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Journal of Virology, April 2000, p. 3423-3426, Vol. 74, No. 7
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Uncoating Kinetics of Hepatitis A Virus Virions
and Provirions
Naomi E.
Bishop* and
David A.
Anderson
Hepatitis Research Unit, Macfarlane Burnet
Centre for Medical Research, Fairfield, Victoria, Australia 3078
Received 6 July 1999/Accepted 22 December 1999
 |
ABSTRACT |
When the growth kinetics of immature hepatitis A virus provirions
and mature virions were monitored, distinct eclipse phases were noted
for both types of particles. Strikingly, uncoating of virions occurred
around 4 h postinfection, while uncoating of provirions occurred
predominantly between 8 and 10 h postinfection. It is proposed
that the heterogenous mixture of infectious hepatitis A virus particles
(virions and provirions) typically present in inocula is responsible
for the normally asynchronous nature of hepatitis A virus uncoating kinetics.
| |
TEXT |
Human hepatitis A virus
(HAV), a member of the family Picornaviridae originally
classified in the Enterovirus genus, is now the type member
of the Hepatovirus genus (12, 31). The
reclassification of HAV was based on a number of differences between
HAV and other enteroviruses, including an extreme stability against
elevated temperature and low pH (25, 26) and a slow and
inefficient replicative cycle in cell culture (see references
27 and 31). The uncoating of HAV
has been demonstrated to be a slow and asynchronous process (3, 5,
9, 30). By contrast, the attachment to and penetration of cells
by HAV appear to be as efficient as for other picornaviruses (3,
8, 29, 32).
Intact, antigenic particles from HAV-infected cells or cell culture
supernatants have recently been demonstrated to be of three types: (i)
virions, containing capsid proteins VP1, VP2, VP3, and possibly VP4 and
viral RNA; (ii) provirions, or "immature" virions, containing VP1,
VP0, VP3, and viral RNA; and (iii) procapsids, which are empty HAV
particles with the same capsid composition as provirions but lacking
RNA (2, 7, 23). Provirions have been implicated as direct
precursors of mature picornavirus virions via the cleavage of VP0 (to
VP2 and VP4) in an RNA-dependent manner (6).
HAV provirions, while infectious, have a lower specific infectivity
than mature virions (6, 7). We proposed that the mixture of
particle types (i.e., of virions and provirions and of particles with
intermediate levels of VP0 and VP2) in viral inocula was responsible
for the asynchronous nature of the HAV uncoating process, on the basis
that provirions might uncoat more slowly than virions. Here we report
that using an HAV inoculum with a high virion content resulted in rapid
as well as synchronous uncoating.
Single-cycle growth kinetics of HAV and PV.
Typical
single-cycle growth kinetics of HAV and poliovirus (PV) are shown in
Fig. 1. An African green monkey cell
line, BS-C-1, was infected with HAV strain HM175A.2 (1, 9)
or PV type 1 (Mahoney), and the accumulation of infectious virus within
cells was determined after various incubation times at 37°C.
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FIG. 1.
Kinetics of HAV and PV replication. Monolayers of BS-C-1
cells were infected for 90 min at 4°C with HAV or PV before being
shifted to 37°C. Infectious virus was assayed throughout the
respective growth cycles by RIFA for HAV or plaque assay for PV.
Cytopathic effect (CPE) was graded on a scale of 0+ to 4+, where 0+
represents a healthy cell monolayer and 4+ is destruction of more than
60% of cells. Much of the PV yield would be in the supernatant from
12 h postinfection.
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In PV-infected cells, eclipse of the inoculated virus was evident at
2 h, reflecting rapid uncoating of the virus, followed
by
logarithmic accumulation of virus to 12 h. From 12 h,
infectious
virus accumulated slowly and much of the virus at this time
was
found in the supernatant. By contrast, eclipse of the HM175A.2
inoculum appeared to be incomplete, with much of the input virus
being
recovered up to 12 h postinfection. From 12 to 24 h, there
was logarithmic virus growth, with cells finally showing distinct
cytopathic effects at 48 h postinfection. Although the final yield
of HAV varied from experiment to experiment, it was typically
50- to
200-fold lower than that of
PV.
From this set of growth curves, it was clear that the growth of HAV was
unusual, as it was slow and lacked a well-defined
eclipse phase. This
phenomenon has also been reported elsewhere
(
3,
5,
10,
30).
However, the exact duration and kinetics
of the HAV uncoating period
vary in different studies, and this
is most likely due in part to the
use of different strains of
HAV and their source, the temperature of
growth, the type of culture
medium, and the cell type and passage
number used. In this regard,
exact kinetics cannot be expected to be
identical.
Kinetics of uncoating of light-sensitive HAV and PV.
To
confirm that the eclipse phase of HAV observed in growth curves was due
to asynchronous uncoating of the virus, we took advantage of the fact
that most picornaviruses can be rendered light sensitive by growing
them in the presence of neutral red, including PV (3,
18-20). When light-sensitive viruses are incubated with
susceptible cells, the viral RNA remains light sensitive only as long
as the RNA is contained inside the capsid (11, 15, 20).
