HPV18 E1^E4 is assembled into aggresome-like compartment and involved in sequestration of viral oncoproteins.

Papillomavirus is the etiological agent for warts and several squamous carcinomas. Skin cancer induced by cottontail rabbit papillomavirus was the first animal model for virus-induced carcinogenesis. The target organ of the virus infection is stratified epithelium and virus replication is tightly regulated by the differentiation program of the host cell. E1^E4 protein is a viral gene product, and although it is considered to be involved in the control of virus replication, little is known about the biological role. We found that HPV18 E1^E4 was assembled into an aggresome-like compartment and was involved in sequestration of virus oncoproteins, which might contribute to the differentiation-dependent lifecycle of papillomavirus.


INTRODUCTION
Papillomavirus is a small virus containing a double-stranded circular DNA as its genome (zur Hausen, 2002). Genomic DNA of typical papillomavirus, human papillomavirus type 16 (HPV16) or HPV18 is ca. 8 kb long and coding six regulatory genes (E1, E2, E4, E5, E6, E7) and two structural genes (L1, L2). Papillomaviruses are found in almost all mammals and also in amniotes. The virus infects to stratified epithelium organ, such as cutaneous or mucosal membrane, and the infection causes various types of hyperplasia. It is known that the infections of some types of papillomaviruses occasionally induce malignant tumors. The cancer formation by the infection of cottontail rabbit papillomavirus (CRPV) was the first animal model of virus-induced carcinogenesis (Campo, 2002).
The replication of papillomavirus is regulated by the differentiation program of the host cell (Doorbar, 2005). The target cell of the virus infection is basal cell of stratified epithelium, in which the virus replication maintains latent status. Cell division of the infected basal cell produces a daughter cell, and the daughter cell is moved to the surface region of the epithelium and proceeds to differentiate. Virus gene expression and genome replication are enhanced in accordance with the cell differentiation, and the productive replication occurs in fully differentiated cells (Sakakibara et al., 2013). The regulatory mechanism of the differentiation-dependent viral replication remains largely unknown.
A variety of mRNAs are produced by alternative splicing in HPV (Schwartz, 2013). About E4 gene, 5 region of E1 is jointed to E4 coding sequence by RNA splicing, then the gene product contains five amino acid residues of E1 at the N-terminus of the protein coded by E4 ORF, which is called "E1ˆE4". By the analysis of the specimens obtained from infected individuals and animals, the expression level of E1ˆE4 appeared to be intense in differentiated layers of the infected lesions (Sterling et al., 1993;Doorbar et al., 1997), suggesting that E1ˆE4 is involved in the productive stage of viral replication. It was reported on CRPV that the E1ˆE4 was required for the viral DNA amplification and the late protein expressions (Peh et al., 2004). E1ˆE4s of HPV16 and HPV31 were reported to be involved in viral genome amplification and cell cycle maintenance in S-phase of differentiated cells (Nakahara et al., 2005;Wilson et al., 2005). HPV16 E1ˆE4 was also reported to be required for viral genome maintenance in undifferentiated basal cells (Nakahara et al., 2005). There was a paper describing that HPV18 E1ˆE4 was participated in viral genome amplification and the late gene expression in differentiated cells, although it was not involved in the viral genome maintenance or the S-phase maintenance of differentiated cells (Wilson et al., 2007). With these findings, E1ˆE4 could be considered to play a role in productive phase of virus replication.
Several biological and biochemical properties of E1ˆE4 were reported previously. HPV16 E1ˆE4 interacts with cytokeratins and collapses the cytokeratin networks spreading in the cytoplasm (Doorbar et al., 1991). Phosphorylation of HPV16 E1ˆE4 by extracellular signal-regulated kinase (ERK) was reported to cause conformational change of E1ˆE4 and promote the interaction with cytokeratins (Wang et al., 2009).
The expression of E1ˆE4 of HPV16 orHPV18 induces G2/M cell cycle arrest (Davy et al., 2002;Nakahara et al., 2002) and the interaction between the E1ˆE4 and Cyclin A/B has been proposed to be involved in the cell cycle arrest (Davy et al., 2005(Davy et al., , 2006. HPV16 E1ˆE4 was also reported to be involved in RNA processing through its association with E4-DEAD box protein (E4-DBP), a putative RNA helicase (Doorbar et al., 2000), in RNA metabolism (Bell et al., 2007), and in mitochondrial function (Raj et al., 2004). There was a report that HPV1 E4 induced the redistribution of nuclear domain 10 (ND10) body, which is a candidate site of the HPV genome replication (Roberts et al., 2003). These biological properties of E1ˆE4 might be involved in the HPV lifecycle, however, their precise roles in virus replication remain to be elucidated.
There is a self-association motif in the C-terminal region of E1ˆE4, and E1ˆE4s form aggregates in the cytoplasm through the motifs (Bryan et al., 1998). It was reported that the aggregate had amyloid-like structure (McIntosh et al., 2008). Several viruses were reported to utilize cytoplasmic aggregates called as"aggresome"for their replication (Wileman, 2007). Although the biological significance of the aggregate formed by E1ˆE4 was unknown, it might contribute to HPV lifecycle.
"Aggresome" was originally defined as a cytoplasmic compartment in which misfolded proteins are assembled (Johnston et al., 1998). Accumulation of misfolded proteins is toxic for cell viability as in the cases of neurological disorders including Parkinson's, Alzheimer's, and Huntchington's diseases. To counteract the toxicity, misfolded proteins are refolded into native structure or eliminated by molecular chaperones or proteasomes, respectively. However, aggregated proteins exhibit resistance to proteolysis. The aggregates are assembled at microtubule organizing center (MTOC) region and form "aggresome", for which the dyneindependent retrograde transport along microtubules is involved. Aggresomes contain polyubiquitinated proteins, molecular chaperones, and histone deacetylase 6 (HDAC6), and are wrapped in vimentin cage. It is considered that aggresomes activate autophagy pathway and they are processed in autophagy-dependent manner (Kopito, 2000).
In order to investigate E1ˆE4 function, we searched for cellular factors that interact with 18E1ˆE4 protein, and vimentin was identified as a candidate. We also found the 18E1ˆE4 aggregates were wrapped with vimentin as "aggresomes." In this report, we present the structure of 18E1ˆE4 aggregate and its possible role in HPV replication.

