FEMS Microbiology Letters 100 (1992) 455-460
© 1992 Federation of European Microbiological Societies 0378-1097/92/$05.00
Published by Elsevier
455
FEMSLE 80057
Why do human hepatitis viruses replicate so poorly
in cell cultures?
S t a n l e y M. L e m o n , L i n d a W h e t t e r , Ki H a C h a n g and E d w i n A. B r o w n
Department of Medicine, The Uniuersity of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
Received 13 July 1992
Accepted 21 July 1992
Key words: Viral hepatitis; Hepatitis A virus; Translation; Picornavirus; Internal ribosomal entry site;
Cell culture
1. S U M M A R Y
2. H Y P O T H E S I S
The five viruses which classically cause hepatitis in man represent diverse families of viruses
and share in common only a striking hepatotropism and substantial restrictions to replication in conventional cell cultures. Hepatitis A
virus is unique among these viruses in that it is
amenable to propagation in cell culture, but
replication of this virus is much slower and less
efficient than replication of other picornaviruses.
This probably reflects less efficient cap-independent viral translation, as well as restrictions at
other points in the replication cycle. We speculate that the significantly restricted replication of
hepatitis viruses in cell culture reflects evolutionary forces controlling their transmission and
propagation through human populations.
In humans, acute viral hepatitis classically occurs due to infection with any of five very distinct
viruses. Conveniently labelled 'hepatitis A'
through 'hepatitis E', these five viruses represent
five diverse families of viruses, some enveloped
and some not, most with RNA genomes but one
with a DNA genome which replicates through a
unique RNA intermediate. Two of these viruses,
hepatitis A virus (HAV) and hepatitis E virus
(HEV) cause only acute, self-limited infections,
while infections with hepatitis B virus (HBV),
hepatitis C virus (HCV) and hepatitis D virus
(HDV) frequently result in viral persistence, occasionally with devastating clinical consequences.
Only two themes seem to be common to all five
of these viruses. One is the striking hepatotropism which sets them apart from other viral
pathogens of man. The other is the fact that each
has been curiously resistant to efforts at propagation in cell cultures.
HAV may be the exception to this rule, because it is amenable to propagation in many types
Correspondence to: S.M. Lemon, Department of Medicine,
The University of North Carolina at Chapel Hill, Chapel Hill,
NC 27599-7030, USA.
456
of primate cell cultures [1,2]. However, one of the
most intriguing features of HAV is its remarkably
slow and nearly always non-cytopathic replication
in cell cultures. N o w classified within the genus
hepatovirus of the family Picornaviridae, HAV
shares many structural and biological attributes
with other picornaviruses such as poliovirus (PV)
(a human enterovirus) or encephalomyocarditis
virus (EMCV) (a murine cardiovirus) [3]. These
include at least a superficially similar, non-enveloped capsid structure, and a positive-sense
RNA genome of approximately 7.5 kb which con-
tains a single large open reading flame encoding
a polyprotein which is post-translationally processed by virally encoded protease(s) into both
structural and non-structural proteins required
for viral replication.
Although the host range of PV is more restricted than EMCV, both of these viruses replicate rapidly in cultured cells. Following attachment, penetration, and uncoating, there is rapid
shut down of host-cell macromolecular synthesis
and initiation of viral replication. Maximum titers
of replicated virus are generally present within 6
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Fig. 1. Secondary structure of the 5'NTR of HAV RNA which was proposed on the basis of phylogenetic comparisons,
thermodynamic predictions, and enzymatic probing with single- and double-strand specific nucleases. The two AUG codons at
which translation is initiated are underlined. Modified from [11].
457
h or less. In contrast, one-step growth studies
with HAV show that uncoating alone may take
up to 12 h, and that replication even of virus
which has been substantially adapted to growth in
cell cultures takes many hours more [4]. Maximum cell culture yields of HAV are not reached
until 70-140 h after infection. Unlike PV and
EMCV which generally cause rapid, cytolytic infection, infection of cell cultures with HAV typically results in viral persistence, with little or no
apparent impact on cellular growth or
metabolism.
What accounts for this marked difference in
these picornaviruses? And why are other hepatitis viruses, such as HBV and HCV, so resistant to
attempts at propagation in cell culture?
