BACKGROUND
3c proteases are the main proteolytic enzymes of picornaviruses.
these enzymes catalyze the processing of polyproteins yielding intermediate and mature viral proteins .
in addition to this major function, 3c proteases can digest host cell proteins.
the cleavage of transcription and translation factors  <cit> , histones  <cit> , cytoskeletal proteins  <cit> , and cell antiviral immunity factors  <cit>  by 3c proteases suppresses the host cell functions and induces its death.
individual expression of 3c proteases of enterovirus  <dig>  <cit> , poliovirus  <cit> , and coxsackievirus  <cit>  induces effects similar to those observed in viral infections.
this advances these enzymes as critical cytotoxic factors of picornaviruses.
in all cases when the cytotoxic effect of 3c proteases was described, these enzymes induced cell death via the caspase-dependent apoptotic pathway  <cit> .
no data on the cytotoxic effect of 3c protease of human hepatitis a virus , which is the only proteolytic enzyme on the virus, are currently available.
at the same time, 3cpro is known to digest host cell proteins of the same functional classes as other targets of 3c proteases.
these proteins include poly-binding protein   <cit> , poly-binding protein  <dig>   <cit> , and mitochondrial factors of cell innate immunity mavs and trif  <cit> .
thus, the induction of apoptosis by 3cpro similar to other 3c proteases could be expected.
however, this study presents the first demonstration that the 3c protease of human hepatitis a virus, unlike other picornaviral 3c proteases, induces caspase-independent cell death.
the 3cpro-induced cell death is accompanied by the accumulation of cytoplasmic vacuoles and depends on the enzyme catalytic activity.
these vacuoles have unique properties and are formed from several organelle types of the endosomal/lysosomal compartment.
the data obtained indicate that 3cpro induces caspase-independent cell death with previously undocumented features.
RESULTS
expression of 3cpro and its catalytically inactive variant
monoclonal cell lines of human lung adenocarcinoma a <dig> tet-off advanced and human lung carcinoma calu- <dig> tet-off advanced constitutively expressing transactivator protein tta were established .
plasmid pbi-egfp was used as the expression vector.
it drives the expression of target and reporter genes under the control of a tta-responsive bidirectional promoter.
enhanced green fluorescent protein  was used as a reporter to identify cells carrying the vector after transient transfection.
protease 3c of human hepatitis a virus  was expressed in intact  and catalytically inactive  variants.
the enzyme was inactivated by a cys172 → ala substitution, which was shown to suppress 3cpro proteolytic activity  <cit> .
the accumulation of 3cpro transcripts was confirmed after the transfection of a <dig> and calu- <dig> cell lines with pbi-egfp/3c and pbi-egfp/3cmut .figure  <dig>
reverse pcr transcription analysis and dynamics of egfp-positive cell accumulation in cultures.
a. transcripts were detected 48 h after a <dig> and calu- <dig> transfection with pbi-egfp , pbi-egfp/3c, encoding intact 3c protease , and pbi-egfp/3cmut, encoding catalytically inactive 3c protease .
glyceraldehyde 3-phosphate dehydrogenase  was used as a reference gene.
b. the proportions of egfp-positive cells were counted in a <dig> and calu- <dig> cultures 144 h post-transfection  with pbi-egfp/3c  and pbi-egfp .
presented values were averaged for at least two independent experiments including four measurements each.
the confidence interval was calculated at 95%.
c. 3cpro-expressing cells 48 h p.t.
normal , cells with normal morphology; rounded/blebbed , round and shrunk cells with smooth or blebbed plasma membrane; vacuolated , vacuolated cells.
d. proportions of egfp-positive cells of different morphology in control  and experimental  cultures.
the values were averaged for at least two independent experiments.
two hundred cells were counted in three replicates for each time point.
the confidence interval was calculated at 95%.
individual expression of 3cpro gene induces morphological changes and cell death
to investigate the effect of 3cpro, a <dig> and calu- <dig> cells were transiently transfected with pbi-egfp/3c  and pbi-egfp lacking the 3cpro gene .
control cultures a549/mock and calu-1/mock demonstrated gradual accumulation of egfp-positive cells .
by the end of the observation period , their proportion amounted to about 30% of total culture cells.
most egfp-positive cells retained the morphology typical for this cell line throughout the observation period .
the proportion of egfp-positive cells in experimental cultures a549/3cpro and calu-1/3cpro reached the maximum 48 h p.t.
and decreased to 1-2% 72 h post-transfection .
only single egfp-positive cells were observed 96 h p.t.
.
a substantial fraction of egfp-positive cells in experimental cultures demonstrated an altered morphology.
there were cells that became round and shrunk, while their plasma membrane could remain smooth or became blebbed .
the morphology of these cells resembled that of apoptotic cells.
in addition, there were cells that remained spread but contained a lot of cytoplasmic vacuoles.
the proportion of different morphological types of egfp-positive cells varied with time .
twenty-four hours p.t., most cells retained normal morphology.
fourty-eight hours p.t., a significant fraction of cells  became vacuolated.
the proportion of vacuolated cells remained unaltered up to 72 h p.t.
the majority of egfp-positive cells had round shape 96 h p.t., while the cells with normal morphology could hardly be observed.
time-lapse microscopy of the cell cultures at 5-min intervals for 96 h demonstrated that vacuolated cells remained spread even many hours after numerous vacuoles emerged.
shortly before detachment of vacuolated cells from the substrate, the vacuoles disappeared and cells became indistinguishable from the round and shrunk cells .
the data obtained suggest that 3cpro induces cell death.
death of a substantial fraction of cells was accompanied by the accumulation of cytoplasmic vacuoles.
finally, all 3cpro-expressing cells before detachment acquired similar apoptotic-like morphology .
catalytically inactive 3cpro induces no cell vacuolization and death
following transformation with pbi-egfp/3cmut, the accumulation dynamics of egfp-positive cells and their morphology were the same in experimental a549/3cmut and calu-1/3cmut and control a549/mock and calu-1/mock cultures .
thus, cell vacuolization and death depend on the proteolytic activity of 3cpro.
3cpro-induced cell death does not include caspase activation
the type of 3cpro-induced cell death was evaluated by the state of the nuclei and chromatin, functional state of mitochondria, phosphatidylserine localization, and activation of caspases in 3cpro-expressing cells.
control a549/mock and calu-1/mock cells had normal nuclear morphology and chromatin state throughout the observation period .
most round/shrunk a549/3cpro and calu-1/3cpro cells demonstrated chromatin condensation and karyorrhexis 48 h p.t., while only some vacuolated cells showed nuclear deformation and partial chromatin condensation at the sites of contact with the vacuoles .
after 72 h, most vacuolated cells demonstrated pronounced chromatin condensation and their nuclei looked fragmented .
at the same time, vacuolated cells demonstrated no plasma membrane blebbing and remained spread.
chromatin in vacuolated and round/shrunk cells was not stained by propidium iodide, which indicated their intact plasma membrane and non-necrotic death.figure  <dig>
characteristics of 3cpro-expressing cells.
calu-1/3cpro culture is shown 72 h p.t.
chromatin was visualized by dna-intercalating dye hoechst  <dig> .
active caspases were detected by fluorescent agent flica .
egfp-positive cells with normal morphology are indicated by cyan arrows.
egfp-positive vacuolated and round cells with condensed chromatin and inactive caspases are indicated by white and green arrows, respectively.
egfp-positive cells with active caspases are indicated by red arrows.
cells demonstrating chromatin condensation and karyorrhexis but not accumulating egfp are indicated by yellow arrows.
egfp is the channel for gfp fluorescence; merged channel shows overlapping signals.
mitochondria were examined and their membrane potential  was evaluated using rhodamine  <dig> , a potential-dependent fluorescent dye  <cit> .
throughout the observation period , mitochondria maintained their mmp and normal size and formed reticulum in control a549/mock and calu-1/mock cells .
the majority of vacuolated cells in a549/3cpro and calu-1/3cpro cultures demonstrated disrupted mitochondrial reticulum and swelling of mmp-maintaining mitochondria 48 h p.t.