CELL CULTURE, TRANSFECTION
HeLa, CV1 and 293T cells were maintained with Dulbecco's modified minimal essential medium (DMEM) supplemented with 10% fetal bovine serum. The cells were transfected with plasmid DNA (5 μg) and herring sperm DNA (5 μg; Roche Diagnostics, GmbH, Mannheim, Germany) by a standard calcium phosphate coprecipitation method.

YEAST TWO-HYBRID SYSTEM
We used ProQuest TM Two-Hybrid System (Invitrogen TM , Life Technologies, Corp, Carlsbad, CA, USA). 18E1ˆE4 cDNA was cloned into pPC86 vector. For cDNA library, we used ProQuest TM Human Fetal Brain cDNA Library (Invitrogen TM , Life Technologies, Corp., Carlsbad, CA, USA). Screening was performed by following manufacturer's instruction.

IMMUNOPRECIPITATION AND IMMUNOBLOT
Total cell lysates were prepared with triple detergent lysis buffer [150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate] supplemented with protease Frontiers in Microbiology | Virology inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) and 1 mM DTT. The cell lysates were centrifuged at 14,000 rpm for 10 min at 4 • C, and the supernatants were used for immunoprecipitation and immunoblot. The supernatants were used as soluble fractions in several experiments. The pellets were resuspended in 2× SDS sample buffer [0.125 M Tris-HCl (pH6.8), 4% SDS, 0.2 M DTT, 20% glycerol, 0.001% bromophenol blue] and used as insoluble fractions. In our experiment, 10 μg of protein could be obtained from ca. 1 × 10 4 cells as soluble fraction. For immunoblot analysis, 10 μg of soluble fraction was loaded into each lane. It was not feasible to measure the protein concentration of insoluble fraction, therefore the portion equivalent to 1 × 10 4 cells was loaded into each lane.

