Differential Regulation of Cellular FAM111B by Human Adenovirus C Type 5 E1 Oncogenes

The adenovirus type 5 (HAdV-C5) E1 transcription unit encodes regulatory proteins that are essential for viral replication and transformation. Among these, E1A and E1B-55K act as key multifunctional HAdV-C5 proteins involved in various steps of the viral replication cycle and in virus-induced cell transformation. In this context, HAdV-C5-mediated dysregulations of cellular factors such as the tumor suppressors p53 and pRB have been intensively investigated. However, cellular components of downstream events that could affect infection and viral transformation are widely unknown. We recently observed that cellular FAM111B is highly regulated in an E1A-dependent fashion. Intriguingly, previous reports suggest that FAM111B might play roles in tumorigenesis, but its exact functions are not known to date. Here, we set out to investigate the role of FAM111B in HAdV-C5 infections. We found that (i) FAM111B levels are upregulated early and downregulated late during infection, that (ii) FAM111B expression is differentially regulated, that (iii) FAM111B expression levels depend on the presence of E1B-55K and E4orf6 and that (iv) a FAM111B knockdown increases HAdV-C5 replication. Our data indicate that FAM111B acts as an anti-adenoviral host factor that is involved in host cell defense mechanisms in productive HAdV-C5 infection. Moreover, these findings suggest that FAM111B might play an important role in the host antiviral immune response that is counteracted by HAdV-C5 E1B-55K and E4orf6 oncoproteins.


Introduction
Human adenoviruses (HAdVs) are non-enveloped, linear dsDNA viruses that mostly cause asymptomatic or mild disease in younger and immunocompetent individuals. However, HAdV infections of immunocompromised patients or patients with pre-existing respiratory or cardiac conditions can cause severe disease courses and are serious health issues [1]. Therefore, it is important to thoroughly study basic mechanisms of HAdV infection as well as the interplay between HAdV gene products and host immunity.
In a yet unpublished transcriptome analysis, we found that cellular FAM111B was strongly upregulated upon transduction with the two HAdV-C5 oncogenes E1A and E1B. The exact function of FAM111B, the second and last member of the "family with sequence similarity 111" gene family, is widely unknown. Interestingly though, a recent study demonstrated that FAM111B co-precipitates with HAdV-C5 E1B-55K [2]. FAM111B contains a predicted trypsin-like cysteine/serine peptidase domain, comprising of two subdomains connected by a linker region. Notably, three specific alterations within the linker region were previously found to induce hereditary fibrosing poikiloderma [3]. Mercier et al. showed that FAM111B is important for fibrosis development, which is a key pathological process in a variety of human diseases [3]. The trypsin-like cysteine/serine peptidase domain of FAM111B shares 45% homology with FAM111A. Mutations in this region were identified to induce Kenny-Caffey syndrome and osteocraniostenosis, which are characterized by impaired skeletal development [4,5]. Intriguingly, the 11q12 locus (and the genes encoding FAM111A and FAM111B in this region, specifically) is associated with prostate cancer susceptibility and serves as a signature to discriminate the risk of metastases [6]. In line with this, more and more recent evidence continues to point towards an involvement of FAM111B in tumorigenesis [7][8][9]. Finally, the SV40 virus large T antigen interacts with FAM111A to inhibit viral replication in restrictive cells and FAM111A restricts vaccinia virus (VACV) replication [10][11][12][13]. However, it was also found that SV40 virus still replicates in a restrictive cell line upon FAM111A depletion [10].
In this report, we set out to investigate the role of FAM111B in the context of HAdV-C5 infection and the regulation of this putative cellular oncogene by HAdV-C5 E1 genes.

Cells and Culture Conditions
H1299 (ATCC no. CRL-5803: ATCC Global Bioresource Center, Manassas, Virginia, VA, USA) and A549 (ACC 107; DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were grown in DMEM (Gibco; Carlsbad, CA, USA) supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin. All cells were kept in incubators at 37 • C in 5% CO 2 . FAM111B knockdown cell lines were generated by lentiviral transduction of A549 cells. Transduced cells were maintained under 2 µM puromycin selection. All cells were tested for mycoplasma contamination on a regular basis.

