ROLE OF VIRION-ASSOCIATED LIPIDS IN HCV INFECTION
FIG. 1. Role of HCV-associated cholesterol in infection. (A) Effect of cholesterol depletion on HCV infectivity. HCVcc particles (⬃2 fmol of the core
protein) were treated with B-CD at 0.1, 1, and 5 mg/ml for 1 h at 37°C. After removal of B-CD, Huh-7 cells were infected with the treated virus particles,after which the core protein content of infected cells at 72 h p.i. was determined as an indicator of infectivity, as previously established (24). (B) Effectof cholesterol replenishment on infectivity. After treatment with 5 mg/ml B-CD, virus was treated either with medium alone or with medium containingexogenous cholesterol for 1 h at 37°C. (C) Effect of cholesterol depletion and replenishment on density gradient profiles of the viral particles. The HCVcctreated with 5 mg/ml B-CD was replenished with exogenous cholesterol (1 mM) and then separated by 10-to-60% sucrose gradient ultracentrifugation.
The core protein in each fraction was measured. The density of each fraction was determined by refractive index measurement. (D) Effects of cholesteroldepletion and replenishment on viral infectivity. Each fraction (see panel C) was infected, and then the core proteins in the cells were measured at 72 hp.i. (E) Effect of cholesterol depletion on the infectivity of HCVpv (genotype 1a) (shaded bars) or the control, VSVdelG-GFP/G (solid bars). The viruseswere preincubated with B-CD for 1 h at 37°C before infection. (F) (Left) The culture medium from HCVcc-producing cells was fractionated as describedabove. For each fraction, the amounts of core and intracellular core (infectivity) are plotted. Peaks of the core (arrow) and infectivity (arrowhead) areindicated. (Center) An aliquot of fraction 8 (peak of the core) was treated with 1 mM cholesterol for 1 h at 37°C. The resultant aliquot and an untreatedaliquot of the fraction were subjected to sucrose gradient ultracentrifugation. The core in each fraction was plotted. (Right) The infectivities of fractions(Fr.) 6 and 8 (see the left panel) with or without cholesterol treatment were determined as shown above. Data are means from four independentexperiments. Error bars, standard deviations.
increasing concentrations (0.1 to 5 mg/ml) of B-CD, which is
evaluated by quantifying the viral core protein in cells at
known to extract cholesterol from membranes (40). The
72 h postinfection (p.i.). Using an immunoassay that pro-
viral samples were then used to inoculate Huh-7 cells after
vides results indicative of HCV infectivity (25), we also
removal of B-CD by ultracentrifugation. Infectivity was
confirmed a good correlation between the core level and
AIZAKI ET AL.
TABLE 2. Depletion of virion-associated cholesterol by B-CD
tivity (Fig. 1F, left). As indicated above, maximum infectivitywas obtained with fraction 6 (1.13 g/ml). In contrast, a major
Radioactivity (cpm) of
fraction of core protein banded at a higher density (1.17 g/ml)
in fraction 8. We hypothesized that fraction 8 contains lipids at
lower levels than those in fraction 6. However, quantification
of lipids, including cholesterol, in the fractions obtained failed,
presumably due to a low sensitivity of detection. Thus, to
a Determined by subtracting the radioactivity of uninfected cells from that of
extend our findings on the involvement of cholesterol, we
HCVcc-infected cells in two experiments.
added exogenous cholesterol to fraction 8, followed by ultra-
Percentage of the radioactivity of the untreated sample.
filtration to remove unincorporated cholesterol. A subsequentdensity gradient profile demonstrated a shift in the core pro-tein peak to 1.13 g/ml (Fig. 1F, center). A concomitant increase
infectious titers (data not shown). As shown in Fig. 1A, core
in the infectivity of the fraction, approaching that of untreated
protein levels following B-CD treatment at 0.1, 1, or 5 mg/ml
fraction 6, was observed (Fig. 1F, right). In contrast, supple-
were reduced by 60, 83, or 98%, respectively, from the levels
mentation of fraction 6 with exogenous cholesterol did not
with the untreated virus. The cholesterol level of HCVcc
alter its infectivity (Fig. 1F, right) or change its density gradient
treated with 5 mg/ml B-CD was found to be ⬃50% of that of
(data not shown). These results suggest that exogenous cho-
untreated virions (Table 2).
lesterol supplementation can reverse deficits in the infectivity
To demonstrate that the reduced infection efficiency of B-
of HCV virions due to low cholesterol content.