IMMUNOFLUORESCENCE ANALYSIS
For IFA, the cells on cover glasses were fixed with 4% paraformaldehyde (PFA) at room temperature for 5 min or cold methanol (for γ-tubulin staining) at −20 • C for 20 min, permeabilized with 0.1% Nonidet P-40/phosphate buffered saline (PBS) followed by blocking with 5% non-fat dry milk. The samples were incubated with each primary antibodies diluted as manufacturer's instruction. Alexa Fluor ® 488 or 546 labeled secondary antibodies were purchased commercially (Molecular Probes ® , Life Technologies Corp., Carlsbad, CA, USA). Fluorescence microscope (Axiovert200 and AxioVision; Carl Zeiss Microscopy GmbH, Jena, Germany) and confocal laser microscope (TCS SP2 AOBS, Leica Microsystems GmbH, Wetzlar, Germany) were used for analysis.

INTERACTION BETWEEN HPV18 E1ˆE4 AND VIMENTIN PROTEINS
To investigate the biological function of HPV E1ˆE4, we searched for cellular factors that interact with HPV18 E1ˆE4 protein (18E1ˆE4). For screening, we used the yeast two-hybrid assay with 18E1ˆE4 as the bait. Among several factors identified from screening, we focused on vimentin, a cytoskeletal protein categorized as a type III intermediate filament. It is known that vimentin is involved in various cellular events, including cell division and signal transduction (Ivaska et al., 2007); therefore, we considered that the interaction between 18E1ˆE4 and vimentin might induce a modification of the cellular structure or function to adapt it in favor of virus replication.
The interaction between 18E1ˆE4 and vimentin was confirmed by the in vitro binding assay ( Figure 1A). We could detect weak but significant interaction between GST-tagged 18E1ˆE4 and vimentin obtained by in vitro translation, indicating the direct binding of 18E1ˆE4 to vimentin. Similar binding activity was also detected between HPV11 E1ˆE4 (11E1ˆE4) and vimentin ( Figure 1A).
Next, we examined the interaction between endogenous vimentin and ectopically expressed 18E1ˆE4 in 293T cells. For the experiment, a FLAG epitope-tag was added at the N-terminus of 18E1ˆE4. The FLAG-18E1ˆE4 was immunoprecipitated with anti-FLAG antibody, and then co-precipitated vimentin was detected by immunoblotting analysis. As shown in Figure 1B, 18E1ˆE4 could interact with endogenous vimentin. Intracellular localizations of 18E1ˆE4 and vimentin were analyzed with CV1 cells, monkey kidney epithelial cells negative for papillomavirus infection. In control cells, vimentin showed filamentous distribution throughout the cytoplasm ( Figure 1C). The ectopically expressed 18E1ˆE4 formed aggregates in cytoplasm, as reported previously ( Figure 1C; Nakahara et al., 2002). In 18E1ˆE4-expressing cells, vimentin was co-localized at the E1ˆE4 aggregates. 11E1ˆE4 could also form aggregates with vimentin ( Figure 1C). The fine localization of 18E1ˆE4 and vimentin was examined with confocal microscopic analysis, and it was found that the aggregate was wrapped by vimentin ( Figure 1D). These results indicated that 18E1ˆE4 and vimentin were associated in vivo, and suggested that 18E1ˆE4 recruited vimentin to its aggregates through this interaction.

