Sequence Analysis of Macaca mulatta TRIM4 and Its Role in the Interferon Pathway

Monkey diseases are becoming increasingly severe, and some may be transmitted to humans through direct and indirect contact. Innate immunity is the first line of defense against foreign microorganisms. It is of great significance to explore the immune characteristics of monkey and human diseases. TRIM4, an important immune molecule in Macaca mulatta, was cloned and its immunological characteristics were preliminarily explored. The results showed that Macaca mulatta TRIM4 was in the same branch as human TRIM4. Overexpression of TRIM4 increased the mRNA levels of interferon (IFN)-alpha, IFN-beta, RIG-I, MAVS, IRF3, IRF7, OAS1, IFIT3, and CCL5, TRIM4 up-regulated the activities of IFN-beta, NF-κB, and ISRE reporter. In contrast, inhibiting TRIM4 expression by small interfering RNA (siRNA) down-regulated the IFN pathway. In summary, Macaca mulatta TRIM4 plays an essential role in the IFN pathway.


INTRODUCTION
Innate immunity plays an important role in the resistance to foreign pathogen invasion. Foreign microorganisms are recognized by pattern recognition receptors (PRRs), including toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), and C-type lectin receptors (CLRs) (1). The body stimulates a series of signaling proteins and pathways and activates multiple cytokines, which further stimulate downstream signaling molecules that act as antiviral molecules and play an essential role in eliminating invading pathogens (2)(3)(4)(5).
Tripartite motif protein (TRIM) is a vital signaling molecule that plays an essential role in many cellular processes, including cell proliferation, differentiation, carcinogenesis, innate immunity, and apoptosis (6). For example, TRIM79α inhibits the replication of Tick-borne encephalitis virus by degrading RNA polymerase (7). TRIM28 inhibits the infection of murine leukemia virus and promotes virus entry into the latent period (8). TRIM52 interacts with the NS2A protein of the Japanese encephalitis virus and degrades the NS2A protein. TRIM41 ubiquitinates and degrades nucleoprotein and inhibits influenza virus replication by interacting with the influenza virus nucleoprotein (9). TRIM11 has been shown to inhibit the invasion, replication, and release of human immunodeficiency virus 1(HIV-1), plays an antiviral role, carries out ubiquitination modification of the protein, and negatively regulates interferon regulatory factor 3(IRF3) and activates and promotes the infection of HSV-1 (10).
Studies have shown that human TRIM4, an essential regulatory molecule of RIG-I, participates in the interferon (IFN) pathway (11). However, TRIM4 has not yet been reported in monkeys. In this study, the TRIM4 gene was cloned from rhesus macaque (Macaca mulata), and the relationship between TRIM4 and IFN was preliminarily explored, providing a theoretical basis for treating diseases.

Phylogenetic Tree Construction
The phylogenetic tree was constructed using the neighborjoining method with MEGA 5.0 (12).

Real-Time PCR
The mRNA expression levels of IFN-alpha, IFN-beta, retinoicacid-inducible gene I (RIG-I), mitochondria antiviral signaling protein (MAVS), interferon regulatory factor (IRF) 3, IRF7, 2 ′ -5 ′ Oligoadenylate synthetases (OAS)1, interferon-induced protein with tetratricopeptide repeats (IFIT3), and CCL5 were determined by real-time PCR on a 7500 Fast Real-time PCR system (Applied Biosystems, Foster City, CA). The PCR procedure and quantification were performed as described in a previous study (13). Total RNA was extracted using TRIzol (Beyotime, Shanghai, China, #R0016), and then 500 ng RNA was reverse transcribed by Hiscript III 1st strand cDNA synthesis kit (+gDNA wiper) (Vazyme, Nanjing, China, #R312), the cDNA was stored for realtime PCR. The volume of real-time PCR reaction was 20 µL, in which 10 µL BeyoFast SYBR Green qPCR Mix (Beyotime, Shanghai, China, #D7265), 6 µL ddH 2 O, 2 µL cDNA, 2 µL upstream and downstream primers were included. The realtime PCR conditions were 95 • C for 2 min, followed by 40 cycles at 95 • C for 15 s and 60 • C for 30 s, the primers are shown in Supplementary Table 1. The relative expression of the target gene was calculated by 2 − Ct relative quantitative method.

Infections
MARC145 cells were infected with the PRRSV-2 BJ-4 strain (GenBank accession no. AF331831) for 1 h. Then, the medium was changed to DMEM with 2% FBS, and samples were obtained at the indicated time points.

Statistical Analysis
There are three biological repeats in all the experiments. Mean ± standard deviation is the method of displaying data, and paired student's t-test and GraphPad Prism 5.0 (GraphPad Software, San Diego, CA) were used to analyze the data. Values of p < 0.05 were considered statistically significant.

Phylogenetic Analysis of TRIM4 From Different Species
First, we cloned the Macaca mulata TRIM4 gene from healthy Macaca mulata liver tissue. TRIM4 gene is 1,425 bp in length and encodes 475 amino acids. Phylogenetic tree analysis showed that Macaca mulata TRIM4 is in the same branch as humans and pan troglodytes, and they share a close genetic relationship (Supplementary Figure 1).