.
at the same time, certain vacuolated cells completely or partially retained their mitochondrial reticulum and normal mitochondrial size .
the presence of such cells suggests that initial vacuoles are formed before the changes in mitochondrial state become apparent.
vacuolated cells demonstrated a substantial decrease in the number of mmp-maintaining mitochondria 72 h p.t.
at the same time, the intensity of mitochondrial staining by rh <dig> in most vacuolated cells was lower than in untransformed cells , which likely indicates decreased mmp.
a similar pattern was observed for round cells with smooth plasma membrane .
no rh123-positive mitochondria were observed in cells with blebbed plasma membrane .figure  <dig>
mitochondrial status in calu- <dig> and a <dig> cells expressing 3cpro.
cells maintaining mitochondrial transmembrane potential were visualized using the potential-dependent fluorescent dye rhodamine  <dig>  a549/mock and calu-1/mock cells 48 h p.t.
.
a549/3cpro and calu-1/3cpro cells: vacuolated, 48 h p.t.
and 72 h p.t.
; round, 72 h p.t.
; with blebbed plasma membrane, 72 h p.t.
.
egfp and rhod are channels for gfp and rhodamine  <dig> fluorescence, respectively; egfp + rhod channel shows overlapping signals.
chromatin condensation, karyorrhexis, decreased mmp, and reduced cell volume are markers of apoptosis, which is usually accompanied by phosphatidylserine exposure on the outer layer of the plasma membrane and induction of caspases  <cit> .
however, phosphatidylserine was undetectable on the surface of a549/3cpro and calu-1/3cpro cells .
the pan-caspase fluorescent reagent flica revealed only single cells with induced caspases 48 h and 72 h p.t.
.
remarkably, all such cells were round, whereas no caspase activation was detected in vacuolated cells and cells with blebbing.
cell culturing with the caspase inhibitor z-vad-fmk did not prevent vacuolization and had no notable effect on the survival of 3cpro-expressing cells.
it should be noted that a549/3cpro and calu-1/3cpro as well as a549/mock and calu-1/mock cultures included a minor fraction of egfp-negative round cells demonstrating chromatin condensation, karyorrhexis, and caspase activation, which indicate their death through caspase-dependent apoptosis .
the capacity of both cell lines to follow the caspase-dependent pathway has been additionally demonstrated using the standard apoptosis-inducing drug doxorubicin .
the data obtained demonstrate that 3cpro induces caspase-independent cell death in both studied lines despite their susceptibility to caspase-dependent apoptosis.
inhibitors of intracellular cysteine proteases and rip <dig> kinase do not suppress 3cpro-induced cell death
cytoplasmic vacuolization accompanies alternative non-apoptotic cell death pathways.
some of them are driven by cysteine proteases cathepsins and calpains  <cit> , which can substitute caspases in their absence  <cit> .
however, the incubation of a549/3cpro and calu-1/3cpro cells with the inhibitor of lysosomal cysteine proteases z-fa-fmk neither prevented vacuolization nor had a notable effect on the survival of transfected cells .
a similar pattern was observed for the caspase inhibitor z-vad-fmk, high concentrations of which  block cysteine cathepsins and calpains apart from caspases  <cit> .
apparently, cathepsins and calpains as well as caspases are not solely responsible for 3cpro-induced cell death.
necroptosis, the best studied subtype of programmed necrosis, is predominantly mediated by rip <dig>  <cit> .
however, the specific rip <dig> inhibitor necrostatin- <dig>  <cit>  had no effect on the vacuolization and survival of a549/3c and calu-1/3c cells either .
3cpro-induced vacuoles are bounded by a single bilayer and contain multimembrane structures
ultrastructural analysis using electron microscopy demonstrated that cytoplasmic vacuoles are bounded by a single bilayer membrane in a549/3cpro and calu-1/3cpro cells .
a portion of vacuoles had no clear boundary and were surrounded by structures resembling endoplasmic reticulum  or golgi cisternae .
certain vacuoles contained multimembrane structures and small vesicles; however, the majority of vacuoles contained no significant quantities of membranous material .
the vacuoles often neighbored smaller vesicles also bounded by a single membrane layer and largely containing no membranous inclusions .
such vesicles were also numerous in the vicinity of the plasma membrane, which points to endocytosis as the mechanism of their formation.
morphological analysis suggests that the vacuoles might be formed from components of the endosomal/lysosomal, er, or golgi compartments.figure  <dig>
ultrastructural analysis of 3cpro-induced vacuoles.
cytoplasmic vacuoles in a549/3cpro and calu-1/3cpro cells were analyzed by electron microscopy.
a. vacuoles bounded by a single membrane layer.
b. vacuoles with blurred boundaries.
c. vesicles neighboring vacuoles.
vesicles inside vacuoles are indicated by triangles; multimembrane formations, m; small vesicles neighboring vacuoles, *.
scale,  <dig>  μm.
mitochondria, endoplasmic reticulum, and golgi are not involved in vacuole formation
the origin and properties of cytoplasmic vacuoles were further studied using a set of vectors encoding fluorescent proteins targeted to different cellular compartments.
mitochondria were studied using a cyan fluorescent protein fused to the mitochondrial targeting signal from human cytochrome c oxidase subunit viii , which visualizes mitochondrial membrane irrespective of the functional state of these organelles  <cit> .
mito-cfp accumulated in mitochondria of 3cpro-expressing cells, but was not observed in vacuolar membranes or lumen .figure  <dig>
statuses of mitochondria, endoplasmic reticulum, and golgi in vacuolated cells.
localization of fluorescent proteins targeted to mitochondria , endoplasmic reticulum , and trans-golgi network  in control a549/mock, calu-1/mock  and vacuolated a549/3cpro, calu-1/3cpro  cells, respectively.
er was visualized using a red fluorescent protein fused to the er retention signal sekdel   <cit> .
in control a549/mock and calu-1/mock cells, er-rfp was found in the granules characteristic for er .
in vacuolated a549/3cpro and calu-1/3cpro cells er-rfp demonstrated homogeneous cytoplasmic localization but was not found in vacuolar lumen and membranes .
a similar pattern was observed for staining by the low-molecular-weight er-tracker blue-white dpx dye specific for er membranes .
the data obtained indicate abnormal er function and its possible degradation.
components of the trans-golgi network were visualized using rfp with the golgi retention signal of human β- <dig> -galactosyltransferase.
in both control culture cells and vacuolated ones, this fluorescent protein accumulated in distinct organelles but not in the vacuolar lumen and membranes .
thus, the data obtained suggest that the vacuoles originate from organelles other than mitochondria, er, and trans-golgi network.
vacuoles have endosomal/lysosomal origin
lysosomes and late endosomes were visualized using the lysosomal-associated membrane protein  <dig>  fused with the fluorescent protein mkate <dig> .
l1-mkate <dig> localized in vesicles dispersed or clustered in the cytoplasm of control a549/mock and calu-1/mock cells 48 h p.t.
three cell types could be recognized in a549/3cpro and calu-1/3cpro cells.
type i cells had no vacuoles and the localization of l1-mkate <dig> was similar to that in control cultures.
type ii cells had single small l1-mkate2-positive vacuoles associated with clusters of l1-mkate2-positive vesicles .
type iii cells contained numerous vacuoles and l1-mkate <dig> localized to the membranes of all vacuoles .
notice that the cytoplasm of vacuolated cells contained far less l1-mkate2-positive vesicles compared to vacuole-free cells.
overall, the data obtained indicate that the vacuoles originate from organelles of the endosomal/lysosomal compartment and that clusters of these organelles can be the sites of vacuole formation.figure  <dig>
origin of 3cpro-induced vacuoles.
vacuolated a549/3cpro and calu-1/3cpro cells expressing fusion proteins l1-mkate <dig> 48 h p.t.
and 72 h p.t.