CD-treated virus was caused by the reduced cholesterol con-
Sphingolipid dependence of HCV infectivity. In addition to
tent of the viral envelope, we attempted to reverse the inhib-
cholesterol, sphingolipid is a major component of eukaryotic
itory effect by adding exogenous cholesterol. Following
lipid membranes. We therefore investigated the functional sig-
treatment of HCVcc with 5 mg/ml B-CD, the drug was washed
nificance of sphingomyelin (SM), the most abundant sphingo-
out, and increasing concentrations of cholesterol were added
lipid, with regard to HCV infectivity. HCVcc was treated for
in an attempt to reconstitute the normal virion cholesterol
1 h with increasing concentrations (0.1 to 10 U/ml) of bacterial
content. The addition of 1 mM cholesterol completely reversed
SMase, which is known to hydrolyze membrane-bound SM to
the virus infectivity (Fig. 1B). After cholesterol was replen-
ceramide. Following ultracentrifugation to remove the SMase,
ished, the viral RNA was restored to a level similar to that in
Huh-7 cells were inoculated with the HCVcc. The amount of
the untreated control.
HCV core protein within the cells was quantified at 72 h p.i.
To investigate the effect of cholesterol on the density of
Figure 2A shows 50 and 90% reductions in HCV infectivity
infectious HCV virions, B-CD-pretreated or untreated viral
after incubation of the virion with 0.1 and 1 U/ml SMase,
samples, as well as cholesterol-replenished treated viral sam-
respectively. We further observed that SMase treatment of
ples, were subjected to sucrose density gradient centrifugation
HCVpv resulted in a progressive loss of infectivity, while
(Fig. 1C). The density of HCVcc core protein at its peak
SMase had no effect on the infectivity of the control virus (Fig.
concentration in untreated virus samples was ⬃1.17 g/ml.
2B). This demonstrates that sphingolipid, like cholesterol,
When virion-associated cholesterol was removed by B-CD, the
plays an essential role in HCV infectivity.
density of HCVcc core protein at its peak concentration was
Requirement for virion-associated cholesterol and sphingo-
shifted to 1.20 g/ml. Addition of exogenous cholesterol to this
lipid during HCV cell entry. These findings support the idea
cholesterol-depleted sample restored a lower-density fraction
that virion-associated cholesterol and sphingolipid may influ-
(1.15 g/ml). Figure 1D illustrates the infectivity of each gradi-
ence viral entry into host cells by altering the interaction be-
ent fraction. Untreated virus had maximum infectivity at ⬃1.13
tween viral particles and a host cell factor(s). Viral entry is a
g/ml (fraction 6), while, as expected, fractions from B-CD-
multistep process including binding of the virion to the cell
treated viral samples exhibited minimal to no infectivity. Re-
surface and internalization into the cytoplasm by endocytosis.
plenishment of depleted virus with cholesterol returned infec-
To examine whether virion-associated cholesterol and SM
tivity to untreated-control levels, and cholesterol-replenished
might play a role in cell binding or postbinding events during
virus had a buoyant density of ⬃1.07 g/ml (fraction 4), suggest-
viral entry, we used a binding assay in which Huh-7 cells pre-
ing that HCV-associated cholesterol is crucial for viral infec-
incubated for 1 h at 4°C were infected with B-CD- or SMase-
tivity and that the effect of a cholesterol-depleting drug is
treated HCVcc. Total RNA was extracted after a 1-h addition
reversible. We further observed that B-CD treatment of a
of the virions at 4°C, followed by quantification of HCV RNA.
pseudotyped VSV containing the E1 and E2 proteins of the
As shown in Fig. 3A, treatment of the virions with either B-CD
HCV genotype 1a isolate H77c (HCVpv) resulted in a pro-
or SMase had little influence on their ability to bind to cells.
