Virus Assembly

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Virus Assembly

• 10/09/2003
• 10/16/2003

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What goes in must come out
From Sherman and Greene Microbes Infect. 4 67
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Getting out

• Non-enveloped viruses - adenovirus, parvovirus -
  most probably simply burst out of a dead or dying
  cell
• Immune system-induced, or direct
• Adenovirus E1B induces collapse of intermediate
  filament network (due to inhibited host cell
  protein synthesis) - also E3 glycoprotein
  disrupts nuclear membrane and lyses cells
• Enveloped viruses cannot afford to do this - they
  need a functional cell for the final stage of
  assembly -- envelopment

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Simple retroviruses

• The replicated genome is in the form of viral
  RNA, and assembly occurs in the cytoplasm
• The RNAs most probably use cellular pathways used
  to export messenger RNA (mRNA) - these are still
  not very clearly defined -but these depend on a
  completely spliced mRNA, i.e RNA-based
• One key feature of retrovirus replication is that
  the much of exported viral RNA is
  unspliced/incompletely spliced - this is unique
  for a virus
• Simple retroviruses have a specialized RNA
  sequence called the constitutive transport
  element (CTE), but still use the machinery for
  exporting mRNAs

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HIV

• The replicated genome is in the form of viral
  RNA, and assembly occurs in the cytoplasm
• For HIV the unspliced RNA is exported via a
  different route, which is protein-based
• The RNA is complex with a viral protein called
  Rev, which contains a nuclear export signal (NES)
  - a stretch of hydrophobic residues
• HIV-1 NES is LQLPPLERLTL consensus
  LxLxxLxxLTL
• Rev binds to a defined RNA sequence (the
  Rev-responsive element RRE) and escorts the RNA
  through the nuclear pore via binding to an
  exportin (karyopherin)
• The cellular pathway used normally handles small
  proteins and RNAs (eg 5S rRNA)

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HIV Rev and nuclear export

• Prior to Rev synthesis only spliced RNA is
  exported
• - When Rev is made it enters the nucleus and
  binds the RRE

From Flint et al Principles of Virology ASM Press
Rev promotes export of unspliced RNA via a
leucine-rich nuclear export signal and an
exportin receptor
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Influenza

• vRNPs undergo assembly in the nucleus and exit
  through the NPC
• The key problem for influenza is regulation of
  import vs. export - ie to direct virus formation
  late in infection
• The matrix (M1) protein plays a key role -maybe
  by binding the vRNPs in the cytoplasm and
  preventing their return.
• Export appears to be exportin-dependent (ie
  protein mediated)
• The NES may reside on NP, or on a small
  non-structural protein NS2 that shares transport
  features with HIV Rev

Leptomycin B blocks influenza and HIV nuclear
export - targets the exportin receptor
From Whittaker Exp. Rev. Mol. Med. 8 February,
http//www-ermm.cbcu.cam.ac.uk/01002447h.htm
Parvovirus NS2 also interacts with exportin and
is required for nuclear export
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Hepatitis B virus

• Almost all viruses are targeted out of the
  nucleus late in infection and never return
• Hepatitis B (Hepadnaviridae) is the exception
• It is an inefficient/slow virus and recycles
  its genome (capsid) back into the nucleus for
  further rounds of replication
• - effectively an amplification of the infection
  within a single cell
• Eventually, the level of the surface glycoprotein
  builds up to such a level that budding from the
  plasma membrane can occur

Herpesvirus Nuclear export is intimately
connected to envelopment - details to follow
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Intracellular Transport
Synthesis of viral proteins on cytoplasmic
ribosomes Assembly of capsids Localization of
proteins to the site of budding Movement of the
virus through the cytoplasm Budding and Release
From Flint et al Principles of Virology ASM Press
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Capsid assembly

• Follows the rules outlined in Virus Structure
• Can occur in the nucleus or the cytoplasm
• Occurs via
• A) assembly from individual proteins
• B) assembly from a polyprotein precursor
• C) occurs with the aid of chaperones eg groEL,
  hsp70 (cellular proteins)

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Capsid assembly - T4 phage

• Head, tail and tail fibers form separately
• Then assembled together
• Many genes required - identified by in vitro
  complementation

From Flint et al Principles of Virology ASM Press
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Capsid assembly - adenovirus

• Nuclear assembly

• from individual proteins -
• Not a polyprotein (polio model)
• L1 protein is crucial - scaffolding function ?
• L4 protein is a viral-encoded chaperone for hexon
  trimer formation