, rab5-ecfp , rab7-eyfp , dsred-rab <dig> , dsred-rab <dig> , dsred-rab7dn , dsred-rab5dn , and lc3-rfp  72 h p.t.
dominant-negative mutants of gtpases are indicated by “dn”.
normally lamp <dig> goes through early endosomes on the way to late endosomes and lysosomes  <cit> .
since 3cpro induces abnormal function of the endosomal/lysosomal compartment, mislocalization of l1-mkate <dig> cannot be excluded.
hence, the observed l1-mkate <dig> localization cannot unambiguously point to the organelle type of the endosomal/lysosomal compartment that gave rise to the vacuoles.
in this context, cytoplasmic gtpases of the rab family specifically associated with membranes of different endosome types  <cit>  were used to visualize individual organelle populations.
fusion proteins rab5-eyfp, rab7-ecfp, dsred-rab <dig>  and dsred-rab <dig> served as markers of early endosomes, late endosomes/lysosomes, and endosomes recycling to the trans-golgi network and plasma membrane, respectively  <cit> .
in vacuolated a549/3cpro and calu-1/3cpro cells, all these markers accumulated in the vacuolar membranes.
in addition, the vacuolar membrane-associated vesicles with the fusion proteins have been revealed .
at the same time, the overexpression of these proteins per se in control a549/mock and calu-1/mock cells induced no vacuole formation or other morphology alterations .
it should be noted that the incubation of a549/3cpro and calu-1/3cpro cells with colchicine, an inhibitor of polymerization of microtubules that mediate the transport of organelles of the endosomal compartment, did not suppress vacuole formation .
thus, 3cpro-induced vacuole formation does not depend on the microtubular activity.
the data obtained indicate that several organelle types of the endosomal/lysosomal compartment are involved in the vacuole formation.
overexpression of dominant-negative rab <dig> and rab <dig> does not suppress vacuole formation
the relationship between 3cpro-induced vacuolization and rab <dig> and rab <dig> functions was evaluated using their dominant-negative mutants rab5/n133i  and rab7/t22n  fused with the fluorescent protein dsred.
the expression level of these gtpases evaluated from dsred fluorescence intensity varied significantly from cell to cell.
accordingly, the cells demonstrating top fluorescence levels were selected for analysis.
a549/3cpro and calu-1/3cpro cells with high levels of rab5/n133i and rab7/t22n proved to contain the vacuoles, and both gtpases were associated with the vacuolar membranes .
the size and morphology of these vacuoles was indistinguishable from those in cells expressing 3cpro alone.
autophagy is not essential for 3cpro-induced vacuolization and cell death
the role of autophagosomes in the 3cpro-induced vacuolization was evaluated using the lc <dig> protein  fused to fluorescent protein mrfp.
the fusion protein was not accumulated in the membranes but localized diffusely in the vacuolar lumen .
this indicates the involvement of autophagosomes in vacuole formation.
autophagosome-mediated formation of vacuoles is observed after using some agents that impair autophagy.
in some cases, such impairments proved to result from the constitutive activation of the erk1/ <dig> signaling pathway  <cit> .
however, the incubation of 3cpro-expressing cells with the inhibitors of this pathway  did not suppress the vacuolization and had no noticeable effect on cell survival.
likewise, no noticeable effect was observed after cell exposure to 3-methyladenine, an inhibitor of class  <dig> phosphatidylinositol 3-kinase and autophagosome formation .
thus, the data obtained indicate that the 3cpro-induced vacuolization and cell death do not depend on autophagy.
vacuolization is not essential for 3cpro-induced cell death
cell incubation with the inhibitor of vacuolar atpase bafilomycin a <dig> , which is often used to suppress autophagy  <cit> , completely blocked the vacuolization but had no effect on cell death .
since bafa <dig> blocks not only autolysosome formation but also endosome fusion  <cit> , this finding in the context of no effect of 3-methyladenine indicates again that the vacuolization results from the fusion of organelles of the endosomal/lysosomal compartment.
the effect of bafa <dig> suggests another important conclusion: the vacuolization event is not essential for 3cpro-induced cell death.figure  <dig>
effect of bafilomycin a <dig> on vacuolization.
a <dig> and calu- <dig> cells transfected with pbi-egfp , pbi-egfp/3c  or pbi-egfp/3cmut  and treated by bafilomycin a <dig>  or equal quantity of vehicle  48 h p.t.
3cpro-induced vacuoles do not have properties of degradative organelles
the 3cpro-induced vacuoles carry markers of degradative organells that normally have acidic content and contain active hydrolases  <cit> .
we tested if the vacuoles have the properties of degradative organelles using fluorescent substrate of cathepsin b  and ph-dependent dye .
in all vacuolated cells, the fluorescent product of magic red hydrolysis was detected in individual vesicles, most of which are localized within the vacuoles .
it was not detected in the vacuolar lumen and cytoplasm of vacuolated cells.
this suggests that active lysosomal proteases are not released to the cytoplasm as observed in certain types of caspase-independent cell death.
cell staining with neutral red demonstrated that the vacuolar lumen is not acidic.
at the same time, acidic vesicles were observed within the vacuoles .
most likely, these vesicles contained active cathepsin b. it is of interest that the vesicles with the fluorescent product of magic red hydrolysis and acidic content were largely observed in smaller vacuoles and were nearly always absent in larger ones.
at the same time, such vesicles were missing or sporadic in cells with numerous large vacuoles.
assuming that larger vacuoles have longer lifetime one can propose that the intravacuolar vesicles gradually lose their degradative properties.figure  <dig>
cathepsin b activity and localization of acidic organelles in vacuolated cells.
a549/3cpro and calu-1/3cpro cells 72 h p.t.
after incubation with the fluorescent substrate of cathepsin b magic red   and ph-dependent fluorescent dye neutral red  .
vacuolization is not a consequence of macropinocytosis hyperstimulation
vacuoles lacking the properties of degradative organelles and accumulating lamp <dig> and rab <dig> in their membranes are observed during cell death resulting from hyperstimulation of macropinocytosis.
this cell death pathway was called methuosis  <cit> .
macropinocytic activity was evaluated in a549/3cpro and calu-1/3cpro cells using fluorescent dye lucifer yellow , which cannot penetrate the cell membrane.
ly could be detected in individual vacuoles of single vacuolated cells incubated with the dye for one or several hours  <dig> or 72 h p.t.
cell incubation for 12 h led to ly accumulation in vacuoles of all vacuolated cells .
these data indicate that the vacuoles can accumulate extracellular fluid.
at the same time, the low rate of extracellular fluid accumulation contradicts possible hyperstimulation of macropinocytosis when detectable ly quantities are accumulated in vacuoles in 10– <dig> minutes  <cit> .figure  <dig>
accumulation of lucifer yellow and rac <dig> in vacuolated cells.
a549/3cpro and calu-1/3cpro cells 72 h p.t.
after incubation with fluorescent dye lucifer yellow   or co-expressing dominant-negative mutant of gtpase rac <dig> .
the constitutive induction of gtpase rac <dig> is a prerequisite for the hyperstimulation of macropinocytosis in methuosis  <cit> .
rac <dig> inhibition or overexpression of dominant-negative rac <dig> prevents macropinocytosis hyperstimulation and methuosis  <cit> .
in our case, the expression of the fusion protein yfp-rac <dig> did not prevent the vacuolization even in cells with high yfp-rac <dig> levels .
similarly, incubation of a549/3cpro and calu-1/3cpro cells with filipin, which prevents the formation of macropinosomes  <cit> , had no effect on the size and number of vacuoles, as well as on cell viability .
overall, the data obtained suggest that 3cpro-induced vacuolization is not a consequence of macropinocytosis hyperstimulation and that the cell death does not follow the methuosis pathway.
discussion
this study presents the first demonstration that 3c protease of human hepatitis a virus can induce cell death dependent on the enzyme proteolytic activity and accompanied by the formation of cytoplasmic vacuoles through the fusion of organelles of the endosomal/lysosomal compartment.
individual expression of many picornaviral proteins  <cit>  including 3c proteases of enterovirus  <dig>  <cit> , poliovirus  <cit> , and coxsackievirus  <cit>  was shown to induce cell death.
in all described cases, 3c protease-induced cell death depended on the enzyme activity and was recognized as caspase-dependent apoptosis according to both morphological and biochemical indications.
cell death induced by 3c protease of human hepatitis a virus  also showed some properties typical for apoptosis: disruption of the mitochondrial reticulum, swelling of mitochondria, loss of mitochondrial membrane potential, chromatin condensation, and karyorrhexis at the background of plasma membrane integrity.
.
considering that the nuclear fragments were sandwiched between the vacuoles, one can propose that the mechanical impact of vacuoles could induce the changes in nuclear morphology.