It has been suggested that the slow growth and
persistence of HAV in cell culture may reflect
inefficient uncoating of the virus [4], poor translation of viral proteins a n d / o r the absence of inhibition of cap-dependent translation of host-cell
mRNAs (as seen with poliovirus) [5], very inefficient processing of the polyprotein into specific
viral proteins [6], or possible sequestration of
plus-strand RNA in nascent capsids leading to
sharp reductions in the amount of template available for RNA replication [7]. To a greater or
lesser extent, there is evidence supporting each of
these hypotheses. The virus appears to do poorly
almost everything it must do to replicate and
increase in number.
Our laboratory has been particularly interested in the translation of viral proteins during
HAV infection. By analogy with other picornaviruses, the lengthy 5' non-translated region
(5'NTR) of HAV should play an important role
in initiating viral translation. The 5'NTR precedes the large open reading frame in the genome
organization, and is approximately 735 bases in
length. Similar 5'NTRs in other picornaviruses
are known to initiate translation by a 5' cap-independent process involving internal entry of the
40S ribosomal subunit at a site many hundreds of
bases downstream of the 5' terminus of the RNA
[8,9]. This function is mediated by a large, complex RNA structure several hundred nucleotides
in length which has been variously termed an
'internal ribosomal entry site' (IRES) or 'ribo-
somal landing pad'. The fact that translation of
the HAV polyprotein begins at the l l t h or 12th
AUG triplet from the 5' end of the genome
suggests that HAV also initiates translation by
internal entry, and that translation does not initiate by scanning from the 5' terminus as proposed
for capped cellular mRNAs by Kozak [10].
Work in our laboratory has shown that the
extensive secondary structure of the HAV 5'NTR
(Fig. 1) shares many features in common with
secondary structures proposed for the 5'NTRs of
cardioviruses (such as EMCV) and aphthoviruses
(another picornaviral genus, which includes the
virus of foot-and-mouth disease) [11]. These
structural features include a 5' terminal hairpin,
followed by a series of putative pseudoknots (two
in HAV and three in EMCV) and a lengthy
single-stranded domain. In HAV RNA, this single-stranded region is approximately 60 bases in
length and is comprised almost entirely of pyrimidines. In EMCV, this region is a somewhat longer,
pure polycytidine track. These structural elements are followed by a series of complex stemloops, some of which bear obvious structural similarities to stem-loops within the IRES of EMCV
[111.
The existence of an IRES within the 5'NTR of
HAV was suggested by studies examining the in
vitro translation of 5' terminally deleted HAV
RNAs [11]. This work indicated that RNA structures present in domain IV and V (Fig. 1) are
inhibitory to initiation of translation via a simple
scanning mechanism, and that in vitro translation
was initiated by two distinct mechanisms (scanning and internal entry). We have since formally
demonstrated the existence of an IRES, located
between residues 154 and 735, by characterizing
translation initiated in vitro by bicistronic constructs in which the HAV 5'NTR, placed between two reporter genes, controls translation of
the downstream reporter gene (E.A. Brown et al.,
unpublished results). However, the HAV IRES,
while functionally present and bearing at least
superficial secondary structuraI similarity to the
EMCV IRES appears to be many-fold less active
both in vitro and in vivo.
When rabbit reticuloyte lysates were programmed with RNA transcripts representing the
458
5 ' N T R s of HAV or EMCV fused to the bacterial
chloramphenicol acetyltransferase (CAT) gene,
comparable levels of CAT expression required
approximately 100-fold higher concentrations of
the HAV message (L. Whetter et al., unpublished
results). Similarly, others have noted that replacement of the HAV 5 ' N T R with the EMCV 5 ' N T R
resulted in a decrease in the number of abberant
translation initiation sites within the H A V
genome in an in vitro translation system [12].
More recently, we compared the H A V and
EMCV IRES elements in continuous monkey
kidney cells which are permissive for H A V and
which constituitively express the bacteriophage
T7 RNA polymerase. When cells such as these
are transfected with plasmid DNA containing the
T7 promoter, cytoplasmic T7 polymerase directs
abundant transcription of RNA. These RNA
transcripts are not capped, however, and their
translation is thus dependent upon the presence
of an IRES element [13]. When these cells were
transfected with plasmid D N A containing the
HAV or EMCV IRES fused to the CAT gene
within a T7 transcriptional unit, the EMCV-directed expression of CAT was 50-100-fold greater
than HAV-directed CAT expression (L. Whetter
et al., unpublished results). The relative efficiencies of the HAV and EMCV IRES elements
appear to mirror differences in the binding of one
or more cellular translation factors by these two
RNA translational control elements.