From Flint et al Principles of Virology ASM Press
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Capsid assembly - poliovirus

• Cytoplasmic assembly
• from a polyprotein precursor. i.e. need for
  protease

Note the requirement for cellular membranes
From Flint et al Principles of Virology ASM Press
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Packaging signals

• RNA signals
• Well understood - e.g. psi sequence in
  retroviruses
• Extensive secondary and tertiary structure in RNA
• Structure (not sequence) recognized
• DNA signals
• Not well understood - T4 phage best understood
• Short repeats, some are also part of
  promoters/enhancers
• Located near ori sequences
• T4 phage - replicated DNA concatamers cleaved and
  packaged by terminase complex

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DNA packaging models - phage
Rotating nut Rotating winch
Grappling arms
Figures courtesy of Carol Duffy
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DNA packaging - herpesvirus

• Herpesvirus replicates by rolling circle
  mechanism -gt concatameric DNA
• Packaging sequence recognized
• DNA is reeled in until a headful is reached
• DNA is cleaved at a specific site in DR1

From Flint et al Principles of Virology ASM Press
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Membrane compartments of the cell
From Flint et al Principles of Virology ASM
Press
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Herpesvirus and nuclear exit/assembly

• Capsid assembly occurs in the nucleus
• The capsid is 100nm in diameter, and cannot by
  deformed or dismantled to fit through the 26 nm
  nuclear pore
• This is a big problem
• The virus buds through the inner nuclear envelope
  (INM)

Then what? - the virus is trapped in the space
between the inner and outer nuclear membrane

• 2 models (see Flint) 1) constitutive
  secretion or 2) most likely - the virus fuses
  with the outer nuclear envelope to release the
  capsid into the cytoplasm for further envelopment

From Flint et al Principles of Virology ASM Press
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Release and (final) envelopment of herpesvirus

• The de-enveloped capsid is now present in the
  cytoplasm
• The final envelope is acquired from Golgi
  membranes
• or late endosomal membranes ??? (see HIV)
• However there is no real Golgi retention motif,
  and glycoproteins are also expressed on the cell
  surface at high level(compare with
  corona/bunyaviruses)
• Release is via exocytosis (the constutitive
  secretory pathway)

Functional evidence for this model gH with
INM/ER localization motif (KKSL) fails to be
incorporated into virions
From Flint et al Principles of Virology ASM Press
Initial envelope Final envelope
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Role of membrane compartments in poliovirus
replication

• Remember, polio is a non-enveloped virus
• The virus sets up a virus factory in the
  cytoplasm - to concentrate its replication
  machinery
• The factory is composed of smooth endoplasmic
  reticulum (ER)

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• To form the factory, poliovirus encodes the 2A
  and 3B proteins, which block ER-Golgi transport

The drug brefeldin A (which blocks ER-Golgi
transport) prevents poliovirus replication
From Flint et al Principles of Virology ASM Press
Note in polarized cells, poliovirus is released
exclusively from the apical surface - not simply
virus bursting out of the cell non-specifically
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Targeting of virus glycoproteins

• A key process for many enveloped viruses
• Glycoproteins must be correctly processed in the
  ER and Golgi complex glycosylation
• Brefeldin A will invariably block infection of
  enveloped viruses, but will not affect genome
  replication (compare with poliovirus)
• In some case, the viral glycoprotein is targeted
  to a specific cellular membrane, eg ER, Golgi,
  Plasma membrane
• retention motifs for ER/Golgi (short amino acid
  sequences)

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From Flint et al Principles of Virology ASM Press
To a large extent, glycoprotein targeting
determines the site of viral envelopment
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Modification of viral glycoproteins

• Glycosylation
• Oligomerization
• Proteolytic processing
• Cleavage of influenza HA0 into HA1 and HA2
• Allows subsequent exposure of fusion peptide
  during pH dependent entry (fusion)
• Carried out by host cell proteases
• Can be a major determinant of influenza virus
  pathogenesis (see later)
• Many other examples (eg HIV gp160 gt gp120/41

From Flint et al Principles of Virology ASM Press
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Specialized cells