3cpro-induced cell death was not accompanied by phosphatidylserine externalization and caspase activation; likewise, it was not blocked by the pan-caspase inhibitor z-vad-fmk.
a similar set of characters has been previously reported for alternative non-apoptotic cell death types  <cit> .
thus, 3cpro induces caspase-independent cell death unlike other picornaviral 3c proteases.
a significant fraction of cells undergoing 3cpro-induced death demonstrated cytoplasmic vacuolization before mitochondrial depolarization and other cell death signs.
vacuolization of different intracellular compartments is a marker of certain pathological states  <cit>  and accompanies caspase-independent cell death  <cit> .
vacuoles are formed from different organelles in different types of caspase-independent cell death, which allows us to use the properties and origin of vacuoles to identify cell death type.
we have shown that the 3cpro-induced vacuolization did not affect endoplasmic reticulum, mitochondria, and golgi and was not blocked by necrostatin- <dig>  an inhibitor of rip <dig> kinase.
this allowed us to exclude the paraptotic and necroptotic pathways  <cit> .
the factors of cell death and cytoplasmic vacuole formation include autophagy abnormalities  <cit> .
the lumen of 3cpro-induced vacuoles proved to contain lc <dig> protein, which testifies to the involvement of autophagosomes in the formation of vacuoles.
cell death and vacuolization associated with abnormal autophagy can result from the constitutive activation of the raf-mek-erk1/ <dig> cascade  <cit> .
nevertheless, the used inhibitors of mek kinases had no effect on cell vacuolization and death.
the suppression of autophagy by 3-methyladenine  also had no effect on the action of 3cpro.
at the same time, the inhibitor of vacuolar atpase bafilomycin a <dig>  completely blocked the vacuolization but not the cell death.
although bafa <dig> is commonly used autophagy suppressor, it also inhibits fusion of endocytic organelles  <cit> .
in the context of the 3-methyladenine effect, the action of bafa <dig> is likely mediated by the inhibition of endocytosis pathway rather than autophagy suppression.
thus, the data obtained demonstrate that autophagy is not essential for 3cpro-induced cell vacuolization and death, and vacuolization is a morphological indication but not the cause of 3cpro-induced cell death.
the vacuolar membranes simultaneously accumulated markers of different types of endocytic organelles, fluorescent proteins fused with the lamp <dig> sorting signal or with gtpases rab <dig>  rab <dig>  rab <dig>  and rab <dig>  under normal homeostatic conditions, the cells have organelles simultaneously containing rab <dig> and rab <dig> , rab <dig> and lamp <dig> , as well as rab <dig> and rab <dig> ; rab11-containing endosomes usually represent a separate population  <cit> .
thus, 3cpro-induced vacuoles are formed from several organelle types of the endosomal/lysosomal compartment.
the vacuolization of endocytic organelles has been shown previously in the following cases.
the inhibition of kinases hvps <dig> and pikfyve, which regulate vesicular transport and sorting, leads to the vacuolization of late endosomes but does not involve other organelles  <cit> .
the vacuoles induced by certain bacterial toxins, e.g., vaca from helicobacter pylori, epsilon toxin from clostridium perfringens, and cards toxin from mycoplasma pneumoniae, also have an endosomal/lysosomal origin  <cit> .
however, the vacuolization involves a single  endosomal type in all known cases.
thus, 3cpro induces the formation of vacuoles with not previously described properties, and all major types of endocytic organelles are involved.
gtpases of the rab family revealed in the vacuolar membranes have many functions in the homeostasis regulation in the endosomal/lysosomal compartment including hetero- and homotypic fusion of endosomes  <cit> .
high levels of constitutively active forms of rab <dig> and rab <dig> are known to induce fusion and vacuolization of early and late endosomes, while the prevalence of their nonfunctional forms conversely blocks endosomal fusion  <cit> .
activity of rab gtpases is required for the vacuolization process of endosomal/lysosomal organelles induced by certain bacterial toxins  <cit> .
at the same time, 3cpro-induced vacuolization is not suppressed by the overexpression of nonfunctional rab <dig> or rab <dig> variants, and thus does not depend on the function of these gtpases.
some properties of 3cpro-induced vacuoles  draw them together with the vacuoles resulting from hyperstimulation of macropinocytosis in methuosis  <cit> .
the formation of giant macropinosomes in methuosis requires the constitutive activation of gtpase rac <dig> and is prevented by the overexpression of its nonfunctional variant or in the presence of filipin, an inhibitor of clathrin-independent endocytosis  <cit> .
however, the overexpression of nonfunctional rac <dig> or cell incubation with filipin did not prevent 3cpro-induced cell vacuolization and death.
hence, 3cpro-induced vacuolization does not depend on macropinocytic activity and cell death does not follow the methuosis pathway.
overall, 3cpro-induced vacuoles have previously undescribed features, and thus 3cpro-induced cell death cannot be assigned to any currently known type of caspase-independent cell death accompanied by vacuolization.
in our opinion, it is of primary interest if the effects of 3cpro described in this work are observed in cells infected by human hepatitis a virus.
apoptotic cell death was observed in studied cases of infection by cytopathogenic forms of the virus  <cit> .
apparently, 3cpro is not the main cytotoxic factors in these cases.
at the same time, a number of viruses can induce either apoptotic or caspase-independent cell death depending on infection conditions.
for instance, caspase-independent cell death is triggered in abortive poliovirus infection  <cit>  or at high infectious dose of west nile virus  <cit> .
accordingly, 3cpro can become the main cytotoxic factor under certain infection conditions.
the cytopathic effect of many viruses is manifested as specific changes in cellular compartments preceding cell death  <cit> .
morphological changes of cells infected with cytopathogenic forms of hepatitis a virus and other picornaviruses include swelling of er cisternae and formation of multilayer membrane structures, vesicular structures, and cytoplasmic vacuoles  <cit> .
certain cytopathic effects of hepatitis a virus are due to viral proteins 2b, 2c, and 2bc  <cit> .
at the same time, no data on the factors underlying the emergence of cytoplasmic vacuoles are currently available.
the results obtained in this work allow us to propose that 3cpro mediates the development of cytopathic cell morphology and the formation of vacuoles in hepatitis a virus infection.
the involvement of picornaviral 3c proteases in cytopathic vacuolization has not been reported previously.
at the same time, a variety of proteins of other viruses can induce vacuolization.
most of them have no enzyme activity, e.g., large surface protein of hepatitis b virus  <cit> , env protein of murine leukemia virus  <cit> , capsid protein vp <dig> of simian vacuolating virus  <dig>  <cit> , oncoproteins e <dig> and e <dig> of human papillomavirus  <cit> , and a38l protein of vaccinia virus  <cit> .
on the other hand, the vacuolization effect was described for ns <dig> proteases of certain flaviviruses.
.
cytoplasmic vacuolization is typical for flaviviral infections by hepatitis c virus  <cit> , west nile virus  <cit> , and dengue virus  <cit>  and is also observed after individual expression of their ns <dig> proteases  <cit> .
the case of bovine viral diarrhea virus  is of particular interest since the infection-induced vacuoles have been characterized.
the properties of vacuoles induced by bvdv and 3cpro are similar: both originate from endosomal/liposomal organelles, have nonacidic content, and their formation is autophagy-independent  <cit> .
note that cytoplasmic vacuolization was observed only after infection with a cytopathogenic biotype, which differs from non-cytopathogenic one by elevated expression of protease ns <dig>  <cit> .
thus, analysis of published and obtained data indicates that the observed effect of 3cpro can reflect the participation of this enzyme in the development of cytopathic morphology of infected cells.
in this context, the mechanism of 3cpro impact on the endosomal/lysosomal compartment and the role of this protease in the cytopathic effect of the human hepatitis a virus require further investigation.
CONCLUSIONS
analysis of the cytotoxic effect of 3c protease of human hepatitis a virus allowed us to demonstrate 3cpro-induced cell death independent of caspases and accompanied by accumulation of cytoplasmic vacuoles.
the cytotoxic and vacuolization effects of 3cpro depend on its catalytic activity.
3cpro-induced vacuoles have unique properties and originate from several organelle types of the endosomal/lysosomal compartment.
the data obtained indicate that 3cpro induces caspase-independent cell death with previously unreported morphological characters.