Conversion from photosensitivity to resistance is generally thought to
be concomitant with virus uncoating and release of dye into the cytoplasm.
Light-sensitive HM175A.2 was prepared by growth in the presence of
0.003% (wt/vol) neutral red in the dark; PV was grown in
the same
manner but with 0.01% neutral red. Less neutral red had
to be used for
the growth of HAV due to the toxic effects of neutral
red in culture
medium over longer culture periods. PV and HAV
cultures were harvested
after 12 and 36 h at 37°C, respectively.
Virus was then purified
as for stock virus (
9), except that
all procedures were
carried out in the
dark.
Serial 10-fold dilutions of light-sensitive HM175A.2 and PV were used
to infect cultures of BS-C-1 cells by adsorption at
4°C in the dark.
Cells were then shifted to 37°C and the proportion
of virus which had
become photoresistant was determined after
exposure of cultures to
light at various intervals. The number
of infectious centers in each
culture was then compared to that
of cultures not exposed to light
(Fig.
2). HAV infectivity was
determined
using a radioimmunofocus assay (RIFA) (
3,
17),
and PV
infectivity was determined by plaque assay.
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FIG. 2.
Kinetics of HAV and PV uncoating. Cultures of BS-C-1
cells were infected with serial dilutions of light-sensitive virus at
4°C in the dark. Cells were shifted to 37°C for various times and
then exposed to light. After exposure, overlay medium was added to
allow the number of particles uncoated during the incubation period to
be estimated by RIFA for HAV or plaque assay for PV. RIFU,
radioimmunofocus-forming units.
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|
While uncoating of PV had occurred within 1 h (Fig.
2B), uncoating
of HAV was not complete until 18 h postinfection (Fig.
2A).
Approximately 25% of HAV particles had uncoated by 6 h
postinfection,
and 50% had uncoated by 12 h postinfection. It
thus appears that
not all input HAV particles are uncoated at the same
time. Together
(Fig.
1 and
2A), these experiments suggest that slow and
asynchronous
release of HAV RNA
occurred.
Infectivity of HAV virions and provirions.
HAV inocula,
whether derived from infected cells or supernatants, contain a mixture
of mature virions and immature provirions (6). HAV
RNA-containing particles with a higher proportion of capsid protein VP2
than VP0 have a greater specific activity than those containing a
higher proportion of VP0 than VP2 (7). Furthermore, when HAV
provirions are converted to virions by autocatalytic cleavage of VP0 to
VP2 at 37°C, infectivity is likewise increased (6). These
experiments indicated that HAV provirions are precursors of mature
virions. Moreover, when HAV provirions (around 130S) are converted to
virions in vitro, they then resediment in linear sucrose density
gradients with the same sedimentation coefficient as mature virions
produced in vivo (160S [data not shown]) (2, 23).
Therefore, VP2-containing particles synthesized from VP0-containing particles in vitro have all the characteristics of HAV virions.
To directly study the relationship between the infectivity of HAV
particles and VP0 content, five separate preparations of
HAV provirions
containing between 50 and 89% VP0 were incubated
at 37°C for
autocatalytic cleavage to occur. Cleavage of VP0 to
VP2 was determined
by Western immunoblotting (
6,
7), and
the VP0 proportion of
the VP0-plus-VP2 sum was expressed as a
percentage. The resulting
decrease in VP0 content and the corresponding
increase
(
n-fold) in infectivity were then calculated (Table
1).
The increase in infectivity of the
cleaved aliquots was converted
to a percent increase in infectivity and
plotted against the change
in VP0 (and thus VP2) content of the HAV
particle. The calculation
used should thus negate the variability in
specific infectivity
that resulted from the use of multiple
preparations, which was
necessary because half of each preparation was
required to give
a reliably quantitatable signal in immunoblotting for
the cleaved
and uncleaved samples. Interestingly, VP0 cleavage led to
an exponential
increase in specific infectivity, with infectivity
increasing
approximately twofold for each 25% of VP0 cleaved (Fig.
3). The
exponential increase in the
specific infectivity of particles
in relation to VP2 content suggests
that multiple sites of cleavage
are required to form infectious
particles.
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FIG. 3.
Relationship between VP0 self-cleavage and specific
infectivity. In five separate experiments, provirions were purified
from infected cells and duplicate aliquots were placed at  70 or
37°C for various times. The percent conversion of VP0 to VP2 was
determined, and the corresponding increase in infectivity was
determined (Table 1). The percent decrease in VP0 content was then
calculated and plotted against the increase in infectivity. It should
be noted that the percent decrease in VP0 content of these preparations
correlated absolutely with the increase in VP2 content in all cases.
The lighter line indicates an exponential curve fit.
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These data provide further evidence that the provirion is a precursor
of the mature virion in picornavirus morphogenesis.