• Polarized cells
• Epithelia
• Neurons

• Influenza buds from apical surface
• VSV buds from basolateral surface

Direction relies on targeting signals on
glycoproteins (and perhaps matrix protein)
From Flint et al Principles of Virology ASM Press
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Virus exit and the cytoskeleton
For virus exit, the virus has to move in the
cytoplasm Herpesviruses can move bi-directionally
on microtubules i.e. via kinesin, as well as
dynein
Movie of psudorabies virus capsids moving
anterograde (cell body is to the left)
Supplementary material for Smith et al . (2001)
Proc. Natl. Acad. Sci. USA 98 (6), 34663470.
From Horton and Ehlers J. Neurosci.,  July
16, 2003, 236188-6199
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Localization of matrix proteins

• Matrix proteins are not merely passive glue
• They play a key role in assembly and budding and
  bind
• A) the nucleocapsid
• B) the envelope (either by direct binding to the
  plasma membrane or via the cytoplasmic tails of
  the viral glycoproteins)

From Flint et al Principles of Virology ASM Press
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Virus maturation needed for exit-based on
proteolytic cleavage

• Three examples
• 1 Poliovirus
• - liberation of VP2 and VP4 from VP0
• -note the poliovirus protease is virus -encoded,
  but non-structural.
• influenza virus
• cleavage of HA0 to HA1 and HA2
• by a cellular protease
• HIV
• processing of Gag
• protease is virus -encoded, and structural.

All needed needed for subsequent virus
entry/uncoating
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Maturation of HIV

• The Gag polyprotein is converted into individual
  polypeptides, which converts the virus particles
  into mature virions
• Uses a viral protease (PR) - the target of HIV
  antiviral drugs
• MA trimerizes, CA dimerizes
• Results in an obvious morpholigical rearrangement
  of the capsid (by electron microscopy) - and
  gives the characteristic cone-shaped structure of
  the HIV core

From Flint et al Principles of Virology ASM Press
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Budding of HIV - I

• Many retroviruses encode late domains as part
  of Gag - e.g. PTAP in p6 domain of HIV-1 Gag

From Freed EO. Trends Microbiol. 2003
Feb11(2)56-9.
The late domain mimics the function of a cellular
protein (Hrs) involved in endosome maturation
From Katzmann et al Nature Reviews Molecular Cell
Biology 3, 893-905 (2002)  
Similar late domains also exist in VSV, Ebola etc
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Budding of HIV - II

• Budding may be at the plasma membrane, or into
  the endosome
• The end result is that instead of forming late
  endosomes (multi-vesicular bodies) the machinery
  is used by the virus in budding
• Topologically backwards budding

HIV has distinct tropism for Mø and T cells. DCs
are immune system cells that internalize virus,
but do not become infected
From Amara and Littman J Cell Biol. 2003 Aug
4162(3)371-5.
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Budding of HIV - II

• The network of protein interactions is getting
  very complex

PTAP YXXø
From von Schwedler et al. Cell. 2003 Sep
19114(6)701-13.
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Virus budding- influenza
Envelope proteins - HA, NA, M2 Matrix protein -
M1 Genome - vRNPs Cellular membrane
RNPs
Cellular PM proteins are excluded Role of lipid
rafts or microdomains
M1
What causes the virus to pinch off ??? - no
late domain
From Flint et al Principles of Virology ASM Press
How does the virus know to package 8 segments ??
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Virus budding- influenza II

• Control of virus budding has been difficult to
  pin down, but seems to reside in specific regions
  of the matrix M1 protein and in the cytoplasmic
  tails of the HA and NA
• - also possibly M2

EM of deformed/-filamentous viruses with defects
in the HA/NA tails
From Flint et al Principles of Virology ASM Press
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Superinfection exclusion

• The inability of a virus to infect a cell that is
  already infected
• Often involves receptor down-regulation
• For influenza (receptor sialic acid) the viral
  neuraminidase (NA) strips the cell of sialic acid
  (neuraminic acid)
• Allows efficient release of virus particles
• The target of the new generation of
  anti-influenza drugs

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The unique (?) case of poxviruses

• Poxviruses (smallpox, vaccinia) have a complex
  maturation process that results in the formation
  of different viral forms (IEV, IMV and EEV)
  surrounded by different membranous envelopes

From Flint et al Principles of Virology ASM Press
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Cytoplasmic movement during maturation -
poxviruses

• The IMV is pushed through the cytoplasm by the
  formation of filamentous actin rocket tails
• The virus has a protein on its surface that
  promotes actin polymerization

Actin mediates cell-cell movement Microtubules
mediate intracellular movement MTs also for
herpesvirus in neurons
From Flint et al Principles of Virology ASM Press
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Reading

• Chapters 12 and 13 of Flint
• Chapter 8 of Fields Virology/Fundamental Virology