Viruses
H5pg4100 harboring deletions in the E3-coding region served as the wt HAdV-C5 virus [16]. The mutant virus H5pm4149 (4x) harbors four stop codons within the E1B coding region and H5pm4154 (E4orf6-) has a stop codon at P66 of E4orf6 to abrogate translation of the respective proteins [17,18]. Viruses were propagated, titrated and used for infections as described previously [19]. Virus yield was analyzed at indicated time points by quantitative E2A immunofluorescence staining. Viral titers were determined as described before and are represented as the number of fluorescence-forming units (FFU)/µL [20].
All protein extracts were incubated in radioimmunoprecipitation assay (RIPA) lysis buffer on ice for 30 min. Total cell lysates were sonicated in a high-intensity cup horn (Branson Ultrasonics, Brookfield, CT, USA) for 45 s (40 pulses; output, 0.6 and 0.8 impulses/second) before insoluble debris was removed by centrifugation (11,000 rpm, 3 min, 4 • C). Protein concentration was measured photometrically with Bradford reagent (Bio-Rad, Hercules, CA, USA). Equal amounts of total protein were separated on 10% SDS-polyacrylamide gels after denaturation (5× SDS sample buffer, 95 • C, 3 min) and subjected to immunoblotting exactly as previously described [21].

In Vitro Translation and Pulldown Assays
FLAG-tagged FAM111B was in vitro translated with the TNT Coupled Wheat Germ Systems (VWR, Radnor, PA, USA) according to the manufacturer's instruction. In vitrotranslated FLAG-tagged FAM111B was purified by immunoprecipitation (IP) with an ANTI-FLAG M2 Affinity Gel (Sigma-Aldrich). Briefly, FLAG-tagged FAM111B was added to the supplied beads together with TBS buffer and protease inhibitors (1% phenylmethylsulfonyl fluoride (PMSF), 0.1% aprotinin, 1 µg/mL leupeptin, 1 µg/mL pepstatin, 25 mM iodacetamide and 25 mM N-ethylmaleimide) and incubated overnight at 4 • C. An aliquot of FLAG-tagged FAM111B was eluted from the beads using 2× Laemmli buffer as controls. The remaining beads coupled with FLAG-tagged FAM111B were incubated with protein lysates prepared in RIPA buffer as described in the previous section for 2 h at 4 • C. Bound proteins were eluted with 2× Laemmli buffer at 95 • C for 5 min.

FAM111B RNA and Protein Levels Are Upregulated in HAdV-C5-Infections
To analyze whether FAM111B transcript and protein levels are differentially regulated during HAdV-C5 infection, we infected the human lung adenocarcinoma cell line A549, that is widely used in HAdV research, with wt HAdV-C5 [16] and first investigated mRNA levels of FAM111B during infection at different time points post infection ( Figure 1A). FAM111B transcript levels increased at 16-24 h post infection (h p.i.) compared to mock ( Figure 1A) but interestingly dropped at 48 h p.i. ( Figure 1A). These results indicate that FAM111B is regulated at the transcriptional level in response to HAdV-C5 infection, implying that FAM111B might belong to the family of immediate early response genes activated upon HAdV infection.
To check whether this is also reflected in FAM111B protein levels, we analyzed FAM111B protein expression by immunofluorescence. We stained HAdV-and mockinfected A549 cells with specific antibodies detecting FAM111B and the viral protein DBP (E2A), which formed nuclear spherical structures representing the sites of viral replication [22]. FAM111B showed diffuse nuclear staining in mock-infected cells excluding the nucleoli and a weak cytoplasmic signal, while FAM111B intensity is reduced in HAdV-C5infected cells ( Figure 1B). However, these findings are in contrast to the elevated FAM111B transcript levels at 24 h p.i. (Figure 1A). We therefore performed a time-resolved analysis to investigate FAM111B protein expression levels during a course of 8-72 h p.i. by immunoblotting ( Figure 1C). Here, FAM111B levels increased early, but decreased later during infection-observations that are consistent with our previous mRNA analyses. The steady increase in FAM111B protein levels at 8-24 h was followed by a decrease at 24-72 h ( Figure 1C).  These results indicate that FAM111B expression is upregulated early in infection. In contrast, FAM111B protein levels are downregulated during the late course of infection. Complete abrogation of FAM111B protein expression late during wt HAdV-C5 infection could point towards potent post-translational regulations that remain to be elucidated. Taken together, these data show that wt HAdV-C5 infection strongly induced mRNA and protein levels of FAM111B at early time points, which decreased in the late phase of the infection.