gressive loss of infectivity, while B-CD had significantly less
It has been shown that CD81 plays an important role in
impact on the infectivity of the control virus VSVdelG-GFP/G
HCV internalization but is not correlated with viral attachment
(7, 33). An anti-CD81 antibody was used as a negative control
The results described above raise the possibility that the
for reduced viral attachment. It is likely that heparan sulfate
infectivity of HCV virions with relatively low levels of incor-
proteoglycan on the target cell surface is needed for the initial
porated cholesterol might be enhanced by supplementation
attachment of HCV (33). Thus, heparinase I was used as a
with exogenous cholesterol. Density gradient fractions of cul-
positive control for reduced HCV attachment to the cells. To
ture supernatants collected from HCV-infected cells were an-
examine the roles of cholesterol and sphingolipid on the
alyzed with regard to the presence of core protein and infec-
HCVcc membrane in viral internalization, a virus-cell mixture
ROLE OF VIRION-ASSOCIATED LIPIDS IN HCV INFECTION
FIG. 2. Effect of SM hydrolysis on viral infectivity. (A) Effect on the infectivity of HCVcc. HCVcc was treated with 0.1, 1, or 10 U/ml SMase
for 1 h at 37°C, after which SMase was removed by ultracentifugation. Huh-7 cells were infected with the treated virus, and the core protein contentof infected cells was determined at 72 h p.i. (B) Effect on the infectivity of HCVpv (genotype 1a) (shaded bars) or the control, VSVdelG-GFP/G(VSV cont) (solid bars). The viruses were preincubated with SMase for 1 h at 37°C before infection. Data are means from four independentexperiments. Error bars, standard deviations.
prepared at 4°C as described above was incubated for 2 h at
ported that HCV core protein is associated with DRMs in
37°C, followed by trypsinization to remove virions that were
cells carrying the full-length HCV replicon. To investigate
surface bound but not internalized (Fig. 3B). We verified that
whether HCV structural proteins are associated with DRMs
94% of surface-bound-viruses were removed by trypsinization
in HCVcc-producing cells, lysates from cells infected with
using CD81-negative Huh-7 subclones. A marked reduction in
HCVcc were subjected to membrane flotation analysis. In
viral RNA levels within cells was detected after pretreatment
the absence of detergent treatment, the majority of the core
of the virus with either B-CD or SMase. These results strongly
(Fig. 4A) and E1 (Fig. 4B) proteins were detected in the
suggest that virion-associated cholesterol and sphingolipid
membrane fractions. After treatment with cold TX-100, sig-
function as key determinants of internalization but not of cell
nificant amounts of both viral proteins were recovered from
the DRM fraction. However, after treatment with TX-100 at
Association of HCV structural proteins with lipid rafts.
37°C, the majority of the E1 and core proteins had shifted to
Cholesterol and sphingolipid are major components of lipid
the detergent-soluble fractions. We also found that HCV
rafts, which can be isolated as detergent-resistant mem-
genotype 1b E1 and E2 can be associated with the lipid raft
branes (DRMs) by treatment with cold TX-100, followed by
in 293T cells transfected with an E1 or E2 expression plas-
equilibrium flotation centrifugation. Matto et al. (30) re-
mid (Fig. 4C) and that the cytoplasmic tails of envelope
FIG. 3. Effects of B-CD or SMase on virus attachment and internalization. (A) Virus attachment to Huh-7 cells was determined at 4°C after treatment
of HCVcc with B-CD (1 or 5 mg/ml) or SMase (1 or 10 U/ml). An antibody (Ab) against CD81 was used, in order to ensure that the antibody did notinhibit HCVcc binding (7, 33). Heparinase was used to reduce HCV attachment to the cell. Viral RNA copies were normalized to total cellular RNA,and the normalized RNA copies in the mock-treated sample (⫺) were arbitrarily set at 100%. (B) Virus internalization was measured in Huh7-25, aCD81-negative subclone (CD81⫺) (3), and Huh7-25-CD81, which stably expresses CD81 (CD81⫹), after treatment of the virions with B-CD or SMase.
After internalization for 2 h at 37°C, cells were exposed to trypsin (trypsin ⫹) or phosphate-buffered saline (trypsin ⫺). Huh7-25 was used to ensure thatsurface-bound virus would be removed by trypsin treatment. The amounts of HCV RNA in Huh7-25 and Huh7-25-CD81 cells infected with untreatedHCVcc were assigned the arbitrary value of 100%, respectively. Results are representative of four independent experiments.
AIZAKI ET AL.
FIG. 4. Compartmentation of HCV structural proteins within DRM fractions. Lysates of HCVcc-infected cells were either treated with 1%
TX-100, either on ice or at 37°C, or left untreated, followed by sucrose gradient centrifugation. (A and B) For each fraction, the amount of coreprotein was determined by an enzyme-linked immunosorbent assay (A), and E1, calnexin, and caveolin-2 were analyzed by Western blotting (B).
