VGLL4 Promotes EV71 Virus Proliferation and Accelerates the Apoptosis of Infected RD Cells

Enterovirus 71 (EV71) is one of the major pathogens causing hand, foot and mouth disease (HFMD) which affects public health increasingly. Apoptosis plays an important role in EV71 infection, but the molecular mechanism involved in EV71 induced apoptosis is not completely clear. VGLL4 is a multifunctional protein in host cells, which has been studied in tumor and cell apoptosis, but has not been reported in pathogen. In this study, the mammalian eukaryotic expression plasmid of VGLL4 fused with HA tag (HA-VGLL4) and the model of overexpression VGLL4 RD cells were successfully constructed. The effect of VGLL4 on the proliferation of EV71 was detected by western-blot assay, uorescence quantitative PCR and cytotoxicity assay (CCK assay), and the mechanism of its effect on the proliferation of the virus was researched. The experimental results showed that VGLL4 may promote the replication of EV71 by promoting the apoptosis of infected cells. VGLL4 can be an important target for prevention and treatment of EV71 infection.


Introduction
Hand foot and mouth disease (HFMD) causes serious health problems in the Asia Paci c region, including China. The epidemiological survey results showed that 1,500 out of 3 million children die of severe illness [10,21]. EV71, a new enterovirus, is one of the main pathogens causing HFMD [18]. The symptoms of EV71 infection are mostly self-limited which are usually characterized by fever, maculopapules and herpes in the mouth and extremities; but a few show neurological symptoms and even death in infants and young children [23]. Therefore, the study of EV71 is of great signi cance.
EV71 virus belongs to the family of small RNA viruses, enteroviruses and is a single plus-stranded RNA virus. The life cycle of the virus mainly includes virus infection, replication and translation of the viral genome and assembly and release of the progeny virus. Studies have shown that EV71 can interact with host proteins such as annexinII, PSGL1, SCARB2 and nucleolin to initiate their infection process [2,11,15,22]. The replication, transcription and translation of EV71 virus also require the participation of multiple host proteins, such as hnRNP K and APOBEC3G, which affect the virus replication by interacting with the virus 5'UTR [9,17]. Reticulon3 is involved in endoplasmic reticulum transcription and translation of virus [6]; SIRT1 can affect the replication, transcription and translation of viruses [1]. Therefore, the interaction between EV71 and the host has been a hot topic in research. EV71 is nonenveloped virus, traditional view is that the virus is released by cytolytic manner which inevitably leads to cell death. And the apoptosis stage induced by virus is very important for virus proliferation. Studies have reported that EV71 can promote cell apoptosis by inducing ROS production, and the virus can also induce cell apoptosis through the interaction between their own proteins and apoptosis proteins [8,19]. Despite extensive research, the mechanism between viral proliferation and apoptosis remains unclear.
Vestigial-like Family Member 4 (VGLL4) is an important transcription cofactor. Relevant studies have shown that it can be a tumor suppressor to in uence the occurrence and development of tumors by promoting apoptosis of tumor cells [4,14,20]. In normal cells, it can also promote apoptosis as an inhibitor of apoptosis protein (IAP)-interacting protein [3,16]. However, the relationship between VGLL4 and viruses has not been studied. In this study, the mammalian eukaryotic expression plasmid of VGLL4 fused with HA tag (HA-VGLL4) and the model of overexpression VGLL4 RD cells were successfully constructed, which were used to studied the relationship between EV71 infection, apoptosis and host protein VGLL4.Our study found that VGLL4 can promote EV71 infected cell apoptosis, which can promote the replication of EV71.

Materials And Methods
Cells culture and antibody RD cells and Vero cells were cultured in Dulbecco's modi ed Eagle's medium (DMEM containing 10% fetal bovine serum (FBS, Gibco) and incubated in a 37℃ environment containing 5% carbon dioxide.
The EV71 VP1 antibody was purchased from Abnova, Taiwan, China; the β-tubulin antibody was purchased from Bioprimacy, Wuhan, China; the Caspase3 antibody was purchased from Proteintech, Wuhan, China; and the HA tag antibody was purchased from CST, USA.

Virus propagation and titration
The EV71 strain BrCr (VR-1775) was purchased from ATCC and was propagated in RD cells. The cells grow to 80%, and then get infected with the virus. After 80% of the cells developed a cytopathic efect (CPE) of viral infection, the cell maintenance supernatant (DMEM containing 1% FBS and 1% 100 μg/ml penicillin-streptomycin) was collected and subjected to three freeze-thaw cycles to lyse the cells. The virus titer was determined by the 50% tissue culture infectious dose (TCID 50 assay).
Transfection and expression of plasmids RD cells were seeded in 6-well plate and cultured in DMEM containing 10% FBS at 37℃ in a 5% CO 2 environment. When the degree of monolayer cell confuence reached 60-70%, plasmids were transfected into cells by using lipofectamine 2000 (Invitrigen, USA) according to the manufacturer's instructions. The expression of plasmids were analysed by Western blot assay.
Western blot assay PBS-washed cell pellets were lysed in 1×RIPA buffer (CST, USA). The samples were centrifuged at 12,000 rpm for 15 min to harvest the supernatant, which were resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes (BioRad, USA). The membranes were blocked with 2% bovine serum albumin (BSA, Sigma-Aldrich, Germany) and then incubated with the appropriate primary antibody overnight at 4℃. The membranes were washed with 1×PBST and incubated with a 1:5000 dilution of anti-rabbit or anti-mouse horseradish-peroxidase-conjugated antibody for 1.5 h. Following incubation, the membranes were washed extensively with 1×PBST. Immunoreactive bands detected using ECL reagents (Advansta, USA) were developed with Image Lab (Bio-Rad, USA .

