Importin alpha 1 is required for the nucleus entry of Fowl Adenovirus serotype 4 Fiber-1 protein

https://doi.org/10.1016/j.vetmic.2022.109351Get rights and content

Highlights

  • F1 protein of FAdV-4 could nucleus localization in LMH cells, and the P26 in NLS was required for F1 protein nucleus entry.

  • We elucidated the time rule of F1 protein subcellular localization in nucleus of LMH cells.

  • The importin alpha 1 is required for F1 protein nucleus entry.

  • F1 protein subcellular localization in nucleus was conducive to the proliferation of FAdV-4.

Abstract

Fiber-1 protein (F1) is the structural protein of Fowl Adenovirus serotype 4 (FAdV-4), which could recondite the receptors of host cytomembrane. In this study, we firstly determined that F1 protein located in nucleus of LMH cells after infection with FAdV-4. We additionally revealed that F1 protein had a classic NLS, and the NLS was required for F1 nucleus entry, which was intently associated to the 26th Pro in NLS. The time rule result indicated that some F1 proteins firstly positioned in the nucleus 6 h posttranfection, and it entirely located in the nucleus 12 h posttranfection, then it ordinarily placed in cytoplasm 18 h posttranfection by means of microscopic fluorescence observation and Western Blotting. Then after transfection with pCI-neo-flag-F1 or infection with FAdV-4, the importin alpha 1 was once investigated whether or not it was required for F1 protein nucleus entry through immunofluorescence and/or Co-IP, results demonstrated that the F1 protein and importin alpha 1 co-located in the nucleus 6 h and 12 h posttranfection. The tiers of F1 protein vicinity in nucleus have been additionally investigated after knockdown expression or overexpression of importin alpha 1, and the results further revealed that importin alpha 1 used to be required for F1 protein nucleus entry. Finally, the function of F1 protein nucleus entry was investigated by qPCR, RT-PCR and Western Blotting, and the results revealed that F1 protein nucleus location was conducive to the proliferation of FAdV-4. In summary, we firstly reveal that the F1 protein of FAdV-4 locates in nucleus infected with FAdV-4, and confirm that importin alpha 1 binds to the NLS of F1 protein to nucleus localization, which promotes the proliferation of FAdV-4.

Introduction

Fowl Adenovirus serotype 4 (FAdV-4) belongs to the avian adenovirus family (Yates and Fry, 1957; Chiocca, 1996). Mirzazadeh et al. (Mirzazadeh et al., 2021) discovered that the number of isolated fowl adenovirus serotype (FAdVs) belonging to wonderful serotypes declined in accordance with a upward push of neutralizing antibodies in birds, and underlining the value of serotype-specific antibodies in the epidemiology of FAdV in breeders. The nucleocapsid of FAdV-4 particles has three predominant structural proteins such as Hexon (Zhang et al., 2021), Penton (Aziz et al., 2019) and Fiber (Schachner et al., 2014). Fibers were reported to regulate the infection and pathogenicity of FAdV-4 (Sohaimi et al., 2018). There are many particular receptors on the host cell, which can be precisely identified by using Fiber protein 1 or 2, so that the FAdV-4 can be precisely adsorbed and sure into the host cell (Tian et al., 2020). When the Fiber-1 (F1) gene is knockdown, the replication of FAdV-4 isn’t lost, however it will lose its specific transduction function as a wild-type virus. Shao et al. (Shao et al., 2019) explored that the two monoclonal antibodies (mAbs) unique concentrated on Fiber-1 generated and the monnted monoclonal antibody (mAb) primarily based sandwich ELISA would grant an environment friendly diagnostics method for detection of FAdV-4/10. However the function of F1 protein of FAdV-4 nevertheless was completely understood.

Nuclear pore complex (NPC) located on the nuclear membrane of eukaryotic cells. Some required-nutrients can shuttle into and out nucleus through NPC (Dingwall and Laskey, 1986). Small molecular compounds (<5 Kilodaltons (kD)) can freely shuttle between cytoplasm and nucleus through NPC. But the macromolecular compounds (>50 kD) could bind to nuclear transport proteins on the nuclear membrane via an transport mechanism. The transport proteins can precisely apprehend the nuclear localization signal (NLS) of macromolecular compounds, then bind with each other. When the compounds enter into the nucleus, they separate (Quimby and Dasso, 2003). Ye et al. (Ye et al., 2020) proven that the localization of nuclear localization signal retinoic acid receptor alpha (NLS-RAR alpha) frequently depended on the NLS from the acid receptor alpha (RAR alpha) element of NLS-RAR alpha, and NLS mutant NLS-RAR alpha which used to be in the cytoplasm did not block differentiation of acute myeloid leukemia (AML) cells, in contrast with it in the nucleus. Two classical NLS have been described: NLS1-503KRKRRP508 and NLS2-606PKKNRLRRKS615, which are useful for the nuclear translocation of breast cancer 1 (BRCA1) (Thakur et al., 1997; Chen et al., 1996; Korlimarla et al., 2013). Lange et al. (Lange et al., 2008) manifested that a proline-tyrosine Nuclear Localization Signal (PY-NLS) is required for nuclear localization and function of the saccharomyces cerevisiae messenger ribonucleic acid (mRNA)-binding protein hypoxic response protein 1 (Hrp1).