The amount of core protein in each lysate (TX-100, 37°C; TX-100, 4°C; Untreated) was assigned the arbitrary value of 100%. M, membrane; NM,nonmembrane; DS, detergent soluble. (C) Lysates of 293T cells expressing HCV E1 or E2 protein were either treated with 1% TX-100, either onice or at 37°C, or left untreated, followed by discontinuous sucrose gradient centrifugation. Each fraction was concentrated in a Centricon YM-30filter unit and subjected to 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting with antibodies againstcalnexin, caveolin-2, Myc (E1), or FLAG (E2). (D) (Top) Structures of HCV envelope genes used. Amino acid positions of HCV are indicated.
Signal sequence, transmembrane (TM), and cytoplasmic tail (CT) domains of VSV G protein are shown. (Bottom) Cell lysates expressing chimericHCV E1 or E2 protein were treated with 1% TX-100 on ice or left untreated, followed by discontinuous sucrose gradient centrifugation. It hasbeen reported that VSV-G is not associated with lipid (39). Calnexin, caveolin-2, and chimeric glycoproteins (chimeric E1 and chimeric E2) wereanalyzed by immunoblotting. Fractions are numbered from 1 to 9 in order from top to bottom (light to heavy).
ROLE OF VIRION-ASSOCIATED LIPIDS IN HCV INFECTION
activity in the lysates of Huh-7 cells transfected with an invitro-transcribed JFH-1 replicon RNA containing a luciferasereporter gene (22) (data not shown). Figure 6B shows theeffects of ISP-1 and HPA-12 on de novo sphingolipid biosyn-thesis by replicon cells. No differences in the inhibitory effectsof each compound were observed in replicon cells derivedfrom HCV-N versus JFH-1. When de novo synthesis of sphin-golipids was examined by metabolic labeling with [14C]serine,ISP-1 almost completely inhibited the production of both cer-amide and SM, while HPA-12 greatly inhibited the synthesis ofSM but not ceramide. Levels of phosphatidylethanolamine andphosphatidylserine, into which serine is incorporated by a
FIG. 5. Effects of B-CD or SMase treatment of cells on HCV
infectivity. Huh-7 cells were either left untreated or treated with B-CD
pathway distinct from that of sphingolipid biosynthesis, were
at 0.1, 1, or 5 mg/ml (A) or with SMase at 0.1, 1, or 10 U/ml (B) prior
not influenced by these drugs. These results suggest that sup-
to HCVcc infection. Intracellular core levels were quantitated 72 h p.i.
pression of HCV RNA replication by inhibitors of sphingolipid
Data are means from four independent experiments. Error bars, stan-
biosynthesis might be dependent on the viral genotype or iso-
dard deviations.
This observation prompted us to investigate whether inhib-
itors of the sphingolipid biosynthetic pathway might have the
proteins are important for their interaction (Fig. 4D). These
ability to prevent HCV virion production. Interestingly, when
data suggest that subpopulations of HCV structural proteins
Huh-7 cells producing JFH-1 HCVcc were treated with ISP-1
are associated with lipid rafts in cells generating the HCV
or HPA-12 under conditions similar to those the replicon cells,
viral core levels in the culture supernatants were greatly re-
Moderate inhibition of HCV infection by B-CD or SMase
duced in a dose-dependent manner. For example, exposure to
treatment of host cells. It has recently been reported that
10 M ISP-1 or 1 M HPA-12 reduced viral core protein levels
cholesterol depletion or SM hydrolysis from the host cell mem-
more than 85% from those for control cells (Fig. 6C). The 50%
brane decreases HCV infection, in part by decreasing the level
inhibitory concentrations of both drugs were less than 0.1 M,
of CD81 on the cell surface (19, 53). The involvement of the
50-fold less than those obtained for the RNA replication of the
lipid environment of the host cell plasma membrane in HCV
HCV-N-replicon. Together, these results suggest that the
infection was investigated in our HCVcc infection system.
sphingolipid biosynthetic pathway plays an important role in
Prior to infection, Huh-7 cells were treated with B-CD or
the production of HCV particles, but not in genome replica-
SMase and then washed with the medium five times. Choles-
tion, in JFH-1-based HCVcc.