FAM111B Expression Levels Are Differentially Regulated by E1A, E1B-55K and E4orf6
Adenovirus E4orf6 and E1B-55K proteins form a functional cullin-based E3 ubiquitin ligase complex to target a number of cellular proteins for proteasomal degradation [18,23,24]. Within the complex, E4orf6 associates via multiple BC boxes with cellular elongins B and C to enable binding of either Cul5 or Cul2 and further components to build up the core ligase complex, and E1B-55K serves as a substrate recognition factor. This complex is formed to degrade cellular proteins, which would otherwise counteract viral replication [25][26][27]. To investigate whether E1B and E4orf6 mediate the degradation of FAM111B during late times of infection, we used HAdV-C5 mutants that do not express E1B-55K or E4orf6 [17,18] (Figure 1C). As infection control and control for the virus mutants, we stained for E1A, E1B-55K and E4orf6 and could detect viral proteins starting at 16 h p.i. While FAM111B protein levels of wt-infected cells were not detectable at 48-72 h p.i., they were substantially reduced in cells infected with virus mutants lacking E1B-55K or E4orf6, most likely due to decreased transcript levels at these stages of infection ( Figure 1C). These data suggest that the E3 ubiquitin ligase complex formed by E1B and E4orf6 together with cellular proteins targets FAM111B at late time points of infection.
Since E1B-55K serves as a substrate recognition factor to target cellular antiviral proteins for proteasomal degradation, we then tested whether E1B-55K interacts with FAM111B. We first in vitro translated FLAG-FAM111B ( Figure 2A) and immobilized FAM111B using FLAG antibody-bound agarose beads. Although a considerable amount of FAM111B was still bound on the agarose beads, the eluate was positive for FAM111B after staining with a FAM111B-specific monoclonal antibody (mAb) ( Figure 2B). For IPs, HAdV-C5-infected or mock-infected A549 whole-cell lysates were incubated with immobilized FLAG-tagged FAM111B. Immunoblot analysis of E1A 12S/13S and E1B-55K proteins after co-IP with the FAM111B-specific mAb revealed that FAM111B interacts only with E1B-55K but not with E1A 12S/13S ( Figure 2C), supporting the idea that FAM111B interacts with E1B-55K and serves as a novel substrate of the viral E3 ubiquitin ligase complex. These data are supported by work from Hung et al., who have previously identified FAM111B as a binding partner of the viral E1B-55K protein [2].
To analyze transcriptional regulation of FAM111B by E1A and E1B, we performed luciferase reporter gene assays under control of the FAM111B promoter. Activity was measured after co-transfection with the respective constructs, demonstrating that FAM111B promoter activity is enhanced upon co-transfection with the E1A-but not the E1B-containing plasmid ( Figure 3A,B). E1A is known to interact with pRB and its family members to activate E2F-dependent transcription and cell cycle entry, and transcriptional regulation occurs through CBP/p300-dependent genes by E1A [28,29]. Moreover, the FAM111B promoter is responsive to E2F-1, E2F-4 and CREB [30]. To investigate whether E1A activates the FAM111B promoter via the pRB/E2F pathway, we first analyzed responsiveness of the FAM111B promoter to E2F-1 ( Figure 3C). Additionally, E1A was co-transfected to investigate whether it has an additive effect on FAM111B promoter activation ( Figure 3D). We found that E2F-1 stimulated transcription of the FAM111B promoter approximately 4-fold higher compared to the FAM111B promoter alone ( Figure 3C). Intriguingly, co-transfection of E2F-1 plus E1A further increased FAM111B promoter activity ( Figure 3D). bilized FLAG-tagged FAM111B. Immunoblot analysis of E1A 12S/13S and E1B-55K proteins after co-IP with the FAM111B-specific mAb revealed that FAM111B interacts only with E1B-55K but not with E1A 12S/13S ( Figure 2C), supporting the idea that FAM111B interacts with E1B-55K and serves as a novel substrate of the viral E3 ubiquitin ligase complex. These data are supported by work from Hung et al., who have previously identified FAM111B as a binding partner of the viral E1B-55K protein [2]. Finally, we performed luciferase assays with E1A mutants deficient in binding to RB or CBP or both ( Figure 3E). As expected, the FAM111B promoter was activated about 4-fold after co-transfection with wt E1A (Figure 3E), while functional inactivation of E1Abinding sites to RB, CBP or both clearly diminished E1A's transcriptional stimulation of the FAM111B promoter ( Figure 3E). To control for the expression levels of the different E1A mutants, whole cell lysates were immuno-stained with an E1A-specific antibody ( Figure 3F). In summary, these results show that binding of E1A to p300/CBP and pRB is important for transcriptional regulation of FAM111B. While E1A seems to modulate FAM111B transcription, E1B might regulate protein levels.

