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Herpesviruses use bidirectional fast-axonal transport to spread in sensory neurons Herpesviruses use bidirectional fast-axonal transport
to spread in sensory neurons
Gregory A. Smith* , Steven P. Gross , and Lynn W. Enquist*
§
*Department of Molecular Biology, Schultz Building, Room 301, Princeton University, Princeton, NJ 08544-1014; and Department of Developmental and
Cell Biology, 2113 BioSci II, University of California, Irvine, CA 92697
Communicated by Thomas E. Shenk, Princeton University, Princeton, NJ, January 18, 2001 (received for review November 17, 2000)
Alpha herpesviruses infect the vertebrate nervous system resulting
in either mild recurrent lesions in mucosal epithelia or fatal en-
cephalitis. Movement of virions within the nervous system is a
critical factor in the outcome of infection; however, the dynamics
of individual virion transport have never been assessed. Here we
visualized and tracked individual viral capsids as they moved in
axons away from infected neuronal cell bodies in culture. The
observed movement was compatible with fast axonal ow medi-
ated by multiple microtubule motors. Capsids accumulated at axon
terminals, suggesting that spread from infected neurons required
cell contact.
M
any viruses spread by infecting nerve terminals and trav-
eling from neuron to neuron in the vertebrate nervous
system. The diseases resulting from these infections are usually
debilitating and often fatal (1). Neurotropic herpesviruses (al-
pha herpesviruses) are an exception in that typical infections are
confined to the peripheral nervous system (PNS) and are largely
asymptomatic. Although the herpesvirus genome remains in the
PNS for the life of the host, and newly reactivated virus made in
neurons can retrace the path of primary infection and return to
the surface to be shed, alpha herpesviruses rarely spread from
the PNS into the central nervous system. In instances when this
does occur, the resulting encephalitis has severe consequences
(2). The mechanism by which alpha herpesvirus restrict their
spread to the PNS is not known. In fact, because viruses are too
small to visualize in living cells, the dynamics of viral transport
has never been directly assessed for any neurotropic virus. Here
we show that newly replicated individual herpesvirus capsids
(125 nm diameter) bearing multiple copies of the green fluo-
rescent protein (GFP) can be visualized and tracked in infected
neurons by laser-scanning confocal time-lapse microscopy. We
demonstrate that viral transport in axons is highly processive
(continues over large distances without stopping) and bidirec-
tional. Capsids travel long distances to ultimately accumulate at
axon terminals, suggesting that virions do not bud out of axon
terminals by a cell autonomous mechanism.
Materials and Methods
GFP-Capsid Virus [Pseudorabies Virus (PRV)-GS443] Construction.
The
PRV-Becker UL35 gene was cloned as a 4.5 kb SalI fragment
(3), along with the upstream UL34 gene, and both strands
containing the two open-reading frames were sequenced
(GenBank accession no. AF301599). The gfp open-reading
frame was inserted into the PRV UL35 gene between codons
two and three, following a strategy previously used with herpes
simplex virus (HSV) type 1 (4). A recombinant virus, PRV-
GS443, carrying the fusion allele in place of the wild-type
UL35 gene, was made by using the pBecker3 infectious
Escherichia coli clone (5).
Neuron Culture and Infection.
Dissociated sensory neurons from
the dorsal root ganglia (DRG) of embryonic day 810 chick
embryos were seeded on 22-mm square glass coverslips pre-
treated with polyornithine at
100 neurons coverslip. The
neurons were cultured for 35 days to allow for axon outgrowth
before infection with the GFP-capsid virus (6). Peripheral
sensory neurons do not have dendrites in situ (7), and only axons
extend from DRG neurons in culture (8, 9). Axons were
identified as long projections that did not taper. Because the
number of neurons on a single glass coverslip was kept low to
Abbreviations: PRV, pseudorabies virus; PNS, peripheral nervous system; DRG, dorsal root
ganglion; GFP, green uorescent protein; h.p.i., hours postinfection; HSV, herpes simplex
virus.
Data deposition: The sequence reported in this paper has been deposited in the GenBank
database (accession no. AF301599). G.A.S. and S.P.G. contributed equally to this work.
§
To whom reprint requests should be addressed. E-mail: lenquist@molbio.princeton.edu.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked advertisement in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
Fig. 1.
Time-lapse recording of an axon of an infected DRG sensory neuron.