 3c proteases are the main proteolytic enzymes of picornaviruses. these enzymes catalyze the processing of polyproteins yielding intermediate and mature viral proteins. in addition to this major function, 3c proteases can digest host cell proteins. the cleavage of transcription and translation factors, histones, cytoskeletal proteins, and cell antiviral immunity factors by 3c proteases suppresses the host cell functions and induces its death. individual expression of 3c proteases of enterovirus, poliovirus, and coxsackievirus induces effects similar to those observed in viral infections. this advances these enzymes as critical cytotoxic factors of picornaviruses. in all cases when the cytotoxic effect of 3c proteases was described, these enzymes induced cell death via the caspase-dependent apoptotic pathway. no data on the cytotoxic effect of 3c protease of human hepatitis a virus, which is the only proteolytic enzyme on the virus, are currently available. at the same time, 3cpro is known to digest host cell proteins of the same functional classes as other targets of 3c proteases. these proteins include poly-binding protein, poly-binding protein, and mitochondrial factors of cell innate immunity mavs and trif. thus, the induction of apoptosis by 3cpro similar to other 3c proteases could be expected. however, this study presents the first demonstration that the 3c protease of human hepatitis a virus, unlike other picornaviral 3c proteases, induces caspase-independent cell death. the 3cpro-induced cell death is accompanied by the accumulation of cytoplasmic vacuoles and depends on the enzyme catalytic activity. these vacuoles have unique properties and are formed from several organelle types of the endosomal/lysosomal compartment. the data obtained indicate that 3cpro induces caspase-independent cell death with previously undocumented features.  expression of 3cpro and its catalytically inactive variant monoclonal cell lines of human lung adenocarcinoma a tet-off advanced and human lung carcinoma calu- tet-off advanced constitutively expressing transactivator protein tta were established. plasmid pbi-egfp was used as the expression vector. it drives the expression of target and reporter genes under the control of a tta-responsive bidirectional promoter. enhanced green fluorescent protein was used as a reporter to identify cells carrying the vector after transient transfection. protease 3c of human hepatitis a virus was expressed in intact and catalytically inactive variants. the enzyme was inactivated by a cys172 → ala substitution, which was shown to suppress 3cpro proteolytic activity. the accumulation of 3cpro transcripts was confirmed after the transfection of a and calu- cell lines with pbi-egfp/3c and pbi-egfp/3cmut.figure reverse pcr transcription analysis and dynamics of egfp-positive cell accumulation in cultures. a. transcripts were detected 48 h after a and calu- transfection with pbi-egfp, pbi-egfp/3c, encoding intact 3c protease, and pbi-egfp/3cmut, encoding catalytically inactive 3c protease. glyceraldehyde 3-phosphate dehydrogenase was used as a reference gene. b. the proportions of egfp-positive cells were counted in a and calu- cultures 144 h post-transfection with pbi-egfp/3c and pbi-egfp. presented values were averaged for at least two independent experiments including four measurements each. the confidence interval was calculated at 95%. c. 3cpro-expressing cells 48 h p.t. normal, cells with normal morphology; rounded/blebbed, round and shrunk cells with smooth or blebbed plasma membrane; vacuolated, vacuolated cells. d. proportions of egfp-positive cells of different morphology in control and experimental cultures. the values were averaged for at least two independent experiments. two hundred cells were counted in three replicates for each time point. the confidence interval was calculated at 95%. individual expression of 3cpro gene induces morphological changes and cell death to investigate the effect of 3cpro, a and calu- cells were transiently transfected with pbi-egfp/3c and pbi-egfp lacking the 3cpro gene. control cultures a549/mock and calu-1/mock demonstrated gradual accumulation of egfp-positive cells. by the end of the observation period, their proportion amounted to about 30% of total culture cells. most egfp-positive cells retained the morphology typical for this cell line throughout the observation period. the proportion of egfp-positive cells in experimental cultures a549/3cpro and calu-1/3cpro reached the maximum 48 h p.t. and decreased to 1-2% 72 h post-transfection. only single egfp-positive cells were observed 96 h p.t.. a substantial fraction of egfp-positive cells in experimental cultures demonstrated an altered morphology. there were cells that became round and shrunk, while their plasma membrane could remain smooth or became blebbed. the morphology of these cells resembled that of apoptotic cells. in addition, there were cells that remained spread but contained a lot of cytoplasmic vacuoles. the proportion of different morphological types of egfp-positive cells varied with time. twenty-four hours p.t., most cells retained normal morphology. fourty-eight hours p.t., a significant fraction of cells became vacuolated. the proportion of vacuolated cells remained unaltered up to 72 h p.t. the majority of egfp-positive cells had round shape 96 h p.t., while the cells with normal morphology could hardly be observed. time-lapse microscopy of the cell cultures at 5-min intervals for 96 h demonstrated that vacuolated cells remained spread even many hours after numerous vacuoles emerged. shortly before detachment of vacuolated cells from the substrate, the vacuoles disappeared and cells became indistinguishable from the round and shrunk cells. the data obtained suggest that 3cpro induces cell death. death of a substantial fraction of cells was accompanied by the accumulation of cytoplasmic vacuoles. finally, all 3cpro-expressing cells before detachment acquired similar apoptotic-like morphology. catalytically inactive 3cpro induces no cell vacuolization and death following transformation with pbi-egfp/3cmut, the accumulation dynamics of egfp-positive cells and their morphology were the same in experimental a549/3cmut and calu-1/3cmut and control a549/mock and calu-1/mock cultures. thus, cell vacuolization and death depend on the proteolytic activity of 3cpro. 3cpro-induced cell death does not include caspase activation the type of 3cpro-induced cell death was evaluated by the state of the nuclei and chromatin, functional state of mitochondria, phosphatidylserine localization, and activation of caspases in 3cpro-expressing cells. control a549/mock and calu-1/mock cells had normal nuclear morphology and chromatin state throughout the observation period. most round/shrunk a549/3cpro and calu-1/3cpro cells demonstrated chromatin condensation and karyorrhexis 48 h p.t., while only some vacuolated cells showed nuclear deformation and partial chromatin condensation at the sites of contact with the vacuoles. after 72 h, most vacuolated cells demonstrated pronounced chromatin condensation and their nuclei looked fragmented. at the same time, vacuolated cells demonstrated no plasma membrane blebbing and remained spread. chromatin in vacuolated and round/shrunk cells was not stained by propidium iodide, which indicated their intact plasma membrane and non-necrotic death.figure characteristics of 3cpro-expressing cells. calu-1/3cpro culture is shown 72 h p.t. chromatin was visualized by dna-intercalating dye hoechst. active caspases were detected by fluorescent agent flica. egfp-positive cells with normal morphology are indicated by cyan arrows. egfp-positive vacuolated and round cells with condensed chromatin and inactive caspases are indicated by white and green arrows, respectively. egfp-positive cells with active caspases are indicated by red arrows. cells demonstrating chromatin condensation and karyorrhexis but not accumulating egfp are indicated by yellow arrows. egfp is the channel for gfp fluorescence; merged channel shows overlapping signals. mitochondria were examined and their membrane potential was evaluated using rhodamine, a potential-dependent fluorescent dye. throughout the observation period, mitochondria maintained their mmp and normal size and formed reticulum in control a549/mock and calu-1/mock cells. the majority of vacuolated cells in a549/3cpro and calu-1/3cpro cultures demonstrated disrupted mitochondrial reticulum and swelling of mmp-maintaining mitochondria 48 h p.t.. at the same time, certain vacuolated cells completely or partially retained their mitochondrial reticulum and normal mitochondrial size. the presence of such cells suggests that initial vacuoles are formed before the changes in mitochondrial state become apparent. vacuolated cells demonstrated a substantial decrease in the number of mmp-maintaining mitochondria 72 h p.t. at the same time, the intensity of mitochondrial staining by rh in most vacuolated cells was lower than in untransformed cells, which likely indicates decreased mmp. a similar pattern was observed for round cells with smooth plasma membrane. no rh123-positive mitochondria were observed in cells with blebbed plasma membrane.figure mitochondrial status in calu- and a cells expressing 3cpro. cells maintaining mitochondrial transmembrane potential were visualized using the potential-dependent fluorescent dye rhodamine a549/mock and calu-1/mock cells 48 h p.t.. a549/3cpro and calu-1/3cpro cells: vacuolated, 48 h p.t. and 72 h p.t.; round, 72 h p.t.; with