COMMENTARY
The Two Envelopes Problem: Recent
Developments in Identifying the Second
Membrane in Herpesvirus Envelopment
Ben Bleasdale, Imperial College, London
Introduction
Virion assembly represents an elaborate
molecular
jigsaw, coordinating
numerous
individual components to construct an infectious
agent. For enveloped viruses, such as HIV,
influenza and Herpes Simplex Virus-1 (HSV1), a
vital piece of this puzzle is the acquisition of a
membrane. Unlike viral proteins, this is not
produced de novo, but derived directly from
existing host membranes. The host’s secretory
system, an extensive network of internal
membranes, is exploited to provide this material.
For HSV1, consensus supports a two-stage
envelopment - with an initial membrane, obtained
during nuclear egress, subsequently discarded.
Current proposals implicate the Trans-Golgi
Network as the secondary envelopment site,
however substantial contention remains.
molecular characteristics permitting identification
via immunofluorescence staining and dyes.
The complex shuttling of luminal cargoes, such as
proteins, has been the focus of decades of research
[2] and is regulated by a family of proteins termed
Rab GTPases [3] coupled with molecular motors
[4]. As a major governor of protein localisation,
the operation of the system and these supporting
proteins remains a focal point.
The Plasma Membrane (PM) forms the parental
membrane in this system, linking the pathway to
the external space. Topologically, the luminal
space of all internal compartments is a noncontinuous extension of extracellular space.
Hence, material entering the lumen at any point
along the pathway can be trafficked to the
external space, via vesicle fusion, without crossing
additional membranes.
Recently, Hollinshead et al [1] have refreshed this
debate, proposing an alternative envelopment
pathway involving endocytic vesicles. The authors
ally complementary experimental approaches,
offering explanations for previously conflicting
results. This latest development brings anti-viral
intervention during HSV1 assembly a step closer
to the clinic.
GLYCOPROTEINS
CAPSID
VIRAL DNA
TEGUMENT
VIRAL ENVELOPE
Sites of Envelopment
The cellular secretory pathway is a dynamic
network of compartments fundamental to protein
processing and localisation. The constituents of
the pathway include the Endoplasmic Reticulum
(ER), Golgi apparatus, Trans-Golgi Network (TGN),
endosomes and a vast family of transitional
vesicles which mediate inter-compartmental
trafficking. Each compartment possesses distinct
~200nm
Fig 1. Anatomy of HSV1 virion.
The virion architecture contains three distinct layers. At
the core, the viral DNA is contained within an icosahedral
capsid which is subsequently coated in a heterogeneous
layer of protein, termed tegument. This is enveloped by
the viral membrane, containing the glycoproteins which
mediate cell-binding and entry.
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COMMENTARY
For cellular cargoes, the route through the system
is dictated by the site of synthesis and the ultimate
destination [5]. Newly-synthesised viral particles
face the same biological problem, with their site of
synthesis and molecular architecture governing
their interaction with the pathway.
Topological Hurdles
For enveloped viruses, including HIV, influenza
and HSV1, the process of egress is dictated by the
requirement for each virion to exit with a
membrane. As viral replication strategies differ
considerably, a range of novel solutions to this
shared problem have evolved. HIV and influenza
acquire a membrane by budding through the PM
[6, 7] whilst hepatitis B and C viruses are
enveloped at internal pre-Golgi membranes [8, 9].
For more complex viruses, such as HSV1, this
process of envelopment has proven to be
convoluted with multiple, sequential wrapping
events [10]. Considerable debate still exists over
the membranes involved in these events.
HSV1 – A Complex Case
The envelopment of HSV1 is dictated by the
architecture of the final virion. The multi-layered
molecular assembly is composed of three distinct
compartments: a DNA-containing capsid, an outer
envelope studded with glycoproteins and an
intervening proteinaceous layer termed the
tegument [11]. This structure (Fig. 1) is assembled
successively around the central DNA cargo
replicated in the nucleus [12] during its
processive egress from the host nucleoplasm
towards the extracellular space.
Conflicting observations over several decades
have resulted in a variety of proposed models for
this process [13, 14, 15]. However, the emerging
consensus favours a two-stage process – termed
the envelopment-deenvelopment-reenvelopment
model [10]. In this, capsids acquire and
subsequently lose a primary envelope during
nuclear egress, leaving capsids exposed in the
cytoplasm. A final envelope is then acquired from
an internal membrane, before egression proceeds
via the cellular secretory system (Fig. 2).
The identity of this second membrane has been a
source of great contention [16] and current
theories favour capsid association with the TGN
[17] – an organelle know to have the capacity to
extend tubules and produce vesicular buds [18].
Previously, light microscopy has shown GFPtagged capsids co-localising with membranes
labelling as TGN [19], leading to the suggestion
that wrapping events were being observed. The
TGN has also been shown to co-localise at light
microscopy resolution with viral glycoproteins,
further supporting this organelle as the site of
secondary wrapping [20].
However, a recent publication by Hollinshead et al
has proposed an alternative source of wrapping
membranes – endocytic tubules drawn directly
from the PM. Their work, employing
complementary techniques, avoids inherent
limitations in previous approaches and leads to an
alternative proposal to TGN-envelopment.