E1ˆE4 WAS ASSEMBLED INTO AGGRESOME-LIKE COMPARTMENT
It is known that cytoplasmic aggregates are organized in cells infected with several viruses; the aggregate is called an"aggresome" (Wileman, 2007). Aggresomes are structures assembled close to the MTOC. They contain molecular chaperones, ubiquitinated www.frontiersin.org proteins, proteasomes, and HDAC6, and are wrapped with a vimentin cage (Rodriguez-Gonzalez et al., 2008). 18E1ˆE4 formed aggregates on the periphery of a nucleus and was associated with vimentin as shown in Figures 1C,D, raising the possibility that the E1ˆE4 proteins were assembled in an aggresome-like compartment.
We examined the intracellular localizations of 40 kDa heat shock protein (Hsp40), HDAC6, and p62, all of which were known to be assembled in the aggresome (Johnston, 2006). From immunofluorescence analysis of CV1 cells, it appeared that these factors were co-localized with the 18E1ˆE4-containing aggregates (Figure 2A). Because ubiquitinated proteins have been known to be recruited to the aggresome (Johnston, 2006), their localizations were also analyzed using anti-ubiquitin antibody. As shown in Figure 2B, ubiquitinated proteins were accumulated in the 18E1ˆE4 aggregates. These observations indicated that the 18E1ˆE4 aggregate had an aggresome-like composition. These results suggested that 18E1ˆE4 formed an aggresome-like compartment, called "18E1ˆE4-aggresome" hereafter.
It is considered that aggresomes are assembled by recruiting their components by retrograde transport through microtubules and are located close to MTOC. We analyzed the localization of γ-tubulin, a component of MTOC ( Figure 2C). In control cells, γ-tubulin appeared at the centrosome as small dots in the perinuclear region. In the cells expressing 18E1ˆE4, γ-tubulin was FIGURE 2 | Intracellular localization of "aggresome"-associated factors. (A) Co-localization of endogenous Hsp40, HDAC6, or p62 (green) with FLAG-18E1ˆE4 (red) in CV1 cells. Nuclei were stained with DAPI. Control shows mock transfected cells. (B) Ubiquitinated proteins (Ub, green) were accumulated in FLAG-18E1ˆE4 aggregates (red) in CV1 cells. (C) γ-tubulin was localized at centrosomes in control cells (control). In FLAG-18E1ˆE4 expressing CV1 cells, γ-tubulin (γ-tub, green) was co-localized at the E1ˆE4 (red). For (A-C), the co-localizations could be observed in most of the cells (≥90%) in that 18E1ˆE4 aggregates were detected. (D) The cell lysates were obtained from the 293T cells transfected with FLAG-18E1ˆE4 expression plasmid. The lysates were used for immunoprecipitation with anti-FLAG antibody, and then the precipitates were analyzed by immunoblot with anti-γ-tubulin antibody.
co-localized at the 18E1ˆE4-aggresome, and the normal centrosome could not be detected in those cells, suggesting that 18E1ˆE4aggresome formation disrupted the normal centrosome or MTOC structure.
The finding that γ-tubulin was co-localized at the 18E1ˆE4aggresome urged us to investigate the interaction between γtubulin and 18E1ˆE4. 18E1ˆE4 with a FLAG-epitope tag at its N-terminus was expressed in 293T cells, and anti-FLAG antibody was used for immunoprecipitation of 18E1ˆE4-containing complexes. The complexes were analyzed by immunoblot detection with anti-γ-tubulin ( Figure 2D). The result indicated the interaction between 18E1ˆE4 and γ-tubulin, which might be involved in the co-localization of γ-tubulin at the 18E1ˆE4-aggresome as observed in Figure 2C.

DYNEIN-DEPENDENT FORMATION OF 18E1ˆE4 AGGRESOME
Misfolded/ubiquitinated proteins are connected to dynein, a motor protein, the association of which is mediated by HDAC6 as a linker molecule (Johnston, 2006). This complex is transported along microtubule filaments to the proximate region of MTOC and forms an aggresome (Kawaguchi et al., 2003). Nocodazole treatment interferes with the polymerization of microtubules and prevents aggresome formation.
Nocodazole treatment of normal HeLa cells induced early M-phase cell cycle arrest and the cells were round (control, Figure 3A). In contrast, 18E1ˆE4-expressing cells were flat (18E1ˆE4, Figure 3A). We reported that 18EˆE4 expression induced G2/M cell cycle arrest and accumulation of aneuploid cells (≥4N; Nakahara et al., 2002), suggesting that the cells were maintained in S and G2 phases of the cell cycle. By nocodazole treatment, the formation of 18E1ˆE4-aggresome was significantly inhibited and small aggregates of 18E1ˆE4 were broadly distributed in the cytoplasm, indicating that the assembly of 18E1ˆE4aggresome required functional microtubule networks. We could detect γ-tubulin in 18E1ˆE4 small aggregates in nocodazoletreated cells (Figure 3B), suggesting that 18E1ˆE4 associated with γ-tubulin in cytoplasm and assembled it to an 18E1ˆE4-aggresome in a microtubule-dependent manner.
A similar experiment was performed with a dynein inhibitor, ciliobrevin D ( Figure 3C). Ciliobrevin D treatment strongly suppressed E1ˆE4-aggresome formation, indicating that dyneindependent transport was involved in E1ˆE4-aggresome formation.
The effect of an HDAC6 inhibitor, tubacin, was also tested ( Figure 3D). HDAC6 is important for aggresome formation by loading the cargo containing misfolded/ubiquitinated proteins onto a dynein motor (Kawaguchi et al., 2003). Tubacin treatment disrupted the E1ˆE4-aggresome and small aggregates containing 18E1ˆE4 were detected in the cytoplasm, as in the cases of nocodazole and ciliobrevin D treatments.
These results suggested that the 18E1ˆE4-aggresome was assembled by dynein-dependent retrograde transport along microtubule filaments.

