Entry of vesicular stomatitis virus VSV

1
Entry of vesicular stomatitis virus (VSV)

• Virus receptor is a lipid (phosphatidyl serine
  PS)
• a unique example ??
• Very wide infection range (all cells have PS) -
  one of the most promiscuous viruses out there
• Fusion etc is similar to influenza..
• Both VSV G and influenza HA are referred to as
  type I fusion proteins
• with two main differences
• The trigger is reversible
• The pH threshold is less stringent (approx. pH
  6.5). Fusion is though to occur from the early
  endosome

2
Type I and type II fusion proteins

• Type I is the most common and understood fusion
  protein
• Influenza, VSV, retrovirus

• Type II fusion proteins are not proteolytically
  activated, have internal fusion peptides and no
  coiled-coil form they are principally b-sheet
• Flavivirus (TBE), and togavirus (SFV)

3
Comparison of type I and type II fusion proteins
From Principles of Virology, Flint et al, ASM
Press
4
Surface representation of dengue virus
5
Endosomes and virus entry

• Endosomes are used by cells for nutrient and
  growth factor uptake
• The virus hijacks the cellular pathway
• One key feature of endosomes is their progressive
  acidification - due the the action of the
  vacuolar H/vATPase
• Endosomes do much more than provide low pH
• Deliver through cortical actin and
  microtubule-mediated transport in the cytosol
• Specific redox/ionic environment
• Defined lipids for fusion/penetration

From Cell Biology, Pollard and Earnshaw, Saunders
6

• The lowered pH causes conformational changes in
  the spike glycoprotein, and the exposure of a
  fusion peptide
• This is the trigger needed for virus entry
• In most cases a pH of around 6.2-6.5 is
  sufficient for fusion
• - fusion occurs in the early endosome
• Entry and infectivity (in cell culture) can be
  blocked by
• 1) addition of a weak base (e.g. NH4Cl) that
  neutralize the endosome
• 2) drugs that target the vH/ATPase (e.g
  bafilomycin A)
• 3) drugs that break down the proton gradient
  (e.g. monensin)
• 4) exposure of the virus to a low external pH

Fusion can be induced at the cell surface by
exposure to low pH
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Poliovirus/Rhinovirus (Picornaviridae)

• Picornaviruses bind to a variety of specific cell
  surface molecules - these are specific proteins
• Binding occurs via canyons (depressions) in the
  virus surface

From Principles of Virology, Flint et al. ASM
Press
Similar viruses can have quite distinct
receptors
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Penetration of non-enveloped viruses

• Rhinovirus/Poliovirus (Picornavirus)
• Although not pH dependent, poliovirus may still
  enter through the endosome

• Interaction of poliovirus with PVR causes major
  conformational changes in the virus - leads to
  the formation of the A particle -physically
  swollen (less dense)

From Principles of Virology, Flint et al, ASM
Press
9

• A particles are now hydrophobic. Viruses have
  apparently lost VP4, and the hydrophobic core is
  exposed on the virus surface
• With a non-enveloped virus, fusion is not
  possible. Instead picornaviruses form a membrane
  pore

Penetration might be controlled by sphingosine, a
lipid present in the pocket -- or (more
likely) by the pocket (canyon) allowing
breathing of the capsid
From Principles of Virology, Flint et al, ASM
Press
Parvoviruses may contain a phospholipase A2
activity in their capsid protein The specific
lipid composition of endosomes may be crucial for
some viruses
10
Picornaviruses as enzymes ?Virus entry as
thermodynamics ??
11
Herpesviruses

• A complex system
• Herpesviruses have 10-12 surface glycoproteins
• Binds initially to heparan sulfate (via gC)
• used by a multitude of different viruses -
  non-specific
• An attachment or capture receptor
• Subsequently binds to a co-receptors that allows
  entry (via gD) - herpesvirus entry mediator -
  specific
• A fusion receptor
• HveA TNF-R
• HveB Nectin2 (Prr 2)
• HveC Nectin1 (Prr 1)
• HveD PVR

• Different herpesviruses use different receptors
• But very different viruses can use the same
  receptor
• e.g. pseudorabies virus and polio virus
• Another example CAR - the coxsackie/adenovirus
  receptor

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Adenovirus

• Entry occurs via clathrin-dependent endocytosis
• Instead of forming a discrete pore, adenovirus
  ruptures or lyses the endosomal membrane
• The trigger is low pH, via the penton base
  protein
• The virus undergoes proteolytic cleavage - by
  virus-encoded proteases

From Principles of Virology, Flint et al, ASM
Press
13
SV40

• Entry occurs via endocytosis
• but in a clathrin-independent manner
• Entry does not depend on low pH
• The virus enters through caveolae - a
  specialized endocytic vesicle that forms upon
  specific cellular signaling induced by virus
  binding
• Receptor is combination of a protein (MHCI) and a
  glycolipid?
• The caveosome containing the virus is delivered
  to the endopalsmic reticulum
• What happens thereafter is a mystery