Nuclear transport is usually regulated via the importin family of nuclear transporters. Importin alpha 1 is coded via karyopherin alpha2 (Kpna2) gene. It could bind to NLS, and composes a complex with nuclear receptor proteins into the nucleus via NPC (Cui et al., 2021). Ye et al. (Ye et al., 2020) determined that NLS-RAR alpha entered into the nucleus by binding to importin beta. Döhner et al. (Döhner et al., 2018) evidenced that importin alpha 1 was also required for nuclear import of Herpes simplex virus proteins in fibroblasts and neurons. Cai et al. (Cai et al., 2020) expounded that deubiquitinase 22 (USP22) promoted interferon regulatory factors 3 (IRF-3) nuclear translocation and antiviral responses by deubiquitinating the importin alpha. Wang et al. (Wang et al., 2016) signified that the NLS at the N-terminus of Yorkie and importin alpha 1 play a key function in Yorkie's nuclear location.

In the present study, we wanted to investigate whether the F1 protein could locate in nucleus, and to reveal the mechanism of F1 protein nucleus location. Furthermore we demonstrated that the proliferation of FAdV-4 used to be promoted when the F1 protein located in nucleus. Results would present an necessary therapeutic targets spot of FAdV-4.

Section snippets

Cell, virus and vectors

The chicken liver cancer cell line (LMH) was grown in RPMI 1640 with 10 % fetal bovine serum (FBS) at 37℃5 % carbon dioxide. When the dish was 100 % full, the supernatant was poured off, and cells were washed with phosphate buffer saline (PBS). Finally, LMH cells were digested with trypsin containing 0.25 % ethylene diamine tetra acetic acid (EDTA) for 1 min and then was subculture.

The FAdV-4 strain NP was isolated and identified by our team (GenBank: KU558760.1. median tissue culture infective

F1 protein subcellular localization in nucleus of LMH cells

It was found that F1 protein had a classic NLS, which was located at 15-46th amino acids and 43-144th bp (Fig. 1A). The whole 1315bp of F1 gene was amplified by PCR, (Fig. 1B). The recombinant plasmid of pCI-neo-flag-F1 was identified by PCR and restriction enzyme digestion, and the results showed that the pCI-neo-flag-F1 was constructed (Fig. 1C, D). After transfection 24 h, the subcellular localization of F1 protein was found in nucleus of LMH cells by Western Blotting (Fig. 1E) and

Discussion

FAdV-4 can cause chicken hepatitis and heatitis and hydro-pericardiumsyndrome (HHS). The disease first occurred in Ankara, Pakistan in 1987 (Kim et al., 2008), then it grew to become popular in many areas (Liu et al., 2016). Before 2013, the wide varietyr of FAdV-4 infection cases in China was scarce, but it increased sharply after 2015, and it became widespread. Acute FAdV-4 infection mainly presents symptoms such as gastric erosion, inclusion body hepatitis, and pericardial effusion. Its

Conclusion

In the present study, we firstly confirmed the nucleus location rule of F1 protein, the importin alpha 1 as a nuclear transporter is required for F1 protein to the nucleus entry and is beneficial to the proliferation of FAdV-4.

Author contribution

Quanxi conceived and designed the whole study, did a critical reading of the manuscript then revising of the submitted manuscript. Ruiling and Qing performed all experiments. Shaohua and Menghan organized the data, then Lihui performed the statistics. Ruiling wrote the manuscript. All authors has reviewed and approved submitted version of this manuscript.

Disclosure statement

The authors declared no potential conflict of interest.

Funding

This work was supported by grants from the China Fujian Natural Science Foundation (grant 2019J01658) and the China Fujian Avian Industry System Program (2019–2022).

Data availability statement

The datasets used during this study are available from the corresponding author on reasonable request.

Declaration of Competing Interest

All the authors have read and approved the manuscript and its submission to Veterinary Microbiology. The authors have no conflicts of interest to declare.

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