Cleavage of VP0, as
the final step in morphogenesis, is likely
to be necessary for
uncoating of the virus particle in endosomes
(
13,
14,
22,
24). In comparison to mature PV virions,
which retain one to
three copies of VP0, PV particles in which
VP0 is not myristylated fail
to cleave VP0 and are noninfectious
(
21), and human
rhinovirus particles with a high VP0 content
have been shown to be
deficient in delivery of RNA to the cell
interior after attachment
(
16). Recent studies of PV empty capsids
have further
defined a role for VP0 cleavage in allowing the extrusion
of VP4 during
structural rearrangements subsequent to binding
(
4). If HAV
uses a similar uncoating mechanism, uncoating may
not occur until VP0
cleavage within particles is complete. It
will be difficult to
determine whether there is a threshold level
of VP2 which must be
reached before viral uncoating can occur.
Particles that fail to cleave
sufficient numbers of VP0 within
endosomes may subsequently be degraded
in lysosomes or perhaps
expelled at the cell
surface.
Effect of VP0 processing on HAV replication in single-cycle growth
curves.
To examine the biological differences between HAV virions
and provirions, single-cycle growth kinetics of HAV were examined following infection of cells with inocula with a known VP0/VP2 content.
Replication of the same pools of HAV was studied with or without prior
autocatalytic cleavage of VP0 at 37°C, resulting in inocula with
different levels of provirions and virions.
Examination of the provirion growth curve in Fig.
4A shows the lack of a distinct eclipse
phase, where an HAV pool containing
approximately equal proportions of
VP0 and VP2 was used as the
inoculum. By contrast, a much clearer
eclipse phase was seen in
cells infected with an inoculum containing
predominantly virions
(formed by self-cleavage of the original pool),
indicating that
the uncoating of HAV virions occurred in a more
synchronized manner
(Fig.
4A; virions), as is typical of other
picornaviruses.
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FIG. 4.
Single-cycle growth curves of HAV virions and
provirions. In two separate experiments, pools of provirions were used
to infect BS-C-1 cells with or without prior self-cleavage of VP0 at
37°C. (A) VP0 content of particles was approximately 52% before
cleavage and 27% after self-cleavage at 37°C for 3 days. (B) VP0
content of particles was 65% before cleavage and 22% after
self-cleavage at 37°C for 4 days. Virions in panel A were diluted 1:5
prior to infection, providing an approximately equal input titer. RIFU,
radioimmunofocus-forming units.
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When single-cycle growth kinetics of HAV provirions (65% VP0 content
in the viral pool) were compared to those of HAV virions
derived from
the same pool after 4 days of self-cleavage at 37°C
(now containing
only 22% VP0), a distinct eclipse phase was detected
in growth cycles
of both HAV provirions and HAV virions (Fig.
4B), although slightly
less clearly defined for the provirion
pool.
The observed differences in growth kinetics are unlikely to reflect the
different multiplicities of infection obtained with
provirion and
virion pools, on the basis that the virion pool
is intrinsically more
infectious. In the experiment whose results
are shown in Fig.
4A, the
virion pool had been diluted fivefold
relative to the provirion pool to
obtain a similar input multiplicity
of infection, and a clear
difference between growth curves was
seen. In the experiment whose
results are shown in Fig.
4B, the
relative multiplicities of infection
were not corrected and yet
the residual infectivity in the virion pool
was seen to vary 16-fold
relative to that in the provirion pool over
the first 4 h
postinfection.
These results indicate that the uncoating of HAV provirions is delayed
relative to that of virions, leading to either (i)
asynchronous
uncoating with a lack of a clearly defined eclipse
when the inoculum
contains both species of particles (Fig.
4A)
or (ii) synchronous but
delayed uncoating when the inoculum contains
a very high proportion of
provirions (Fig.
4B). The protracted
time within the cell, during which
provirions are presumably converted
to virions prior to uncoating, is
likely to result in the lower
specific infectivity of these
particles.
The intriguing question is why HAV produces high numbers of provirions,
which are slow to uncoat, in contrast to other picornaviruses.
We
propose this is related to the target of infection in vivo,
the liver.
Hepatocytes are unusual because they lack a direct
excretory mechanism
to mediate viral exocytosis from the same
membrane surface
(
28). Therefore, in order for the host to excrete
viral
particles, HAV must be transported by transcytosis back
to the apical
surface, where the particles can enter the bile
canaliculi and begin
the pathway for excretion from the host.
If virus particles were
capable of fusing with transcytotic vesicles
during cross-cellular
transport, they would not reach the apical
membrane and would not be
available for excretion and subsequent
dissemination.
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ACKNOWLEDGMENTS |
N.E.B. was supported by a Commonwealth Postgraduate Research Award,
and D.A.A. was supported by the BHP Community Trust. This work was
supported in part by the Macfarlane Burnet Centre for Medical Research
and the World Health Organization Programme for Vaccine Development.
We thank J. Mills for critical reading of the manuscript, S. Locarnini
and S. Borovec for helpful discussions, and P. Edwards and D. Hugo for
excellent technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Present address: School of
Biological Sciences, Division of Biochemistry, 2.205 Stopford Building,
Oxford Rd., Manchester M13 9PT, United Kingdom. Phone: 44 161 275 5955. Fax: 44 161 275 5082. E-mail:
nebishop{at}fs2.scg.man.ac.uk.
| |
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Journal of Virology, April 2000, p. 3423-3426, Vol. 74, No. 7
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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