terol depletion from Huh-7 cells by B-CD at 1 or 5 mg/mlinhibited HCV core levels by 20 and 75%, respectively, com-
pared to levels in untreated cells (Fig. 5A). We also found thathydrolysis of SM by SMase at 1 or 10 U/ml on the cells,
In this study, we demonstrated the role of HCV virion-
respectively, led to moderate reduction of the viral infection,
associated cholesterol and sphingolipid in viral infectivity. Al-
by 20 or 55% of the infection level of the untreated control
though dependence on virion-associated cholesterol for virus
(Fig. 5B). There was no influence on cell viability under the
entry has been shown for a number of viruses (4, 6, 28, 49), this
conditions of these treatments (data not shown). These find-
is the first study to demonstrate the importance of envelope
ings, compared with the results in Fig. 1A and 2A, suggest that
cholesterol in a virus belonging to the family Flaviviridae. Fur-
the raft-like environment on the plasma membrane likely
thermore, to our knowledge, the functional role of virion mem-
serves as a portal for HCV entry, but HCV virion-associated
brane-associated SM has not been examined in viruses. Our
cholesterol and sphingolipid more readily play more critical
previous studies using Chinese hamster ovary cell mutants
roles in viral infection.
deficient in SM synthesis have demonstrated that reduction of
Inhibitors of the sphingolipid biosynthetic pathway sup-
cellular SM levels enhances cellular cholesterol efflux in the
press the production of HCVcc, but not RNA replication, for a
presence of B-CD (9, 12). Thus, it may be possible that SM
JFH-1-derived replicon. In the course of studying the involve-
plays a role in the retention of cholesterol on HCV particles
ment of lipid metabolism in the HCV life cycle, we observed
due to interaction between cholesterol and SM. The finding
that inhibitors of the sphingolipid biosynthetic pathway, includ-
that B-CD or SMase treatment of HCVcc markedly inhibited
ing ISP-1 and HPA-12, which specifically inhibit serine palmi-
virus internalization but not cell attachment (Fig. 3) suggests
toyltransferase (31) and ceramide trafficking from the ER to
that HCV membrane-associated cholesterol and sphingolipid
the Golgi apparatus (55), influenced subgenomic replicons de-
are crucial for the interaction of viral glycoproteins with the
rived from the HCV-N isolate (genotype 1b), but not those
virus-receptor/coreceptor required for cell entry. Cholesterol
derived from JFH-1. A dose-dependent decrease in HCV
depletion or sphingolipid hydrolysis might induce a conforma-
RNA copy numbers among HCV-N replicon cells was ob-
tional change in the viral envelope, resulting in instability of
served upon exposure to ISP-1 or HPA-12, as previously re-
the virion structure. Since the cholesterol/phospholipid ratios
ported (43, 52). In contrast, these compounds had little or no
of membranes affect bilayer fluidity, the maturation of viral
effect on viral RNA accumulation in JFH-1 replicon cells (Fig.
envelopes with high cholesterol/phospholipid ratios via associ-
6A). Furthermore, these compounds did not affect luciferase
ation with rafts may be important for the stability of HCV
AIZAKI ET AL.
FIG. 6. Anti-HCV effects of inhibitors of the sphingolipid biosynthetic pathway. Subgenomic replicon cells derived from HCV isolate N or
JFH-1, as well as HCVcc-producing cells, were treated with ISP-1 (0.1, 1, or 10 M), HPA-12 (0.1, 1, or 10 M) or alpha interferon (IFN) (100U/ml) for 72 h. HCV RNA titers in the replicon cells (A) and the HCV core protein content of the culture medium of infected cells (C) weredetermined. Data are means from four independent experiments. Error bars, standard deviations. (B) De novo synthesis of sphingolipid in theabsence or presence of ISP-1 (10 M) and HPA-12 (10 M) was monitored in duplicate by metabolic labeling with [14C]serine for 2 h at 37°C.
