Department of Microbiology & Immunology, C5141 Veterinary Medical Center, Cornell University, Ithaca, NY 14853, USA1
Author for correspondence: Gary Whittaker. Fax +1 607 253 3384. e-mail grw7{at}cornell.edu
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Abstract |
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Introduction |
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Entry into a host cell is of fundamental importance in infection and pathogenesis for all viruses. Animal viruses generally enter by one of two mechanisms, either via the endocytic pathway of the cell, or by fusion at the plasma membrane. Uptake through endocytosis is an essential component of the route of entry of viruses such as influenza virus and Semliki Forest virus, but other viruses, including herpesviruses and many retroviruses, generally enter by direct fusion at the plasma membrane [reviewed in Marsh & Helenius (1989) and Marsh & Pelchen-Matthews (1994)
]. For most viruses that enter by endocytosis, the virus is believed to be trafficked towards the late endosome and is therefore part of the lysosome-targeted pathway of endocytosis, which results in progressive acidification of the vesicle and the fusion and/or uncoating of the virus particle. The alternative endocytic pathway, whereby endosomes do not reach a low pH and are recycled back to the cell surface, does not seem to be a common route of productive virus entry.
The entry of influenza virus A has been extensively studied with regard to binding, fusion and uncoating (Hernandez et al., 1996 ; Whittaker et al., 1996
). Following initial interaction of the virus haemagglutinin (HA) with its sialic receptor, the virus is believed to enter the cell by receptor-mediated endocytosis a process dependent on cellular dynamin (Roy et al., 2000
). Fusion of the virus envelope with the endosomal membrane then occurs in a compartment having an pH equivalent to the late endosome: approximately pH 5·5 (Stegmann et al., 1987
; Yoshimura & Ohnishi, 1984
). Exposure of HA to low pH is essential to trigger its fusion activity (Skehel et al., 1995
; White et al., 1982
). Once released into the cytoplasm, the virus uncoats following a low-pH-triggered release of the matrix protein, M1, and the genomic ribonucleoproteins (vRNPs) then rapidly enter the nucleus, via nuclear pore complexes (Bui et al., 1996
; Martin & Helenius, 1991
).
The acidification occurring as part of endosome maturation is essential for two events in the virus life-cycle. In addition to being a trigger for HA-mediated fusion, the low pH environment is transferred into the interior of the virus, via the M2 ion channel present in the virus envelope (Pinto et al., 1992 ; Sugrue & Hay, 1991
). It is the M2-mediated acidification of the virus interior that is the target for the anti-influenza drug amantadine. The mechanism of action of amantadine is to block the M2 ion channel, so preventing the interior of the virus from encountering low pH (Bukrinskaya et al., 1982
; Hay et al., 1985
; Martin & Helenius, 1991
). This results in prevention of release of the influenza virus matrix protein (M1) from the vRNPs during virus uncoating (Bui et al., 1996
), with the end result that the vRNPs do not enter the nucleus, and replication cannot occur. Amantadine is effective only against strains of influenza A virus, and is ineffective against influenza viruses B and C [see Lamb & Pinto (1997)
for a review].
Based on the action of certain protein kinase inhibitors, such as H7 and staurosporine, the entry mechanisms of several enveloped viruses, including rhabdoviruses, alphaviruses, poxviruses and herpesviruses, have been proposed to require cellular protein kinase C (PKC) (Cirone et al., 1990 ; Constantinescu et al., 1991
). These inhibitors have not been reported to have any effect on entry of orthomyxoviruses, such as influenza virus although H7 has been widely reported as a PKC inhibitor that has effects late in infection and affects influenza viral mRNA and vRNP nuclear export, and late protein production (Bui et al., 2000
; Kurokawa et al., 1990
; Martin & Helenius, 1991
; Vogel et al., 1994
).