A laser scan of the entire eld was completed every 0.184 s (i.e.,
5.4 frames s)
by using an argon laser with a 488-nm excitation, beginning at 13 h.p.i. Every
fteenth frame over a 15.121-s interval is shown. Several GFP punctae are
visible (numbered 1 8), two of which were moving (numbers 1 and 3). The
neuronal cell body was immediately outside the eld of view to the right;
therefore, the moving punctae were undergoing anterograde transport. For
a complete time-lapse recording, see Movie 1 in the supplemental data
(www.pnas.org). (Bar
5 m.)
3466 3470
PNAS
March 13, 2001
vol. 98
no. 6
www.pnas.org cgi doi 10.1073 pnas.061029798 prevent axons from contacting each other, much of the coverslip
was exposed to the viral inoculum. We found that the majority
of the input virions bound directly to the coverslip, as assessed
by GFP emissions, and never came into contact with the cultured
neurons. To infect all neurons on a single coverslip, each sample
was incubated with
1
10
5
plaque-forming units of GFP-
capsid virus for 1 h (incubating with fewer plaque-forming units
resulted in less than 100% of neurons becoming infected; data
not shown). Unbound virus was then washed away before
incubation was allowed to continue.
Confocal Microscopy.
For time-lapse recording of living cells,
individual coverslips of infected neurons were sealed onto a
glass slide in Hepes-buffered media (pH 7.4) by using a 1:1:1
mixture of Vaseline, beeswax, and lanolin. GFP emissions from
infected neurons were then imaged at 37°C with a Zeiss 510
laser-scanning confocal microscope fitted with a heated stage
and a heated 63
1.4 n.a. oil objective. Excitation was at 488
nm with an argon laser, and up to 1,000 frames were captured
per recording. For immunofluorescence, coverslips of infected
neurons were fixed, permeabilized, and reacted with a mouse
monoclonal anti-gB (M2) antibody (10). The secondary
antibody was a goat anti-mouse conjugated to Alexa 546
(Molecular Probes). GFP capsids were excited with a 488-nm
argon laser, and Alexa 546 was excited with a 543-nm HeNe
laser.
Quantitation of GFP Fluorescence.
Individual GFP punctae ob-
served in samples of isolated capsids were measured for total
emission intensity by summing the values of all its pixels after
first correcting pixel values by subtracting background emission.
The GFP emissions of punctae seen in the axons of living
neurons were measured in the same way as above, except that the
pixel values were corrected by subtracting the emission back-
ground of the axon, which developed notable background flu-
orescence during infection.
Fig. 2.
Comparison of GFP-emission intensities of punctae in axons and isolated capsids. A prole of 200 GFP-emission intensities from punctae observed by
confocal microscopy (Bottom Inset) are plotted as a histogram. On the basis of electron microscopy observations (Top Inset; capsids appear as hexagons), the
emission proles corresponding to individual intact GFP capsids (open bars) were estimated by assigning the lowest and highest emission values to fragmented
capsids and capsid clusters respectfully (hatched bars). Two minor peaks in the upper hatched region may reect clusters of two and three capsids (asterisks).
The emissions of the axon punctae from Fig. 1 are overlaid on the histogram with numbers corresponding to those from Fig. 1. Top Inset width
2.0 m; Bottom
Inset width
7.5 m.
Smith et al.
PNAS
March 13, 2001
vol. 98
no. 6
3467
MICROBIOLOGY Results
A mature herpes virion comprises a DNA genome surrounded
by an icosahedral capsid shell made up of four virally encoded
proteins. An assortment of additional viral proteins, collectively
called the tegument, surround the capsid, which in turn is
enclosed by a lipid bilayer. Herpesvirus assembly and transport
out of infected cells are coupled, and only the capsid proteins are
known to be continually associated with the viral genome during
intracellular transport. By fusing GFP to a capsid protein, GFP
signal remains associated with the DNA genome of the virus
during assembly and exit from infected cells.
We first cloned and sequenced the UL35 gene from PRV
(GenBank accession no. AF301599). This gene is homologous to
the HSV UL35 gene that encodes the VP26 capsid protein.
Fusion of GFP to the N terminus of HSV and PRV VP26 does
not inhibit VP26 assembly into capsids or substantially affect
viral growth and spread in culture [(4); data not shown].
Additionally, because there are 900 copies of VP26 per capsid,
individual GFP capsids produced sufficient fluorescence emis-
sions to be imaged with the short exposure times necessary to
perform rapid time-lapse microscopy.
Neurons were seeded at low density to prevent axons from
fasicul