blebbed plasma membrane, 72 h p.t.. egfp and rhod are channels for gfp and rhodamine fluorescence, respectively; egfp + rhod channel shows overlapping signals. chromatin condensation, karyorrhexis, decreased mmp, and reduced cell volume are markers of apoptosis, which is usually accompanied by phosphatidylserine exposure on the outer layer of the plasma membrane and induction of caspases. however, phosphatidylserine was undetectable on the surface of a549/3cpro and calu-1/3cpro cells. the pan-caspase fluorescent reagent flica revealed only single cells with induced caspases 48 h and 72 h p.t.. remarkably, all such cells were round, whereas no caspase activation was detected in vacuolated cells and cells with blebbing. cell culturing with the caspase inhibitor z-vad-fmk did not prevent vacuolization and had no notable effect on the survival of 3cpro-expressing cells. it should be noted that a549/3cpro and calu-1/3cpro as well as a549/mock and calu-1/mock cultures included a minor fraction of egfp-negative round cells demonstrating chromatin condensation, karyorrhexis, and caspase activation, which indicate their death through caspase-dependent apoptosis. the capacity of both cell lines to follow the caspase-dependent pathway has been additionally demonstrated using the standard apoptosis-inducing drug doxorubicin. the data obtained demonstrate that 3cpro induces caspase-independent cell death in both studied lines despite their susceptibility to caspase-dependent apoptosis. inhibitors of intracellular cysteine proteases and rip kinase do not suppress 3cpro-induced cell death cytoplasmic vacuolization accompanies alternative non-apoptotic cell death pathways. some of them are driven by cysteine proteases cathepsins and calpains, which can substitute caspases in their absence. however, the incubation of a549/3cpro and calu-1/3cpro cells with the inhibitor of lysosomal cysteine proteases z-fa-fmk neither prevented vacuolization nor had a notable effect on the survival of transfected cells. a similar pattern was observed for the caspase inhibitor z-vad-fmk, high concentrations of which block cysteine cathepsins and calpains apart from caspases. apparently, cathepsins and calpains as well as caspases are not solely responsible for 3cpro-induced cell death. necroptosis, the best studied subtype of programmed necrosis, is predominantly mediated by rip. however, the specific rip inhibitor necrostatin- had no effect on the vacuolization and survival of a549/3c and calu-1/3c cells either. 3cpro-induced vacuoles are bounded by a single bilayer and contain multimembrane structures ultrastructural analysis using electron microscopy demonstrated that cytoplasmic vacuoles are bounded by a single bilayer membrane in a549/3cpro and calu-1/3cpro cells. a portion of vacuoles had no clear boundary and were surrounded by structures resembling endoplasmic reticulum or golgi cisternae. certain vacuoles contained multimembrane structures and small vesicles; however, the majority of vacuoles contained no significant quantities of membranous material. the vacuoles often neighbored smaller vesicles also bounded by a single membrane layer and largely containing no membranous inclusions. such vesicles were also numerous in the vicinity of the plasma membrane, which points to endocytosis as the mechanism of their formation. morphological analysis suggests that the vacuoles might be formed from components of the endosomal/lysosomal, er, or golgi compartments.figure ultrastructural analysis of 3cpro-induced vacuoles. cytoplasmic vacuoles in a549/3cpro and calu-1/3cpro cells were analyzed by electron microscopy. a. vacuoles bounded by a single membrane layer. b. vacuoles with blurred boundaries. c. vesicles neighboring vacuoles. vesicles inside vacuoles are indicated by triangles; multimembrane formations, m; small vesicles neighboring vacuoles, *. scale,  μm. mitochondria, endoplasmic reticulum, and golgi are not involved in vacuole formation the origin and properties of cytoplasmic vacuoles were further studied using a set of vectors encoding fluorescent proteins targeted to different cellular compartments. mitochondria were studied using a cyan fluorescent protein fused to the mitochondrial targeting signal from human cytochrome c oxidase subunit viii, which visualizes mitochondrial membrane irrespective of the functional state of these organelles. mito-cfp accumulated in mitochondria of 3cpro-expressing cells, but was not observed in vacuolar membranes or lumen.figure statuses of mitochondria, endoplasmic reticulum, and golgi in vacuolated cells. localization of fluorescent proteins targeted to mitochondria, endoplasmic reticulum, and trans-golgi network in control a549/mock, calu-1/mock and vacuolated a549/3cpro, calu-1/3cpro cells, respectively. er was visualized using a red fluorescent protein fused to the er retention signal sekdel. in control a549/mock and calu-1/mock cells, er-rfp was found in the granules characteristic for er. in vacuolated a549/3cpro and calu-1/3cpro cells er-rfp demonstrated homogeneous cytoplasmic localization but was not found in vacuolar lumen and membranes. a similar pattern was observed for staining by the low-molecular-weight er-tracker blue-white dpx dye specific for er membranes. the data obtained indicate abnormal er function and its possible degradation. components of the trans-golgi network were visualized using rfp with the golgi retention signal of human β- -galactosyltransferase. in both control culture cells and vacuolated ones, this fluorescent protein accumulated in distinct organelles but not in the vacuolar lumen and membranes. thus, the data obtained suggest that the vacuoles originate from organelles other than mitochondria, er, and trans-golgi network. vacuoles have endosomal/lysosomal origin lysosomes and late endosomes were visualized using the lysosomal-associated membrane protein fused with the fluorescent protein mkate. l1-mkate localized in vesicles dispersed or clustered in the cytoplasm of control a549/mock and calu-1/mock cells 48 h p.t. three cell types could be recognized in a549/3cpro and calu-1/3cpro cells. type i cells had no vacuoles and the localization of l1-mkate was similar to that in control cultures. type ii cells had single small l1-mkate2-positive vacuoles associated with clusters of l1-mkate2-positive vesicles. type iii cells contained numerous vacuoles and l1-mkate localized to the membranes of all vacuoles. notice that the cytoplasm of vacuolated cells contained far less l1-mkate2-positive vesicles compared to vacuole-free cells. overall, the data obtained indicate that the vacuoles originate from organelles of the endosomal/lysosomal compartment and that clusters of these organelles can be the sites of vacuole formation.figure origin of 3cpro-induced vacuoles. vacuolated a549/3cpro and calu-1/3cpro cells expressing fusion proteins l1-mkate 48 h p.t. and 72 h p.t., rab5-ecfp, rab7-eyfp, dsred-rab, dsred-rab, dsred-rab7dn, dsred-rab5dn, and lc3-rfp 72 h p.t. dominant-negative mutants of gtpases are indicated by “dn”. normally lamp goes through early endosomes on the way to late endosomes and lysosomes. since 3cpro induces abnormal function of the endosomal/lysosomal compartment, mislocalization of l1-mkate cannot be excluded. hence, the observed l1-mkate localization cannot unambiguously point to the organelle type of the endosomal/lysosomal compartment that gave rise to the vacuoles. in this context, cytoplasmic gtpases of the rab family specifically associated with membranes of different endosome types were used to visualize individual organelle populations. fusion proteins rab5-eyfp, rab7-ecfp, dsred-rab and dsred-rab served as markers of early endosomes, late endosomes/lysosomes, and endosomes recycling to the trans-golgi network and plasma membrane, respectively. in vacuolated a549/3cpro and calu-1/3cpro cells, all these markers accumulated in the vacuolar membranes. in addition, the vacuolar membrane-associated vesicles with the fusion proteins have been revealed. at the same time, the overexpression of these proteins per se in control a549/mock and calu-1/mock cells induced no vacuole formation or other morphology alterations. it should be noted that the incubation of a549/3cpro and calu-1/3cpro cells with colchicine, an inhibitor of polymerization of microtubules that mediate the transport of organelles of the endosomal compartment, did not suppress vacuole formation. thus, 3cpro-induced vacuole formation does not depend on the microtubular activity. the data obtained indicate that several organelle types of the endosomal/lysosomal compartment are involved in the vacuole formation. overexpression of dominant-negative rab and rab does not suppress vacuole formation the relationship between 3cpro-induced vacuolization and rab and rab functions was evaluated using their dominant-negative mutants rab5/n133i and rab7/t22n fused with the fluorescent protein dsred. the expression level of these gtpases evaluated from dsred fluorescence intensity varied significantly from cell to cell. accordingly, the cells demonstrating top fluorescence levels were selected for analysis. a549/3cpro and calu-1/3cpro cells with high levels of rab5/n133i and rab7/t22n proved to contain the vacuoles, and both gtpases were associated with the vacuolar membranes. the size and morphology of these vacuoles was indistinguishable from those in cells expressing 3cpro alone. autophagy is not essential for 3cpro-induced vacuolization and cell death the role of autophagosomes in the 3cpro-induced vacuolization was