Each HA trimer folds back on
itself like jackknife, forcing two
3
 [MCR 228]
[Chapter 4: Virus Entry]
membranes closer in the middle of the
cluster
o Step 5: When later of bound water
molecules that prevents fusion is
eliminated in small local spot in the
middle of HA cluster, two bilayers
proceed to fuse.
o Hemifusion (outermost bilayer leaflets
fuse first) then the inner bilayer halves
o Step 6: narrow aqueous channel of
internal cavities of the virus and the
cytosol form
o After flickering, fusion channel remains
permanently open and expands
o Nucleocapsids are now exposed and
can move to the cytosol
• Class I fusion proteins (retroviruses,
paramyxoviruses, filoviruses)
o Reveals a trimeric spike standing
perpendicular to the viral envelope
o Dominated by long central alpha-helical
coiled coils (has 2 distinct
conformations)
• Class II fusion proteins (togaviruses,
flaviviruses, rhabdo and hepadna)
o Fusion-activating changes involve major
alterations in the interactions between
folded domains and protein subunits in
oligomers
o Undergo only minor conformational
changes at secondary structure
Non-enveloped viruses penetrate by membrane
lysis or pore formation
Adenoviruses – best-studied example that penetrate by
acid-activated, virus-dependent endosome lysis
• Virion is subjected to changes prior to activation
• Fibers dissociate, penton base proteins bind to
arg-gly-asp sequences in the integrin
(coreceptor)
• When virion is exposed to low pH, it converts to
lytic form
• Endosome membrane ruptures, and virion and
other contents of endosome is released in the
cytosol
other non-enveloped viruses pass thru cell membrane
barriers without lysis
• Picornaviruses (polioviruses) undergo pH-
independent entry but still depend on endocytosis
• Binding to receptor triggers an initial
conformational change -> loosen up capsid wall->
makes particles hydrophobic -> these “A-
particles” interact hydrophobically with
[Bernadette Pedro]
endosomal membrane -> induce formation of
membrane channel which viral RNA can use to
slip in cytosol -> leaves empty capsid in
endosomal lumen -> remains is RNA-free “B-
particle”
Virions and capsids are transported within the cell
in vesicles or on microtubules
Virus particles or capsids that travel in extremely long
distances inside cell
• Herpes simplex type 1 and rabies virus
Diffusion of macromolecular assemblies in the cytosol is
severely limited due to molecular crowding by organelles,
cytoskeleton, and macromolecules
Virions can be transported:
• inside endocytic vesicles (prior to penetration) –
has the advantage that cell provides all the
systems and signals needed to move the
incoming vesicle in the direction of nucleus
• or as free nucleocapsids within cytoplasm (after
pene)
molecular motors used include
• dynein – microtubule-dependent motor
o moves particles along microtubules from
cell periphery in the direction of
microtubule-organizing center, close to
the nucleus
o Ex. Herpesvirus and adenovirus capsids
use it for retrograde transmission
• dynactin – adaptor complex
Import of viral genomes into the nucleus
Animal, plant and DNA-containing virus – nucleus as site
of replication
• can establish latent state in nucleus and integrate
their genes in host chromosomes
• problem: incoming virus must deliver its genome
and necessary viral proteins to nucleus
• a number of viruses interact with nuclear
targeting receptors such as importins
(karyopherins), and their genomes enter thru
nuclear pore complexes
Dissociation of nuclear membrane during mitosis
• in retroviruses (except lentivirses)
• no means of entering the intact interphase
nucleus
• capsids (“preintegration complexes”) which
contain proviral DNA made by reverse
transcription and other viral proteins must wait in
cytoplasm until cell division
4
 [MCR 228]
[Chapter 4: Virus Entry]
• during cell division, nuclear envelope
disintegrates -> preintegration complexes can
access nucleus when nuclear envelope reforms
• proviral DNA can integrate into cell genome in
reformed nucleus
• infection is only possible in cells undergoing
mitosis
o explains why terminally differentiated
(non-dividing) cells in the body are not
usually targets for retrovirus infection
Import after partial disassembly in the cytoplasm
• in Lentiviruses (HIV1)
• direct their preintegration complexes to enter the
nucleus via nuclear pores
• matrix and Vpr proteins in the complexes interact
with importins
• each of the 8 distinct genome RNAs of influenza
virus is individually packaged thru nuclear pores
• after release of complexes in cytoplasm by fusion
with endosome membrane -> interact with
importins -> transported to and thru nuclear pore
complexes
Import after disassembly at the nuclear pore
• in Adenoviruses and herpesviruses
o large isometric capsids that are too
bulky to pass thru nuclear pores
• capsids bind to fibers that extend from
cytoplasmic face of pore -> capsids are
disassembled -> viral DNA and associated
proteins enter thru nuclear pore
• adenovirus capsids disintegrate in the process
while intact empty herpesvirus capsids remain
unbound to the pore fibers
Transport of intact virions thru nuclear pore complex
• heap B virus capsids (34 nm diameter) and
simian virus 40 capsids (48 nm) and
parvoviruses (20 nm) can pass intact thru nuclear
pores
The many ways in which viral genomes are
uncoated and released
Non-enveloped picornaviruses
• release genome RNA directly in cytosol, leaving
capsid within endosomal vesicle
• virus entry and uncoating are the same event
Hepa B, Parvoviruses, and Polyomaviruses
• uncoating of DNA genome takes place after
capsids have been transported into nucleus thru
nuclear pores
[Bernadette Pedro]
• uncoating of herpesvirus and adenovirus
genomes takes place at nuclear pore, as capsids
are disassembled there
• in adenovirus, viral cysteine protease is activated
by entry into reducing cytosolic environment
• DNA genomes of most of these viruses are
complexed with cell or viral proteins that compact
the DNA and neutralize its charge
Togaviruses
• Uncoating of viral RNA occurs only minutes after
low pH-induced fusion of viral envelope with an
endosomal membrane
• Ribosomal 60s serves as cellular uncoating
factors
Influenza A viruses
• Encode M2 protein that forms membrane
channels allowing free passage of H+ ions at low
pH
• Small amounts of M2 proteins are incorporated
into the virion envelope
• When virions are present in acidifying
endosomes, interior of particle is also exposed to
low pH, and this releases individual
nucleoproteins from their association with each
other and with M1 protein
• Upon fusion of viral envelope with endosomal
membrane, genomes remain associated with
nucleocapsid proteins at all times
Reoviruses
• Enter cytosol by a pH-independent mechanism
thru PM or endosomal membrane in which an
outer layer of capsid proteins is removed but
inner capsid remains intact
• Uncoating is incomplete, viral DS RNA is never
released to cytosol
5