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Reovirus

• The rare example of a virus requiring the
  lysosome
• Reoviruses have a complex double capsid, which
  is very stable to low pH (gastro-intestinal
  viruses rotavirus)
• The lysosomal proteases degrade the outer capsid
  to form a subviral particle i.e degradation by
  cellular proteases
• The subsequent penetration step is unknown

From Principles of Virology, Flint et al, ASM
Press
15
Nuclear Import

• Why replicate in the nucleus?What are the
  benefits?
• DNA viruses - need cellular DNA polymerase and/or
  accessory proteins (eg topoisomerase) -
• All DNA viruses replicate in the nucleus
• exception Pox viruses (even these will not
  replicate in an enucleated cells or cytoplast)
• Almost all RNA viruses replicate in the
  cytoplasm, and most will replicate in a cytoplast
• Principal exceptions retroviruses (these have
  a DNA intermediate) and influenza virus (has a
  spliced genome)

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What are the problems with nuclear replication?

• An additional barrier during genome transport
• The nucleus of a eukaryotic cell is surrounded by
  a double lipid bilayer - the nuclear envelope.
• The nuclear envelope is studded with transport
  channels - the nuclear pores

From Flint et al Principles of Virology ASM Press
17
Parvovirus

• Possibly the simplest example of nuclear entry
• Small icosahedral DNA virus (18-26nm diameter)
• Enters through endosomes (pH-dependent)
• VP1 contains a nuclear localization signal (NLS)
• Basic amino
  acids
• The NLS binds to cellular receptors (karyopherins
  or importins) that carry proteins into the
  nucleus

From Flint et al Principles of Virology ASM Press
But, the NLS is hidden on the inside of the
capsid Therefore a conformational change must
occur to expose the NLS
18
Adenovirus

• Contains NLSs on its capsids, binds microtubules
• But,
• The functional size limit of the nuclear pore is
  26 nm
• The virus is therefore transported as far as the
  pore.
• It docks to the nuclear pore and then undergoes
  final disassembly, and the DNA is injected into
  the nucleus - with DNA binding proteins attached
• A similar scenario occurs for herpesviruses
  (150-200nm)

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The problem of cytoplasmic transport

• Assume the virus in question has undergone
  receptor binding and penetration - ie the
  virus/capsid in the the cytoplasm.
• The cytoplasm is viscous and the nucleus is often
  a long distance from the site of entry.
• This is especially true for specialized cells
  such as neurons

mm
mm
From Sodeik, Trends Microbiol 8 465
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Microtubules and virus entry
VSV/Rabies, influenza Adenovirus Herpesvirus
From Sodeik, Trends Microbiol 8 465

• To facilitate transport viruses often bind to the
  cytoskeleton and use microtubule-mediated motor
  proteins for transport, i.e. dynein

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Herpesvirus

• After fusion the tegument (most of it) is shed -
  phosphorylation dependent
• Contains NLSs on its capsids, binds microtubules
  via dynein
• The virus is therefore transported as far as the
  pore.
• It docks to the nuclear pore and then undergoes
  final disassembly, and the DNA is injected into
  the nucleus

Note the capsid is empty - no dark center on EM
From Whittaker Trends Microbiol 6 178
22
Influenza virus

• The nucleoprotein (NP) contains NLSs and the
  RNPs are small enough to translocate across the
  nuclear pore
• The key to influenza nuclear import is the
  pH-dependent dissociation of the matrix protein
  (M1) from the vRNPs.
• This relies of the M2 ion channel in the virus
  envelope, the target of amantadine

From Whittaker Exp. Rev. Mol. Med. 8 February,
http//www-ermm.cbcu.cam.ac.uk/01002447h.htm
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Retroviruses

• Simple complex
• Simple retroviruses (oncoretroviruses) can only
  replicate in dividing cells, e.g. Rous sarcoma
  virus (RSV), avian leukosis virus (ALV).
• Nuclear entry occurs upon mitosis - the nuclear
  envelope breaks down and the virus is passively
  incorporated into the new nucleus
• This is relatively inefficient and restrictive
  for virus tropism
• Complex retroviruses (lentiviruses) have evolved
  mechanism for nuclear entry in non-dividing
  cells, e.g. HIV

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HIV

• Once in the cytoplasm the RNA genome is reverse
  transcribed into a DNA copy - the
  pre-integration complex (PIC)
• There may or may not be a role for microtubules
  (most likely they are involved)
• The PIC is a large (Stokes radius 28nm)
  nucleoprotein complex that contains several
  proteins, including
• integrase (IN) matrix (MA) and Vpr
• Each of these three proteins seems to play a role
  in transporting the very large PIC to and across
  the nuclear pore

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Further reading

• Chapters 4 and 5 of Flint et al.
• Chapter 4 of Fields Virology
• Brief overview on cellular virus receptors,
  Mettenleiter TC, Virus Research 82 (2002) 3-8
• Cool movies -- http//trimeris.com/science/hivfusi
  on.html