Looking Beyond the TGN
The authors propose that enveloping membrane is
supplied from the PM, via cellular endocytic
processes. The creation of large numbers of
endocytic tubules was observed by combining
Electron Microscopy (EM) studies with the use of
the
soluble-phase
marker
Horse-Radish
Peroxidase (HRP). This exclusively labelled
compartments recently derived from the external
space, termed endocytic tubules, which were
observed throughout infected cells, clustering
around the Microtubular Organising Centre
(MTOC) along with capsids in various stages of
envelopment. The molecular resolution of EM, and
novel use of HRP-labelling, led to the conclusion
that envelopment material was from an
externally-exposed source, rather than an existing
internal membrane population such as the TGN.
These newly-enveloped virions are then
proposed to traffic back to the PM via normal
endosome recycling pathways, shepherded by
cellular Rab GTPases (Fig. 2). Supporting this are
targeted Rab depletion studies showing that, in
the absence of specific Rab proteins, virion
output from cells drops dramatically and capsids
accumulate cytoplasmically.
2
COMMENTARY
Addressing previous observations of TGN and
capsid co-localisation, the authors draw
attention to the resolution limits of light
microscopy, compared to EM studies. They
highlight the densely-populated MTOC region –
where TGN, Golgi and large amounts of
endosomal material amass – as an environment
where co-localisation of TGN markers and
capsids could be misleadingly apparent when
using insufficient resolving power. Contrastingly,
the authors’ use of higher resolution confocal
microscopy, coupled with molecular resolution
EM, support no co-localisation of these markers.
Further Avenues
This novel proposal has reopened investigations
into the identity of the second membrane. It
highlights the strengths of combining
complementary techniques, an approach that
offers great potential for future work. In the
immediate future, extending this approach with
existing investigative assets, such as labelled
tegument and glycoprotein systems, is likely to
yield further results. This progression is vital, as
synthesising a complete model for HSV1
assembly will require a united understanding of
all three viral components – capsid, tegument
and glycoprotein-studded membrane.
The use of supporting data from cell biology
studies, in this case Rab proteins, highlights the
benefits of expanding from traditional imaging
approaches to inter-disciplinary resources. The
Fig 2. Two hypothesised envelopment routes for HSV1 capsids.
Label 1 shows DNA-containing capsids produced in the nucleoplasm, these bud through the
Inner Nuclear Membrane acquiring a primary envelop. This envelop is subsequently lost
through fusion with the Outer Nuclear Membrane (Label 2) leaving the capsid unenveloped
in the cytoplasm. Two proposals are then represented. In the TGN Pathway, the capsid and
tegument coalesce with the TGN (Label 3a), acquiring a new envelope by budding into the
lumen. The wrapped virion is then passed via the lumen of the secretory pathway (Label
4a) until fusion of the vesicle with the PM releases the virion (Label 5a). Alternatively, in
the Endocytic Tubule Pathway, membrane is drawn into the cell from the PM (Label 3a),
these tubules then coalesce and wrap the capsids (Label 4a), wrapped virions are then
trafficked in endocytic vesicles to the surface and release by fusion (Label 5a).
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COMMENTARY
importance of Rab proteins in other viral
lifecycles has already been documented [21, 22,
23], suggesting significant potential for the
continuation of such studies with HSV1.
Examining these cellular proteins under the
pressure of viral assembly may also prove
fruitful in uncovering fundamental principles
relating to their normal operation.
This proposal also implicates cellular endocytic
processes, including the clathrin-dependant
endocytotic mechanism, as a controller of viral
egress warranting further attention. Clathrin in
particular has already been implicated in viral
morphogenesis elsewhere [24] and the role of
subverted host endocytic systems in HSV1
assembly poses many new questions.
The study of such cellular accomplices carries
excellent potential for medically translatable
results. The ability to target a cellular
component, rather than viral factors, with antiviral intervention can reduce the risk of
mutational escape. Anti-viral drugs targeting
envelopment
are
established,
including
BrefeldinA against polio virus [24], but the
limitations of these [25] highlight the continued
need for a deeper understanding of the interplay
of host and viral systems. Recent approaches for
other viruses have proved revealing, with
promising leads [26, 27] and HSV1 may well
benefit from such an approach.
Finally, expanding the reach of interdisciplinary
collaboration promises substantial rewards.
Considering the role of topological changes in
membrane distribution and availability in the
proposed model, employing a mathematical
approach may be revealing. Studies have
established the ability to examine topological
characteristics in living cells [28] and extending
such work to infected cells may yield useful,
quantitative data on the subversion of the
secretory system.
Concluding Remarks
Hollinshead et al have successfully emphasised
the importance of combining different
approaches to avoid the limitations of individual
techniques. The complexity of the HSV1
wrapping process, co-opting cellular systems,
means that continuing to embrace new sources
of data is likely to be a key feature of future
work.
The intertwined nature of viral assembly and
the secretory system offers a window of
opportunity to discover fundamentals within
both processes. The innovative matching of cell
biology approaches with classical virology
techniques has proven revealing so far, and the
importance of cellular partners such as Rabs and
clathrin is likely to be an expanding field of
research.
The cell represents a clamorous environment
from which to isolate and study a single process,
such as viral wrapping. As such, interdisciplinary
approaches uniting complementary data offer
the best chance piece together the jigsaw, having
already successfully reopened the debate on the
HSV1 envelopment problem.
Key Words: HSV1, assembly, envelopment,
endocytic tubules
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