PKC is a large superfamily of related proteins which carry out diverse regulatory roles in many key cellular processes (Mellor & Parker, 1998 ; Toker, 1998
). Current data on the role of PKC in virus entry have relied on the use of the kinase inhibitors H7 and staurosporine, but these are known to be non-selective in their action and inhibit a wide range of different protein kinases (Bradshaw et al., 1993
; Garland et al., 1987
; Quick et al., 1992
). As such, their use in determining a role for PKC must be treated with caution. Recently, a new generation of PKC inhibitors, the bisindolylmaleimides, has been described (Toullec et al., 1991
). The bisindolymaleimides are thought to block the ATP-binding site on the catalytic domain of PKC and are highly specific, appearing to inhibit all PKC isozymes with similar potency.
In the present study we used the highly specific PKC inhibitor bisindolylmaleimide I.HCl and examined its effect on the entry and replication of influenza virus. We found that virus replication was inhibited in a dose dependent and reversible manner, and that the block in replication occurred very early in infection, apparently during virus endocytosis and uncoating. These results suggest that activity of PKC is crucial for influenza virus entry, and may be a target for future antiviral therapy.
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Methods |
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Bisindolylmaleimide I.HCl was obtained from Calbiochem. Unless stated otherwise, cells were pre-treated for 10 min with bisindolylmaleimide I.HCl at the concentration stated and maintained in bisindolylmaleimide throughout the infection.
Virus infections. Influenza A virus (strain A/WSN/33) (Stuart-Harris, 1939 ) was obtained from A. Helenius (ETH-Zürich, Switzerland). Stocks of virus were prepared from the supernatant of infected MadinDarby bovine kidney (MDBK) cells and plaque titred on Madin-Darby canine kidney (MDCK) cells (Whittaker et al., 1995
).
Influenza B virus (strain B/Yamagata/83) was kindly provided by Peter Palese (Mt Sinai School of Medicine, NY, USA). Stocks of virus were grown in MDCK cells at 34 °C and were plaque titred on MDCK cells.
For infection (15 p.f.u. per cell), virus stocks were first diluted in RPMI 1680 medium containing 0·2% BSA and buffered to pH 6·8 with 10 mM HEPES (RPMI-BSA). Virus was adsorbed for 90 min at 0 °C and cells were then washed and maintained in Mv-1 growth medium containing 2% FBS. Infections were carried out in a 5% CO2 incubator. For infections at high m.o.i. (approximately 150200 p.f.u. per cell), virus stocks were diluted in RPMI-BSA, adsorbed for 90 min at 0 °C and cells washed and maintained in Mv-1 growth medium without sodium bicarbonate, containing 2% FBS, and buffered to pH 7·3 with 20 mM HEPES. Infections were then carried out by quickly shifting the cells to a water-bath set to 37 °C. A protein synthesis inhibitor (1 mM cycloheximide; Sigma) was added to all high m.o.i. experiments to ensure that only signal from input virus was detected, i.e. to exclude any possibility of signal arising from newly synthesized virus protein.
Production of radioactive virus.
MDBK cells were infected with 15 p.f.u. per cell influenza virus, and 167 µCi/ml 35S Pro-mix (Amersham) was added at 6 h p.i. Virus was harvested from the supernatant at 20 h pi, and concentrated on a 3060% sucrose step-gradient. The peak radioactive fractions containing virus (approximately 1x104 c.p.m./10µl) were pooled, and stored at -80 °C.
Virus binding assay.
Approximately 1x105 cells were washed with ice-cold RPMI-BSA and pre-chilled for 15 min on ice, and approximately 1 p.f.u. per cell influenza virus bound; 5x103 c.p.m. of radioactive virus was mixed with unlabelled virus (in RPMI-BSA) to give the desired m.o.i. Cells were incubated on ice for 90 min and washed extensively with RPMI-BSA to remove unbound virus. Cells were then lysed in SDSPAGE sample buffer, and radioactivity was determined by scintillation counting.
Assay of virus infectivity.