FAM111B Knockdown Increases HAdV-C5 Gene Expression
Finally, we set out to analyze the role of FAM111B during HAdV-C5 infection in more detail. Therefore, we used shRNA knockdowns (KD) of FAM111B in A549 cells, in comparison to control cells that express scrambled shRNA ( Figure 4A). First, the KD was confirmed by immunoblotting as FAM111B shRNA reduced steady-state levels of the FAM111B protein, while no effect in FAM111B levels was observed with the control shRNA ( Figure 4A). Next, cell growth analyses revealed that A549s replicate slightly better with depleted FAM111B, although this was not statistically significant ( Figure 4B). Finally, we infected A549-derived FAM111B KD cells and the respective control cell line with wt HAdV-C5 (MOI 5 and 20) and performed virus yield analysis at time points 24 h, 48 and 72 h p.i. (Figure 4C,D). The FAM111B KD enhanced progeny virus production by up to 3-fold at 48 h p.i. These results suggest that FAM111B may function as a host restriction factor that affects virus replication and virus progeny production.   ter with depleted FAM111B, although this was not statistically significant ( Figure 4B). Finally, we infected A549-derived FAM111B KD cells and the respective control cell line with wt HAdV-C5 (MOI 5 and 20) and performed virus yield analysis at time points 24 h, 48 and 72 h p.i. (Figure 4C,D). The FAM111B KD enhanced progeny virus production by up to 3-fold at 48 h p.i. These results suggest that FAM111B may function as a host restriction factor that affects virus replication and virus progeny production.

Conclusions
This study demonstrates that FAM111B expression is transcriptionally activated at early times after HAdV-C5 infection. This upregulation is mediated by E1A on a transcriptional level, resulting in elevated FAM111B expression dependent on E1A's CBP/RBbinding motifs in an E2F-dependent manner. While FAM111B protein levels increase early during infection, they are downregulated at later time points, likely mediated by the E3 ubiquitin ligase complex composed of E1B and E4orf6. Taken together, our data strongly indicate that FAM111B is a novel member of a growing list of cellular restriction factors that are activated in response to HAdV infection during the immediate early phase of the infection. This, together with the findings that the related FAM111A protein restricts SV40 and VACV replication, suggests that members of nuclear trypsin-like proteases act as restriction factors to antagonize replication of DNA viruses. Future investigations will aim to further reveal its impact on HAdV replication and also possible contributions of FAM111B to viral transformation. These data could be applied to other viral oncogenes and viral infections in general that manipulate key cellular pathways. Finally, deeper insights into the exact functions of antiviral restriction factors might potentially contribute to the identification of new therapeutic antiviral strategies.