Results
Transfection of HA-VGLL4 recombinant plasmid in RD cell RD cells transfected with pHA-VGLL4 or the control vector were lysed with RIPA at 24h, and western blot assay were performed to evaluate the transfection e ciency of the plasmid in cells. The plasmid was successfully transfected and performed high transfection e ciency into RD cells (Fig 1).
VGLL4 promotes the expression of EV71 virus protein RD cell transfected with pHA-VGLL4 or the control vector were infected with EV71 at an MOI of 10 3 TCID 50 /mL. The cells were lysed with RIPA and western blot assay were performed to detect the expression of EV71 VP1 protein at 24 h post infection. The results showed that EV71 VP1 protein were highly low expressed, which means that VGLL4 could promote EV71 proliferation in RD cells (Fig 2).
VGLL4 facilitates the replication of EV71 genomic RNA Because we speculated that VGLL4 might enhance EV71 replication real-time quantitative RT-PCR was performed to detect the mRNA level of the VP1 gene of EV71. RD cells were transfected with the pHA-VGLL4 or the control plasmid; at 24h post-transfection, the cells were infected with EV71 at an MOI of 10 3 TCID 50 /mL and then harvested at 24h post infection. The results of real-time quantitative RT-PCR showed that the amount of VP1 mRNA in RD cells overexpressing VGLL4 was signi cantly increased at 24h post infection compared with the control group (Fig 3).

VGLL4 upregulated virus titer in cell maintenance supernatant
To further con rm that VGLL4 could facilitate propagation of EV71. The above RD cell maintenance supernatant were collected and the viral titers were calculated by the TCID 50 assay. The results showed that the titer of EV71 were 10 5.54 TCID 50 /mL in cells transfected with pHA-VGLL4 and 10 4.83 TCID 50 /mL in cells transfected with control vector, which means the production of viral particles were increased by VGLL4 (Fig 4).

VGLL4 promotes EV71-induced apoptosis
Because VGLL4 is an inhibitor of apoptosis protein (IAP)-interacting protein [3], we speculated that VGLL4 might enhance EV71 replication by accelerate the cell apoptosis. RD cells were transfected with pHA-VGLL4 plasmid or the control plasmid; at 24h post transfection, the cells were infected with EV71 at an MOI of 10 3 TCID 50 /mL. At 24h post infection. The cell viability was evaluated by cytotoxicity assay and the cell lysates were analyzed by western blot assay. As showed in gure, the cell viability was signi cant reduced in VGLL4 overexpression cells infected with EV71 (Fig 4). Cleaved caspase3 are considered to be hallmarks of apoptosis. As showed in Fig 5, the cleaved caspase3 was up-regulated in VGLL4 overexpression cells infected with EV71. Thus, VGLL4 increases EV71-induced apoptosis, which may be bene cial for the virus production. Discussion EV71 infection mainly occurs in infants and children under 5 years old, which can cause a serious public health [5,7,12]. However, the interaction between the virus and the host remains unclear. In this paper, we identi ed the roles of VGLL4 in EV71 replication.
With the completion of the Human Genome Project, more and more genes have been discovered, most of which have unknown functions. Using expression system to express target genes in mammalian cells is an important means to study gene function and its interaction. In this study, a recombinant plasmid expressing VGLL4 with HA label was successfully inserted into mammalian eukaryotic expression plasmid and was expressed in RD cells. This lays the foundation for the following functional research.
To detect VGLL4 in uence on EV71 proliferation, VGLL4 was overexpressed and EV71 was infected in RD cells. Western blot assay was to test the EV71 VP1 expression and real-time quantitative RT-PCR was to detect the EV71 RNA replication. At the same time, TCID 50 assay was performed to detect the virus titer in RD cells maintain supernatant. All above experiments fully proved that VGLL4 can promote the proliferation of EV71.
Apoptosis is closely related to EV71 replication. The effect of VGLL4 on the cellular viability of EV71 infected cells were evaluated by cytotoxicity assay. The results showed that VGLL4 can inhibit the cellular viability of EV71 infected cells. Therefore, VGLL4 may play an important role in EV71 inducing apoptosis.
Since cleaved-caspase-3 is hallmark of apoptosis and cleaved-caspase-3 has been reported to promote the proliferation of EV71 [13]. The effect of VGLL4 on EV71 virus induced cleaved caspase3 was detected by western blot assay. The results showed that cleaved caspase3 levels were signi cantly increased in VGLL4 overexpressing cells.
In conclusion, evidence is provided to demonstrate that VGLL4 can promote EV71 infected cell apoptosis, which can promote the replication of EV71. Thus, VGLL4 may be a potential therapeutic target for anti-EV71.