PROTEASOME INHIBITOR AUGMENTED E1ˆE4-AGGRESOME FORMATION
In the cytoplasmic region, proteasomes are located around the centrosome, close to cytoskeletal networks and on the surface of the endoplasmic reticulum (ER), and the centrosome region is considered as the major site for proteasome-dependent proteolysis, called the proteolysis center (Wójcik and DeMartino, 2003). It was reported that inhibition of proteasome function accelerated aggresome formation in the centrosome region (Johnston et al., 1998), which is considered as one of the hallmarks of aggresomes.
We examined the effect of MG132, a proteasome inhibitor, on cells expressing 18E1ˆE4, and found that MG132 treatment augmented 18E1ˆE4-aggresome formation ( Figure 4A). This observation was consistent with the idea that 18E1ˆE4 formed aggresome-like compartment.
www.frontiersin.org  The expression levels of 18E1ˆE4 were examined in MG132treated cells. As reported previously (Nakahara et al., 2002), most of 18E1ˆE4 was found in the insoluble fraction of cell lysate (Figure 4B), which was corresponding to 18E1ˆE4-aggresome formation. With MG132, 18E1ˆE4 in the insoluble fraction was increased significantly, reflecting the augmentation of aggresome formation. Surprisingly, 18E1ˆE4 in the soluble fraction was also increased, suggesting that some portion of 18E1ˆE4 was processed in proteasome-dependent manner ( Figure 4B).
18E1ˆE4 proteins were assembled into aggresomes as insoluble fraction of cell lysate, indicating that the factors recruited to 18E1ˆE4-aggresomes might be sequestrated as insoluble materials. As shown in Figures 2C,D, γ-tubulin was associated with 18E1ˆE4 and recruited to the aggresomes. We examined the effect of 18E1ˆE4 expression on the protein levels of γ-tubulin ( Figure 4C). The amounts of soluble γ-tubulin were reduced by 18E1ˆE4 expression. On the contrary, those in the insoluble fraction were increased, suggesting that γ-tubulin was sequestrated into the 18E1ˆE4-aggresome as insoluble material, which might reduce active fraction of γ-tubulin and disturb normal centrosome/MTOC formation as shown in Figure 2C.

18E1ˆE4 AGGRESOME WAS INVOLVED IN THE TURN OVER OF HPV ONCOPROTEINS
As described above, 18E1ˆE4 could sequestrate γ-tubulin in the aggresome. In considering the involvement of 18E1ˆE4aggresome in HPV replication, we examined the possibility that the aggresome contributed to sequestration of other viral proteins.
In CV1 cells, FLAG-epitope tagged 18E5, 18E6, or 18E7 was expressed with or without 18E1ˆE4, and then the expression level was monitored by immunoblotting analysis (Figure 5A). Although the expression of E5 was not affected, those of E6 and E7 in the soluble fraction were significantly reduced by 18E1ˆE4 and accumulation of those proteins in insoluble material was observed. This result suggested that E6 and E7 were sequestrated in 18E1ˆE4aggresomes. Nocodazole treatment blocked the effect of 18E1ˆE4 (Figure 5B), suggesting that the 18E1ˆE4-aggresome formation was involved in sequestration of 18E6 and18E7. www.frontiersin.org In the cells expressing 18E1ˆE4, E6 and E7 were co-localized at 18E1ˆE4-aggresomes ( Figure 5C). The localization of 18E5 was not altered by 18E1ˆE4 expression. These observations indicated that major viral oncoproteins, E6 and E7, were recruited to the 18E1ˆE4-aggresome and sequestrated in insoluble materials.