evaluated using the lc protein fused to fluorescent protein mrfp. the fusion protein was not accumulated in the membranes but localized diffusely in the vacuolar lumen. this indicates the involvement of autophagosomes in vacuole formation. autophagosome-mediated formation of vacuoles is observed after using some agents that impair autophagy. in some cases, such impairments proved to result from the constitutive activation of the erk1/ signaling pathway. however, the incubation of 3cpro-expressing cells with the inhibitors of this pathway did not suppress the vacuolization and had no noticeable effect on cell survival. likewise, no noticeable effect was observed after cell exposure to 3-methyladenine, an inhibitor of class phosphatidylinositol 3-kinase and autophagosome formation. thus, the data obtained indicate that the 3cpro-induced vacuolization and cell death do not depend on autophagy. vacuolization is not essential for 3cpro-induced cell death cell incubation with the inhibitor of vacuolar atpase bafilomycin a, which is often used to suppress autophagy, completely blocked the vacuolization but had no effect on cell death. since bafa blocks not only autolysosome formation but also endosome fusion, this finding in the context of no effect of 3-methyladenine indicates again that the vacuolization results from the fusion of organelles of the endosomal/lysosomal compartment. the effect of bafa suggests another important conclusion: the vacuolization event is not essential for 3cpro-induced cell death.figure effect of bafilomycin a on vacuolization. a and calu- cells transfected with pbi-egfp, pbi-egfp/3c or pbi-egfp/3cmut and treated by bafilomycin a or equal quantity of vehicle 48 h p.t. 3cpro-induced vacuoles do not have properties of degradative organelles the 3cpro-induced vacuoles carry markers of degradative organells that normally have acidic content and contain active hydrolases. we tested if the vacuoles have the properties of degradative organelles using fluorescent substrate of cathepsin b and ph-dependent dye. in all vacuolated cells, the fluorescent product of magic red hydrolysis was detected in individual vesicles, most of which are localized within the vacuoles. it was not detected in the vacuolar lumen and cytoplasm of vacuolated cells. this suggests that active lysosomal proteases are not released to the cytoplasm as observed in certain types of caspase-independent cell death. cell staining with neutral red demonstrated that the vacuolar lumen is not acidic. at the same time, acidic vesicles were observed within the vacuoles. most likely, these vesicles contained active cathepsin b. it is of interest that the vesicles with the fluorescent product of magic red hydrolysis and acidic content were largely observed in smaller vacuoles and were nearly always absent in larger ones. at the same time, such vesicles were missing or sporadic in cells with numerous large vacuoles. assuming that larger vacuoles have longer lifetime one can propose that the intravacuolar vesicles gradually lose their degradative properties.figure cathepsin b activity and localization of acidic organelles in vacuolated cells. a549/3cpro and calu-1/3cpro cells 72 h p.t. after incubation with the fluorescent substrate of cathepsin b magic red and ph-dependent fluorescent dye neutral red. vacuolization is not a consequence of macropinocytosis hyperstimulation vacuoles lacking the properties of degradative organelles and accumulating lamp and rab in their membranes are observed during cell death resulting from hyperstimulation of macropinocytosis. this cell death pathway was called methuosis. macropinocytic activity was evaluated in a549/3cpro and calu-1/3cpro cells using fluorescent dye lucifer yellow, which cannot penetrate the cell membrane. ly could be detected in individual vacuoles of single vacuolated cells incubated with the dye for one or several hours or 72 h p.t. cell incubation for 12 h led to ly accumulation in vacuoles of all vacuolated cells. these data indicate that the vacuoles can accumulate extracellular fluid. at the same time, the low rate of extracellular fluid accumulation contradicts possible hyperstimulation of macropinocytosis when detectable ly quantities are accumulated in vacuoles in 10– minutes.figure accumulation of lucifer yellow and rac in vacuolated cells. a549/3cpro and calu-1/3cpro cells 72 h p.t. after incubation with fluorescent dye lucifer yellow or co-expressing dominant-negative mutant of gtpase rac. the constitutive induction of gtpase rac is a prerequisite for the hyperstimulation of macropinocytosis in methuosis. rac inhibition or overexpression of dominant-negative rac prevents macropinocytosis hyperstimulation and methuosis. in our case, the expression of the fusion protein yfp-rac did not prevent the vacuolization even in cells with high yfp-rac levels. similarly, incubation of a549/3cpro and calu-1/3cpro cells with filipin, which prevents the formation of macropinosomes, had no effect on the size and number of vacuoles, as well as on cell viability. overall, the data obtained suggest that 3cpro-induced vacuolization is not a consequence of macropinocytosis hyperstimulation and that the cell death does not follow the methuosis pathway. discussion this study presents the first demonstration that 3c protease of human hepatitis a virus can induce cell death dependent on the enzyme proteolytic activity and accompanied by the formation of cytoplasmic vacuoles through the fusion of organelles of the endosomal/lysosomal compartment. individual expression of many picornaviral proteins including 3c proteases of enterovirus, poliovirus, and coxsackievirus was shown to induce cell death. in all described cases, 3c protease-induced cell death depended on the enzyme activity and was recognized as caspase-dependent apoptosis according to both morphological and biochemical indications. cell death induced by 3c protease of human hepatitis a virus also showed some properties typical for apoptosis: disruption of the mitochondrial reticulum, swelling of mitochondria, loss of mitochondrial membrane potential, chromatin condensation, and karyorrhexis at the background of plasma membrane integrity.. considering that the nuclear fragments were sandwiched between the vacuoles, one can propose that the mechanical impact of vacuoles could induce the changes in nuclear morphology. 3cpro-induced cell death was not accompanied by phosphatidylserine externalization and caspase activation; likewise, it was not blocked by the pan-caspase inhibitor z-vad-fmk. a similar set of characters has been previously reported for alternative non-apoptotic cell death types. thus, 3cpro induces caspase-independent cell death unlike other picornaviral 3c proteases. a significant fraction of cells undergoing 3cpro-induced death demonstrated cytoplasmic vacuolization before mitochondrial depolarization and other cell death signs. vacuolization of different intracellular compartments is a marker of certain pathological states and accompanies caspase-independent cell death. vacuoles are formed from different organelles in different types of caspase-independent cell death, which allows us to use the properties and origin of vacuoles to identify cell death type. we have shown that the 3cpro-induced vacuolization did not affect endoplasmic reticulum, mitochondria, and golgi and was not blocked by necrostatin- an inhibitor of rip kinase. this allowed us to exclude the paraptotic and necroptotic pathways. the factors of cell death and cytoplasmic vacuole formation include autophagy abnormalities. the lumen of 3cpro-induced vacuoles proved to contain lc protein, which testifies to the involvement of autophagosomes in the formation of vacuoles. cell death and vacuolization associated with abnormal autophagy can result from the constitutive activation of the raf-mek-erk1/ cascade. nevertheless, the used inhibitors of mek kinases had no effect on cell vacuolization and death. the suppression of autophagy by 3-methyladenine also had no effect on the action of 3cpro. at the same time, the inhibitor of vacuolar atpase bafilomycin a completely blocked the vacuolization but not the cell death. although bafa is commonly used autophagy suppressor, it also inhibits fusion of endocytic organelles. in the context of the 3-methyladenine effect, the action of bafa is likely mediated by the inhibition of endocytosis pathway rather than autophagy suppression. thus, the data obtained demonstrate that autophagy is not essential for 3cpro-induced cell vacuolization and death, and vacuolization is a morphological indication but not the cause of 3cpro-induced cell death. the vacuolar membranes simultaneously accumulated markers of different types of endocytic organelles, fluorescent proteins fused with the lamp sorting signal or with gtpases rab rab rab and rab under normal homeostatic conditions, the cells have organelles simultaneously containing rab and rab, rab and lamp, as well as rab and rab; rab11-containing endosomes usually represent a separate population. thus, 3cpro-induced vacuoles are formed from several organelle types of the endosomal/lysosomal compartment. the vacuolization of endocytic organelles has been shown previously in the following cases. the inhibition of kinases hvps and pikfyve, which regulate vesicular transport and sorting, leads to the vacuolization of late endosomes but does not involve other organelles. the vacuoles induced by certain bacterial toxins, e.g., vaca from helicobacter pylori, epsilon toxin from clostridium perfringens, and cards toxin