Cer, ceramide; PE, phosphatidylethanolamine; PS, phosphatidylserine.
particles. Replenishing the viral membrane with cholesterol
studies have demonstrated budding at the plasma membrane
following treatment with 5 mg/ml B-CD successfully restored
(13, 36, 37, 41), and it has been proposed that the site of
viral infectivity to the same level as that of untreated virus (Fig.
budding may be virus and cell type dependent (27). We dem-
1), suggesting that reversible B-CD-induced changes in HCV
onstrate here that subpopulations of HCV structural proteins
structure might critically influence viral infectivity. However,
partition into cellular detergent-resistant, lipid-raft-like mem-
we were unable to restore viral infectivity by replenishing cho-
brane fractions in HCVcc-producing cells (Fig. 4) and that
lesterol after pretreatment of the virion with concentrations of
inhibitors of the sphingolipid biosynthetic pathway block HCV
B-CD exceeding 10 mg/ml (data not shown). Under these
virion production (Fig. 6). Furthermore, a large proportion of
conditions, it is likely that large holes in the viral membrane
HCV E2 protein incorporated into HCVcc is endoglycosidase
destroy the virus, a result that cannot be reversed by supplying
H resistant (data not shown). Thus, membrane compartments
containing cholesterol- and sphingolipid-rich microdomains
How are cholesterol and sphingolipid involved in the HCV
may be involved in HCV virion maturation. Another explana-
virion during the process of virus maturation? Like most pos-
tion for the recruitment of these lipids to the HCV membrane
itive-stranded RNA viruses, HCV is thought to assemble at the
may be an association between the virus and very-low-density
ER membrane. However, Miyanari et al. (32) reported that
lipoprotein (VLDL) or low-density lipoprotein. Recently,
lipid droplets are important for HCVcc formation. These au-
Huang et al. (16) demonstrated a close link between HCV
thors have shown that the characteristics of lipid-droplet-asso-
production and VLDL assembly, suggesting that an HCV-
ciated membranes in Huh-7 cells differ from those of ER
VLDL complex is generated and secreted from cells.
membranes. In the case of flaviviruses, for which the mecha-
Recent reports have demonstrated that CD81-mediated
nism of viral assembly and budding remains unclear (15), a few
HCV infection is partly dependent on cell membrane choles-
ROLE OF VIRION-ASSOCIATED LIPIDS IN HCV INFECTION
terol (19) and SM (53). We further characterized the role of
4. Bender, F. C., J. C. Whitbeck, H. Lou, G. H. Cohen, and R. J. Eisenberg.
lipid on the plasma membrane in viral infectivity and found
2005. Herpes simplex virus glycoprotein B binds to cell surfaces indepen-
dently of heparan sulfate and blocks virus entry. J. Virol. 79:11588–11597.
that cholesterol depletion by B-CD, as well as hydrolysis of SM
5. Blanchard, E., D. Brand, S. Trassard, A. Goudeau, and P. Roingeard. 2002.
by SMase, moderately inhibits HCV infectivity (Fig. 5). These
Hepatitis C virus-like particle morphogenesis. J. Virol. 76:4073–4079.
6. Chazal, N., and D. Gerlier. 2003. Virus entry, assembly, budding, and mem-
results suggest that cholesterol and sphingolipid in the plasma
brane rafts. Microbiol. Mol. Biol. Rev. 67:226–237.
membrane environment may assist HCV entry, while HCV
7. Evans, M. J., T. von Hahn, D. M. Tscherne, A. J. Syder, M. Panis, B. Wolk,
virion-associated cholesterol and sphingolipid appear to play
T. Hatziioannou, J. A. McKeating, P. D. Bieniasz, and C. M. Rice. 2007.
Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry.
critical roles in viral infection.
We previously demonstrated that HCV RNA and nonstruc-
8. Ezelle, H. J., D. Markovic, and G. N. Barber. 2002. Generation of hepatitis
tural proteins are present in DRM structures, likely in the
C virus-like particles by use of a recombinant vesicular stomatitis virus
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context of a lipid-raft structure, and that viral RNA is likely
9. Fukasawa, M., M. Nishijima, H. Itabe, T. Takano, and K. Hanada. 2000.
synthesized at a raft membrane structure in cells containing the
Reduction of sphingomyelin level without accumulation of ceramide in Chi-
genotype 1b HCV replicon (2, 10, 46). Here we observed that
nese hamster ovary cells affects detergent-resistant membrane domains andenhances cellular cholesterol efflux to methyl--cyclodextrin. J. Biol. Chem.