DISCUSSION
It was reported that ectopically expressed HPV E1ˆE4 formed aggregates in cytoplasm (Doorbar et al., 1991), although the function of the aggregate remained to be clarified. In this paper, we described that 18E1ˆE4 was assembled into an aggresomelike compartment (18E1ˆE4-aggresome) and was involved in the sequestration of viral oncoproteins.

AGGRESOME-LIKE COMPARTMENT FORMATION BY 18E1ˆE4.
We found that 18E1ˆE4 interacted with vimentin and recruited it to the 18E1ˆE4 aggregates (Figure 1), which inspired us to consider that 18E1ˆE4 was assembled into an aggresome-like compartment because aggresomes are known to be wrapped by vimentin.
There is a report that 16E1ˆE4 could interact with cytokeratins 8/18 (CK8/18) but not with vimentin (Wang et al., 2004). We therefore analyzed the interaction between 18E1ˆE4 and endogenous vimentin both in vivo and in vitro (Figures 1B,C), although they used an in vitro binding assay with recombinant vimentin and 16E1ˆE4. The different experimental condition could be the cause of the controversial observations.

Frontiers in Microbiology | Virology
Aggresomes are assembled to process misfolded/ubiquitinated proteins that are not well handled by the ubiquitin-proteasome pathway or the chaperone-dependent refolding system (Goldberg, 2003). It is known that aggresomes incorporate molecular chaperones, ubiquitinated proteins, p62 and HDAC6 (Rodriguez-Gonzalez et al., 2008). We confirmed that these molecules were recruited to the 18E1ˆE4-aggregate (Figures 2A,B). This observation strongly suggested that 18E1ˆE4 aggregate had an aggresomelike structure.
Aggresome formation is dependent on microtubules and dynein. Microaggregates of misfolded proteins are transported to MTOC along microtubules in a dynein-dependent manner (Johnston, 2006). Dynein is a motor protein and microaggregates are linked to dynein through HDAC6 (Kawaguchi et al., 2003). We examined the effects of nocodazole, an inhibitor of microtubule polymerization; ciliobrevin D, a dynein inhibitor; and tubacin, a HDAC6 inhibitor, on 18E1ˆE4 aggregate formation, and found that all of the inhibitors could efficiently interfere with aggregate formation (Figure 3). This result supported the possibility that 18E1ˆE4 was assembled in the aggresome-like compartment, 18E1ˆE4-aggresome. We are currently investigating a role of the interaction between 18E1ˆE4 and vimentin in aggresome formation.

DISRUPTION OF MTOC BY 18E1ˆE4
Aggresomes are known to be assembled close to MTOC (Johnston et al., 1998). We examined the localization of γ-tubulin, a component of MTOC, in 18E1ˆE4-expressing cells, and found that it was co-localized at the 18E1ˆE4-aggresome ( Figure 2C). Direct interaction was found between 18E1ˆE4 and γ-tubulin (Figure 2D), by which γ-tubulin might be recruited to the E1ˆE4-aggresome. Even though nocodazole treatment inhibited E1ˆE4-aggresome formation, colocalization of 18E1ˆE4 and γ-tubulin could be detected ( Figure 3B). It was also found that regular centrosome or MTOC formation was disrupted in 18E1ˆE4 expressing cells ( Figure 2C). Proper assembly of MTOC is essential for mitotic events (Bettencourt-Dias and Glover, 2007), and the disturbance of MTOC formation by 18E1ˆE4 might contribute to the G2/M cell cycle arrest induced by 18E1ˆE4.