from mycoplasma pneumoniae, also have an endosomal/lysosomal origin. however, the vacuolization involves a single endosomal type in all known cases. thus, 3cpro induces the formation of vacuoles with not previously described properties, and all major types of endocytic organelles are involved. gtpases of the rab family revealed in the vacuolar membranes have many functions in the homeostasis regulation in the endosomal/lysosomal compartment including hetero- and homotypic fusion of endosomes. high levels of constitutively active forms of rab and rab are known to induce fusion and vacuolization of early and late endosomes, while the prevalence of their nonfunctional forms conversely blocks endosomal fusion. activity of rab gtpases is required for the vacuolization process of endosomal/lysosomal organelles induced by certain bacterial toxins. at the same time, 3cpro-induced vacuolization is not suppressed by the overexpression of nonfunctional rab or rab variants, and thus does not depend on the function of these gtpases. some properties of 3cpro-induced vacuoles draw them together with the vacuoles resulting from hyperstimulation of macropinocytosis in methuosis. the formation of giant macropinosomes in methuosis requires the constitutive activation of gtpase rac and is prevented by the overexpression of its nonfunctional variant or in the presence of filipin, an inhibitor of clathrin-independent endocytosis. however, the overexpression of nonfunctional rac or cell incubation with filipin did not prevent 3cpro-induced cell vacuolization and death. hence, 3cpro-induced vacuolization does not depend on macropinocytic activity and cell death does not follow the methuosis pathway. overall, 3cpro-induced vacuoles have previously undescribed features, and thus 3cpro-induced cell death cannot be assigned to any currently known type of caspase-independent cell death accompanied by vacuolization. in our opinion, it is of primary interest if the effects of 3cpro described in this work are observed in cells infected by human hepatitis a virus. apoptotic cell death was observed in studied cases of infection by cytopathogenic forms of the virus. apparently, 3cpro is not the main cytotoxic factors in these cases. at the same time, a number of viruses can induce either apoptotic or caspase-independent cell death depending on infection conditions. for instance, caspase-independent cell death is triggered in abortive poliovirus infection or at high infectious dose of west nile virus. accordingly, 3cpro can become the main cytotoxic factor under certain infection conditions. the cytopathic effect of many viruses is manifested as specific changes in cellular compartments preceding cell death. morphological changes of cells infected with cytopathogenic forms of hepatitis a virus and other picornaviruses include swelling of er cisternae and formation of multilayer membrane structures, vesicular structures, and cytoplasmic vacuoles. certain cytopathic effects of hepatitis a virus are due to viral proteins 2b, 2c, and 2bc. at the same time, no data on the factors underlying the emergence of cytoplasmic vacuoles are currently available. the results obtained in this work allow us to propose that 3cpro mediates the development of cytopathic cell morphology and the formation of vacuoles in hepatitis a virus infection. the involvement of picornaviral 3c proteases in cytopathic vacuolization has not been reported previously. at the same time, a variety of proteins of other viruses can induce vacuolization. most of them have no enzyme activity, e.g., large surface protein of hepatitis b virus, env protein of murine leukemia virus, capsid protein vp of simian vacuolating virus, oncoproteins e and e of human papillomavirus, and a38l protein of vaccinia virus. on the other hand, the vacuolization effect was described for ns proteases of certain flaviviruses.. cytoplasmic vacuolization is typical for flaviviral infections by hepatitis c virus, west nile virus, and dengue virus and is also observed after individual expression of their ns proteases. the case of bovine viral diarrhea virus is of particular interest since the infection-induced vacuoles have been characterized. the properties of vacuoles induced by bvdv and 3cpro are similar: both originate from endosomal/liposomal organelles, have nonacidic content, and their formation is autophagy-independent. note that cytoplasmic vacuolization was observed only after infection with a cytopathogenic biotype, which differs from non-cytopathogenic one by elevated expression of protease ns. thus, analysis of published and obtained data indicates that the observed effect of 3cpro can reflect the participation of this enzyme in the development of cytopathic morphology of infected cells. in this context, the mechanism of 3cpro impact on the endosomal/lysosomal compartment and the role of this protease in the cytopathic effect of the human hepatitis a virus require further investigation.  analysis of the cytotoxic effect of 3c protease of human hepatitis a virus allowed us to demonstrate 3cpro-induced cell death independent of caspases and accompanied by accumulation of cytoplasmic vacuoles. the cytotoxic and vacuolization effects of 3cpro depend on its catalytic activity. 3cpro-induced vacuoles have unique properties and originate from several organelle types of the endosomal/lysosomal compartment. the data obtained indicate that 3cpro induces caspase-independent cell death with previously unreported morphological characters. 
3c proteases, the main proteases of picornaviruses, play the key role in viral life cycle by processing polyproteins. in addition, 3c proteases digest certain host cell proteins to suppress antiviral defense, transcription, and translation. the activity of 3c proteases per se induces host cell death, which makes them critical factors of viral cytotoxicity. to date, cytotoxic effects have been studied for several 3c proteases, all of which induce apoptosis. this study for the first time describes the cytotoxic effect of 3c protease of human hepatitis a virus, the only proteolytic enzyme of the virus. individual expression of 3cpro induced catalytic activity-dependent cell death, which was not abrogated by the pan-caspase inhibitor and was not accompanied by phosphatidylserine externalization in contrast to other picornaviral 3c proteases. 3cpro, apart from other picornaviral 3c proteases, induces caspase-independent cell death, accompanying by cytoplasmic vacuolization. 3c proteasehepatitis a viruscytoplasmic vacuolizationcaspase-independent cell deathissue-copyright-statement© the author 2015 3cpro-induced vacuoles have unique properties and are formed from several organelle types of the endosomal/lysosomal compartment. the data obtained demonstrate previously undocumented morphological characters of the 3cpro-induced cell death, which can reflect unknown aspects of the human hepatitis a virus-host cell interaction. a substantial fraction of dying cells demonstrated numerous non-acidic cytoplasmic vacuoles with not previously described features and originating from several types of endosomal/lysosomal organelles. the lysosomal protein lamp and gtpases rab rab rab and rab were associated with the vacuolar membranes. the cell survival was also not affected by the inhibitors of cysteine proteases and rip kinase, critical enzymes involved in non-apoptotic cell death.
 3c proteases, the main proteases of picornaviruses, play the key role in viral life cycle by processing polyproteins. in addition, 3c proteases digest certain host cell proteins to suppress antiviral defense, transcription, and translation. the activity of 3c proteases per se induces host cell death, which makes them critical factors of viral cytotoxicity. to date, cytotoxic effects have been studied for several 3c proteases, all of which induce apoptosis. this study for the first time describes the cytotoxic effect of 3c protease of human hepatitis a virus, the only proteolytic enzyme of the virus.  individual expression of 3cpro induced catalytic activity-dependent cell death, which was not abrogated by the pan-caspase inhibitor and was not accompanied by phosphatidylserine externalization in contrast to other picornaviral 3c proteases. the cell survival was also not affected by the inhibitors of cysteine proteases and rip kinase, critical enzymes involved in non-apoptotic cell death. a substantial fraction of dying cells demonstrated numerous non-acidic cytoplasmic vacuoles with not previously described features and originating from several types of endosomal/lysosomal organelles. the lysosomal protein lamp and gtpases rab rab rab and rab were associated with the vacuolar membranes. the vacuolization was completely blocked by the vacuolar atpase inhibitor and did not depend on the activity of the principal factors of endosomal transport, gtpases rab and rab as well as on autophagy and macropinocytosis.  3cpro, apart from other picornaviral 3c proteases, induces caspase-independent cell death, accompanying by cytoplasmic vacuolization. 3cpro-induced vacuoles have unique properties and are formed from several organelle types of the endosomal/lysosomal compartment. the data obtained demonstrate previously undocumented morphological characters of the 3cpro-induced cell death, which can reflect unknown aspects of the human hepatitis a virus-host cell interaction. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords 3c proteasehepatitis a viruscytoplasmic vacuolizationcaspase-independent cell deathissue-copyright-statement© the author 2015 