Mv-1 cells were infected with influenza virus (approximately 1 p.f.u. per cell) in RPMI-BSA buffered with 10 mM HEPES and virus was allowed to adsorb at 4 °C for 90 min. Cells were washed and incubated in Mv-1 growth medium containing 10 mM HEPES, pH 7·3, and 2% FBS for 2 h at 37 °C. Cells were then washed with 0·1 M glycine, 0·1 M NaCl, pH 3·0, for 1 min to remove any uninternalized virus from the surface of cells. The cells were then washed and incubated once more in Mv-1 growth medium containing 10 mM HEPES, pH 7·3, and 2% FBS and infection allowed to proceed until 12 h post-infection. Viral supernatants were then collected and stored at -80 °C.
To quantify the amount of virus released, supernatants were serially diluted in RPMI-BSA and adsorbed to a confluent monolayer of MDCK cells in a six-well plate for 60 min at 37 °C. The cells were then rinsed with RPMI and media replaced with DMEM, 0·2% BSA, 2 µg/ml TPCK-trypsin, including 1% agarose, and incubated at 37 °C for 36 h to permit plaque formation.
SDSPAGE and Western blotting.
For analysis by SDSPAGE, cell monolayers were washed with PBS, and scraped into ice-cold PBS with a rubber policeman. Cells were lysed in SDSPAGE sample buffer and subjected to SDSPAGE on 12% acrylamide gels. Samples were transferred to nitrocellulose by semi-dry blotting and membranes blocked in 5% non-fat dry milk. Samples were analysed using the anti-influenza polyclonal antibody IBO (Whittaker et al., 1995 ) (1:500 dilution for 2 h at room temperature). By Western blot analysis, IBO reacts strongly with influenza virus nucleoprotein (NP; 55 kDa) and more weakly with matrix protein (M1; 27 kDa) (G. Whittaker, unpublished observation). As secondary antibodies we used anti-rabbit alkaline phosphatase (Bio-Rad; 1:5000 dilution for 30 min at room temperature). Blots were developed using standard alkaline phosphate reagents. As a control antibody, we used an anti-calnexin polyclonal antibody at a dilution of 1:500 (kindly provided by A. Helenius, ETH Zürich), which reacts with the cellular protein calnexin (90 kDa by SDSPAGE). Blots were scanned and stored using Adobe Photoshop. Adobe Illustrator was used for final layout of the figures.
Indirect immunofluorescence microscopy.
Immunofluorescence microscopy was carried out essentially as described previously (Whittaker et al., 1995 ). Briefly, cells were fixed with 3% paraformaldehyde in PBS for 15 min, quenched with 50 mM NH4ClPBS and permeabilized for 5 min with 0·1% Triton X-100/PBS. After blocking in 10% goat serum, cells were incubated with primary and secondary antibodies for 30 min each and mounted in Mowiol. Influenza A virus NP was detected using the monoclonal antibody H10, L16-4R5 (ATCC). Influenza virus neuraminidase was detected using the mouse monoclonal antibody H17, L17-5R17 (kindly provided by J. Yewdell, National Institutes of Health, Bethesda, MD, USA). Influenza B virus NP was detected using the mouse monoclonal antibody 23A7 (kindly provided by Wendy Barclay, University of Reading, UK). As secondary antibody we used Oregon Green 514-labelled goat anti-mouse IgG (Molecular Probes). Hoechst 33258 (Molecular Probes) was used at a concentration of 1 µg/ml. Cells were viewed using a Zeiss Axioskop fluorescence microscope fitted with a UV filter and a long pass 520 nm filter and either a 40x or 63x objective lens. Images were photographed using Ektachrome 400 film (Kodak) and scanned into Adobe Photoshop. Adobe Illustrator was used for final layout of the figure.
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Results |
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In most cases expression of both NP and NA was completely abolished in the presence of bisindolylmaleimide I, i.e. the inhibitor had an all-or-nothing effect. However, in some cells treated with low levels of bisindolylmaleimide I (5 µM) there was a nuclear signal for NP (Fig. 1F, arrows). This most probably represents a general slowing of virus replication rather than any specific block in virus trafficking. This is in contrast to cells treated with H7, which show NP expression, but with retention of both the NP and vRNPs in the nucleus (Martin & Helenius, 1991
; Bui et al., 2000
). Overall, bisindolylmaleimide I seems to block influenza virus infection in a mechanistically quite different manner to the more extensively studied protein kinase inhibitor H7.