POSSIBLE ROLE OF 18E1ˆE4 AGGRESOME IN VIRUS REPLICATION
Although it is known that aggresome formation has a protective role against bacterial and protozoal infections (Wileman, 2007), several viruses are reported to utilize aggresomes for their replication processes (Wileman, 2007). Nucleocytoplasmic large DNA viruses (NCLDV), including poxviruses, African swine fever virus (ASFV), iridoviruses and phycodnaviruses, have been reported to utilize aggresomes as compartments for the accumulation of host and viral proteins, where virus replication and virion assembly are accelerated. It has been proposed that infection with a retrovirus or herpes virus produces an aggresome-like structure in the perinuclear region, which is utilized as a virus assembly site (Wileman, 2007). These findings suggested that the 18E1ˆE4-aggresome had a functional role in virus replication.
As shown in Figures 2C,D, 18E1ˆE4 bound to γ-tubulin and recruited it to aggresome-like compartment. This sequestration of γ-tubulin might cause disruption of normal centrosome/MTOC organization. We considered that the 18E1ˆE4-aggresome might be involved in sequestration of other viral proteins, especially of the viral oncoproteins. We examined the effect of 18E1ˆE4 on the expression levels of 18E5, 18E6, and 18E7 ( Figure 5A). Although the expression level of E5 did not altered by 18E1ˆE4, those of E6 and E7 in soluble fraction were severely reduced. E6 and E7 were found in insoluble fraction and co-localized at 18E1ˆE4-aggresomes (Figures 5A,C). These observations suggested that 18E1ˆE4 sequestrated E6 and E7 into the inactive aggregate and reduced active fractions of them. We could not detect direct binding activity of 18E1ˆE4 to 18E6 or 18E7 (data not shown), and therefore it will be necessary to clarify the mechanism by which E6 and E7 are recruited to the aggresome.
Most 18E6 and 18E7 are partitioned in soluble fraction as shown in Figure 5A. The amounts of these proteins in soluble fraction were significantly reduced by 18E1ˆE4 expression, although those in insoluble fraction were increased modestly. The result suggested that 18E1ˆE4 expression reduced the total amounts of these oncoproteins in the cells possibly by accelerating their turn over. We are now investigating the effect of 18E1ˆE4 expression on total amounts of the viral oncoproteins.
In lesions infected with cutaneous-type HPVs, HPV1, HPV4, and HPV63, E1ˆE4 aggregate could be detected in upper layers of the warts as intracytoplasmic inclusion bodies (Egawa, 1994). In the case of HPV16 infection, it was reported that inclusion bodies of E1ˆE4 were found in differentiated layers of cervical intraepithelial neoplasia grade 1 (CIN1) lesions (Doorbar et al., 1997;Doorbar, 2005). These observations suggest that the E1ˆE4aggresome functions in the upper layers of the infected lesion.
Here we propose a model of E1ˆE4 function in viral replication. In basal and parabasal cells of HPV-infected lesions, viral oncoproteins, E6 and E7, are expressed from the viral early promoter. This suppresses cell differentiation and promotes cell proliferation (Nguyen et al., 2003;Ueno et al., 2006), which is required for expanding the population of infected cells. As cellular differentiation progresses, the viral late promoter is activated and directs the expression of E1ˆE4. E1ˆE4 causes G2/M cell cycle arrest and activates endoreduplication (Nakahara et al., 2005). This cellular condition favors genome amplification and gene expression of the virus. Then the high-level expression of E1ˆE4 induces aggregate formation in upper layers of the lesion, where the E1ˆE4-aggresome sequestrates E6 and E7, suppresses their inhibitory effect on cellular differentiation and induces terminal differentiation. Terminal differentiation is required for capsid protein expression and virion assembly, although the underlying mechanism remains unknown (Sakakibara et al., 2013).
It was reported that the formation of E1ˆE4 aggregates disrupted cytokeratin networks and might be helpful for virion egress from keratinized cells (Doorbar et al., 1991). This idea is very attractive for an E1ˆE4 function, and it is important to verify these E1ˆE4 functions in an animal infection model, histological analysis of human samples, or an organotypic raft culture system. www.frontiersin.org