Overexpression of TRIM4 Activates the IFN Pathway
To study the immunological characteristics of TRIM4, we first explored the role of TRIM4 in the IFN pathway. TRIM4 plasmid was transfected into MARC145 cells, after 48 h, the  mRNA expression of IFN-alpha, IFN-beta, RIG-I, MAVS, IRF3, IRF7, OAS1, IFIT3, and CCL5 was determined by qPCR, and the poly(I:C) control group was set up. First, TRIM4 was well expressed in cells (Figure 1A), then the results indicated that poly(I:C) and TRIM4 increased the mRNA levels of IFN-alpha, IFN-beta, RIG-I, MAVS, IRF3, IRF7, OAS1, IFIT3, and CCL5 ( Figure 1B). Furthermore, we cotransfected TRIM4 and promoter plasmids (IFN-beta, NF-κB, and ISRE) into MARC145 cells and measured their promoter activity at 48 h post-transfection. The results indicated that TRIM4 increased the activity of these promoters (Figure 1C). Western blotting results showed that poly(I:C) and TRIM4 significantly increased the levels of phosphorylated IRF3, phosphorylated IRF7 and phosphorylated NF-κB p65 levels ( Figure 1D).

TRIM4 SiRNA Inhibits the IFN Pathway
Next, we used siRNA to verify the effect of TRIM4 in the IFN pathway; TRIM4 siRNA was transfected into MARC145 cells, and its knockdown effect on TRIM4 expression levels was confirmed by western blot. The results indicated that TRIM4 siRNA effectively knocked down TRIM4 expression (Figure 2A). Then, we stimulated the MARC145 cells with poly(I:C) for 9 h, and measured the mRNA levels of IFN-alpha, IFN-beta, RIG-I, MAVS, IRF3, IRF7, OAS1, IFIT3, and CCL5 by qPCR. The results indicated that the mRNA levels of IFN-alpha, IFN-beta, RIG-I, MAVS, IRF3, IRF7, OAS1, IFIT3, and CCL5 were significantly decreased after TRIM4 siRNA transfection (Figure 2B). On the other hand, different promoter plasmids and TRIM4 siRNA were co-transfected into MARC145 cells; after 24 h, the cells were stimulated with poly(I:C) for 9 h, and the promoter activities were tested. The results showed that the activities of IFN-beta, NF-κB, and ISRE promoters decreased significantly after transfection with TRIM4 siRNA (Figure 2C). Western blotting results showed that si-TRIM4 significantly reduced the protein levels of phosphorylated IRF3, phosphorylated IRF7 and phosphorylated NF-κB p65 ( Figure 2D).

Effect of Different Stimuli on TRIM4
To investigate the effect of porcine reproductive and respiratory syndrome virus (PRRSV) infection on TRIM4 expression, we infected MARC145 cells with PRRSV at different multiplicities of infection (MOI). The results showed that 0.01, 0.1, and 1 MOI PRRSV infection increased the Macaca mulata TRIM4 mRNA levels and protein levels (Supplementary Figure 2A). The changes in TRIM4 at different time points of viral infection were further analyzed. The results showed that the expression of TRIM4 was the highest at 36 h post-infection, which was increased by approximately 7-fold, western blotting results showed that the protein level of TRIM4 increased (Supplementary Figure 2B). Further experimental results show that over-expression of TRIM4 inhibit PRRSV replication (Supplementary Figure 2C).

DISCUSSIONS
Viral pathogen-associated molecular patterns (PAMPs) are recognized by PRRs, inducing type I IFN and downstream proteins, which play a pivotal role in initiating the antiviral immune response. Type I IFN mediated by PRRs is regulated by post-translational modifications, especially phosphorylation and ubiquitination (2).
TRIM protein is expressed in cellular physiological processes, such as cell differentiation, apoptosis, tumor genesis, and innate immunity (6). An increasing number of studies have shown that TRIM proteins, such as TRIM5 (19), TRIM21  immune response. However, whether Macaca mulata TRIM4 regulates immune response is still unknown.
The immunological characteristics and nucleic acid information regarding Macaca mulata TRIM4 were reported first in this study. The above experiments further confirmed that Macaca mulata TRIM4 is closely genetically related to human TRIM4, and monkeys are a practical animal model to study human diseases. Sequence analysis showed that TRIM4 has the RING, B-Box, and coil-coil domain; these indicate that Macaca mulata TRIM4 may be functionally similar to human TRIM4.
Expression of TRIM4 increases after PRRSV infection in MARC145 cells, indicating that TRIM4 plays a role in the immune regulation of PRRSV. PRRSV activates the expression of TRIM4, prompting an immune response in the host and inhibiting virus replication. The results showed that TRIM4 overexpression inhibits PRRSV replication, indicating that TRIM4 played an antiviral role as a host antiviral factor.
Poly(I:C) is a simulated RNA analog, and after poly(I:C) stimulation, the expression of TRIM4 increased, which indicated that TRIM4 played a role in the immune regulation of poly(I:C).
These results indicate that TRIM4 responses to RNA virus regulation, the poly(I:C) results coincide with PRRSV results.
Poly(dA: dT) is a simulated DNA analog, and after poly(dA: dT) stimulation, the expression of TRIM4 increased, which indicated that TRIM4 functions in the immune regulation of poly(dA: dT). These results indicate that TRIM4 functions in the regulation of DNA viruses.
Studies on TRIM4 have shown that TRIM4 interacts with transient receptor potential melastatin 8 and regulates its channel function through K423-mediated ubiquitination (29). TRIM4 can interact with RIG-I, which is regulated by K63-linked polyubiquitination and induces CARD tetramers and facilitates RIG-I multimerization and filamentation, thereby activating RIG-I (11). TRIM4 competes with TRIM25 and interacts with RIG-I-CARD. We speculate that Macaca mulata TRIM4 may contribute to similar regulatory mechanisms and participate in the monkey IFN pathway. Macaca mulata TRIM4 may play an antiviral role, and these mechanisms will be studied in the future.
In summary, Macaca mulata TRIM4 is a crucial regulatory molecule in the IFN pathway and functions a vital role in revealing the monkey immune response.

CONCLUSIONS
Macaca mulata TRIM4 positively regulates IFN-beta in the IFN pathway.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The animal study was reviewed and approved by Foshan University.  Supplementary Table 1 | Primers used in this study.