To confirm and extend the results of immunofluorescence microscopy, we infected Mv-1 cells with influenza virus (WSN), treated them with various amounts of bisindolylmaleimide I, and analysed cell lysates by SDSPAGE and Western blotting (Fig. 2). Infection with influenza virus was assayed using the anti-influenza polyclonal antibody IBO. As expected, in the absence of bisindolylmaleimide I, lysates of influenza virus-infected cells showed strong reactivity with NP and weaker reactivity with M1, and lysates of mock-infected Mv-1 cells showed no reactivity. Addition of 5 µM bisindolylmaleimide I reduced levels of NP and M1 slightly, and addition of 10 µM bisindolylmaleimide I resulted in further reductions in the expression of NP and M1. Following addition of 20 µM bisindolylmaleimide I, levels of both NP and M1 were suppressed markedly, and both proteins were only barely detectable by Western blot. As a loading control, we assayed a duplicate blot with an anti-calnexin antibody and showed that essentially identical levels of protein were present in each lane. These experiments confirm that influenza virus replication is inhibited in a dose-dependent manner by bisindolylmaleimide I.
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In untreated cells both influenza A and B virus NP was expressed and in most cells showed a predominant localization to the cytoplasm (Fig. 4A and C
). In the presence of 10 µM bisindolylmaleimide I influenza virus NP expression was markedly inhibited, with less than 1% of cells expressing NP. These experiments show that influenza B virus is also susceptible to inhibition by bisindolylmaleimide at micromolar concentrations.
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Bisindolylmaleimide reversibly blocks virus entry into the nucleus
Because the effects of bisindolylmaleimide on influenza virus infection appeared to occur at early times of infection, we examined the effects of the inhibitor on virus entry. We infected Mv-1 cells with a high m.o.i. of virus and treated infected cells with bisindolylmaleimide I. We then assayed cells 60 min after infection by immunofluorescence microscopy. In these experiments cycloheximide was added to ensure that all signal was due to input virus and not replicating virus. Fig. 6 clearly shows that in the absence of bisindolylmaleimide, vRNPs efficiently enter the nucleus by the 60 min time-point, and cells showed a bright and distinct nuclear signal with little or no fluorescent signal from vRNPs present in the cytoplasm (Fig. 6D
). However, on addition of 20 µM bisindolylmaleimide I, the nuclear signal from vRNPs was completely lost, and instead vRNPs were visible in weak punctate areas of the cytoplasm (Fig. 6E
). In bisindolylmaleimide-treated cells we also observed low levels of red-orange fluorescence due to the presence of the drug (Fig. 6E
), but as the cells were exposed to the inhibitor for much shorter periods of time the fluorescent signal was much weaker than in earlier experiments; compare Fig. 6
to Figs 1
, 4
and 5
. Overall, these experiments clearly show that bisindolylmaleimide I blocks the entry mechanism of influenza virus within the first 60 min of infection during virus entry.
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Effects of bisindolylmaleimide on virus binding
To determine whether the presence of bisindolylmaleimide affected infection due to impaired virus binding, radiolabelled influenza virus (WSN) was mixed with an excess of unlabelled virus and adsorbed at 0 °C onto Mv-1 cells pretreated with 20 µM bisindolylmaleimide I, or left untreated. Virus was allowed to bind for 90 min in the presence or absence of bisindolylmaleimide. Cell-associated counts were then determined. No significant difference in virus binding was detected in the presence or absence of bisindolylmaleimide (Fig. 7). These data show that bisindolylmaleimide has no effect on influenza virus binding to cells.