This is a puzzling result, as the advantage
which the virus might gain in maintaining such an
inactive IRES is not immediately clear. The conventional explanation would be that the cell type
in which the experiment was carried out differs in
some vital function (presumably a 5'NTR-binding
protein) from the hepatocyte in which the virus
replicates in vivo. Similar explanations are usually
put forth to explain difficulties in propagating
hepatitis viruses in cell cultures. However, perhaps the in vivo and in vitro situations are not so
dissimilar. Even in primary hepatocyte explants,
the replication of HAV and other hepatitis viruses
such as HBV and HCV is not particularly impressive [1,14]. An alternative hypothesis is that the
selective forces influencing the spread of viruses
such as HAV within human populations have
forced the virus to evolve toward such a poor
replicative posture.
The normal liver in adult humans weighs between 1400 and 1600 grams, and thus the total
mass of hepatocytes represents an immense
reservoir of cells which are permissive for the
hepatitis viruses. Infection of the liver with a
virus having the replication properties displayed
by PV or EMCV in cell cultures would probably
result in rapid, overwhelming hepatic failure and
death. However, non-enveloped hepatitis viruses
such as HAV and HEV depend upon their secretion into bile by a relatively normally functioning
liver in order to be shed in feces and transmitted
to other individuals. Transmission of the other,
enveloped hepatitis viruses appears to be even
less efficient and probably depends largely upon
the maintenance of a pool of chronically infected,
viremic carriers. In either case, acute destruction
of the liver by rapid, cytolytic virus replication
offers few opportunities for transmission of a
virus, and its subsequent propagation through the
human population. Thus we can postulate that
the inefficient translational activity of the HAV
IRES, and possibly other H A V replicative functions, may reflect the adaptation of this RNA
virus to a relatively unique epidemiologic niche.
Consistent with this hypothesis, the replication
of HAV in cultured cells has no apparent impact
on host-cell macromolecular synthesis. In contrast, replication of PV and EMCV both result in
efficient host-cell shut-off. In the case of PV, this
appears to be mediated through a viral protease,
2A, which indirectly promotes the cleavage of the
p220 component of the cap-binding complex,
eIF4F, thereby preventing cap-dependent translation of cellular m R N A s [15]. With EMCV, shutoff may be due to sequestration of the eucaryotic
translation.initiation factor e I F 2 / 2 B by certain
structural elements within the EMCV IRES [16].
In contrast, although HAV translation appears to
occur through a cap-independent mechanism similar to these other picornaviruses, HAV has not
evolved any mechanism to interfere with host-cell
translation.
Although often not included in discussions of
hepatitis viruses, the flavivirus responsible for
yellow fever presents an interesting contrast. Here
459
is a virus which typically causes rapid, overwhelming hepatitis and, frequently, death in humans,
following an incubation period which is much
briefer than that of the classical forms of viral
hepatitis. Unlike other hepatitis viruses, yellow
fever virus (YFV) almost certainly causes liver
injury by a direct viral induced cytopathology. In
marked contrast to the other hepatitis viruses,
YFV also replicates well and induces cytopathology in a wide variety of cell lines and does not,
for example, demonstrate the very restricted
replication phenotype of wild-type HAV. However, yet another, very important difference is the
mechanism of transmission of YFV. This virus is
transmitted and biologically amplified through a
biting arthropod vector, the culicine mosquito,
such that production of an acute viremia in infected primates is sufficient to maintain virus
transmission under the proper epidemiologic conditions.
While teleological arguments have inherent
limitations, the bulk of experimental data are
consistent with the hypothesis that the most important human hepatitis viruses, HAV, HBV and
HCV, have all evolved toward a level of intrahepatic replication which optimizes the probability of
transmission to other-human hosts. Disease
caused by these viruses appears to be largely if
not exclusively immunopathologic in nature, and
not due to direct viral cytopathic effects. This has
important implications for understanding the biology and molecular biology of these viruses. In
addition, this view of the hepatitis viruses suggests that attempts to develop live, attenuated
virus vaccines, in contrast to the dramatic success
of the attenuated yellow fever vaccine, may prove
particularly difficult with viruses such as HAV.
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