ISP-1 and HPA-12 suppress HCVcc production, but not viral
RNA replication, by the JFH-1 replicon (Fig. 6). Impairment
10. Gao, L., H. Aizaki, J. W. He, and M. M. Lai. 2004. Interactions between viral
nonstructural proteins and host protein hVAP-33 mediate the formation of
of particle assembly and maturation, rather than suppression
hepatitis C virus RNA replication complex on lipid raft. J. Virol. 78:3480–
of genome replication, by these drugs may account for the
inhibition of HCV production in the JFH-1 system. Viral RNA
11. Guo, J. T., V. V. Bichko, and C. Seeger. 2001. Effect of alpha interferon on
the hepatitis C virus replicon. J. Virol. 75:8516–8523.
replication of the HCV-N replicon, however, was efficiently
12. Hanada, K., T. Hara, M. Fukasawa, A. Yamaji, M. Umeda, and M. Nishi-
inhibited by these compounds, as found in previous reports
jima. 1998. Mammalian cell mutants resistant to a sphingomyelin-directed
(43). The virus strain specificity of the anti-HCV activity of
cytolysin. Genetic and biochemical evidence for complex formation of theLCB1 protein with the LCB2 protein for serine palmitoyltransferase. J. Biol.
cyclosporine has recently been demonstrated: JFH-1 replica-
tion is less sensitive to cyclosporine than replication of geno-
13. Hase, T., P. L. Summers, K. H. Eckels, and W. B. Baze. 1987. An electron
and immunoelectron microscopic study of dengue-2 virus infection of cul-
type 1b strains. Furthermore, the requirement for interaction
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with a cellular replication cofactor, cyclophilin B, differs among
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HCV strains (18). It appears that ISP-1 and HPA-12 are fur-
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ther examples of diverse effects on HCV strain replication.
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In summary, our data here demonstrate important roles of
16. Huang, H., F. Sun, D. M. Owen, W. Li, Y. Chen, M. Gale, and J. Ye. 2007.
cholesterol and sphingolipid in HCV infection and virion mat-
Hepatitis C virus production by human hepatocytes dependent on assemblyand secretion of very low-density lipoproteins. Proc. Natl. Acad. Sci. USA
uration. Specifically, mature HCV particles are rich in choles-
terol. Depletion from HCV or hydrolysis of virion-associated
17. Ikeda, M., M. Yi, K. Li, and S. M. Lemon. 2002. Selectable subgenomic and
genome-length dicistronic RNAs derived from an infectious molecular clone
SM results in a loss of infectivity. Moreover, the addition of
of the HCV-N strain of hepatitis C virus replicate efficiently in cultured
exogenous cholesterol restores infectivity. In addition, choles-
Huh7 cells. J. Virol. 76:2997–3006.
terol and sphingolipid on the HCV membrane play key roles in
18. Ishii, N., K. Watashi, T. Hishiki, K. Goto, D. Inoue, M. Hijikata, T. Wakita,
N. Kato, and K. Shimotohno. 2006. Diverse effects of cyclosporine on hep-
virus internalization, and portions of structural proteins are
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2007. Initiation of hepatitis C virus infection is dependent on cholesterol andcooperativity between CD81 and scavenger receptor B type I. J. Virol.
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that agents capable of modifying virion-associated lipid con-
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Nagayama, T. Tanaka, and T. Wakita. 2001. Sequence analysis of hepatitis
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We thank M. Matsuda, M. Sasaki, S. Yoshizaki, T. Shimoji, M.
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Source: http://3gmed.co.th/images/3g_med/products/viroblock_SA/Critical_Role_of_Virion-Associated_Cholesterol_and_Sphingolipid_in_Hepatitis_C_Virus_Infection.pdf
Postgrad Med J 2001;77:759–764 Hypokalaemia and hyperkalaemia A Rastergar, M Soleimani compartments. Humans, as carnivorous ani- Disturbances in potassium homoeostasis mals, consume large amount of potassium presenting as low or high serum potas- intermittently. Dietary potassium, which is sium are common, especially among hos-
Laboruntersuchungen im Institut für Klinische Chemie und Labormedizin (IKL) - Krankenhaus Dresden-Friedrichstadt - Direktor: Prof. Dr. Dr. med. Th. Demant Kommentiertes Leistungsverzeichnis Letzte Aktualisierung: 09.02.2011 Zum Gebrauch des Leistungsverzeichnisses . Notfallparameter. Leistungsverzeichnis des Instituts: 1.