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Fig. 8 shows immunofluorescence microscopy of cells 60 min after infection with a high m.o.i. of influenza virus WSN. In the presence of NH4Cl at 20 mM, a concentration known to prevent endosome acidification and influenza virus infection, no vRNPs were detectable at pH 7·8. However, at pH 6·5 the weak base-effect of NH4Cl was neutralized by the external medium and vRNP entry into the nucleus was observed. In contrast, bisindolylmaleimide I (20 µM) inhibited virus entry independent of the pH of the external medium, showing that it is not acting non-specifically as a weak base at micromolar concentrations.
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Discussion |
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At present, our data show that bisindolylmaleimide inhibits influenza virus entry within the first 60 min of infection at some point prior to entry of vRNPs into the nucleus. Interestingly, the point of action at which the anti-influenza drug amantadine is observed to act is also at the stage of vRNP nuclear import (Bukrinskaya et al., 1982 ; Kato & Eggers, 1969
; Martin & Helenius, 1991
). It is now well-established that at micromolar concentrations amantadine acts by blocking the M2 ion channel of influenza A viruses (Pinto et al., 1992
; Sugrue & Hay, 1991
), and in the presence of the drug pH-dependent virus uncoating and nuclear transport do not occur. Thus, amantadine and bisindolylmaleimide appear to inhibit the virus life-cycle at similar points. However, it is well established that amantadine has no effect on replication of influenza B viruses at micromolar concentrations because the function of M2 is replaced by a different protein (NB) in influenza B viruses [see Lamb & Pinto (1997
) for a review]. Our experiments with influenza B virus clearly show that bisindolylmaleimide inhibits replication of this virus in addition to influenza A virus. Overall, our results demonstrate that amantadine and bisindolylmaleimide appear to have different targets in the entry pathway and that bisindolylmaleimides represents a new class of influenza virus entry inhibitors. Although we cannot exclude an effect on fusion or nuclear import at the present time, we feel that the most likely point at which bisindolylmaleimide has its inhibitory effect is in the process of endocytosis.
Several groups have suggested a role for PKC in endocytosis. Activators of PKC such as phorbol esters stimulate receptor-mediated endocytosis of the HIV receptors CD4 and CXCR4 36-fold (Pelchen-Matthews et al., 1993 ; Signoret et al., 1997
), and in the case of CD4 appear to divert receptor from the endosomal recycling pathway to a late-endosomal compartment (Pelchen-Matthews et al., 1993
). Phorbol esters are thought to promote endocytosis either by increasing PKC-dependent phosphorylation to the internalization motif located in the cytoplasmic tail of the receptor (Signoret et al., 1997
) or by promoting endosomeendosome fusion (Aballay et al., 1999
). In many cases, the increased levels of endocytosis induced by PMA can be abrogated by addition of PKC inhibitors such as staurosporine or calphostin C. PKC inhibitors have also been shown to block the endocytosis of certain hormone receptors into cells, although the mechanism of action is incompletely understood (Ferrari et al., 1999
). More specifically, a role for one specific isotype of PKC (PKC
) has been shown in lysosome-targeted endosomes. PKC
is targeted to late endosomes, and a dominant negative mutant of PKC
severely inhibits endocytosis by the lysosome-targeted pathway but not the recycling pathway (Sanchez et al., 1998
).
It remains to be determined whether bisindolylmaleimide drugs have effects on entry of other viruses in addition to influenza virus. Based on a predicted site of action in the endocytic pathway, it is possible that bisindolylmaleimide I will be inhibitory for other pH-dependent viruses such as vesicular stomatitis virus or Semliki Forest virus. Experiments to address effects of bisindolylmaleimide on the endocytosis of these viruses are currently in progress. One of the most effective ways of preventing virus infection and disease is to prevent the initial entry of virions into their target cells (Dimitrov, 1997 ; Marsh & Pelchen-Matthews, 1994
). Further studies on the precise mechanism of action of bisindolylmaleimide with respect to virus entry will significantly add not only to our knowledge of virus entry into cells and possible antiviral therapy, but also to our understanding of the cellular mechanisms of endocytosis.
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Acknowledgments |
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Footnotes |
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References |
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Received 19 June 2000;
accepted 4 August 2000.