miR-142-3p suppresses porcine reproductive and respiratory syndrome virus (PRRSV) infection by directly targeting Rac1

https://doi.org/10.1016/j.vetmic.2022.109434Get rights and content

Highlights

  • miR-142–3p remarkably impairs PRRSV infection in porcine alveolar macrophages (PAMs).

  • miR-142–3p directly targets Ras-related C3 botulinum toxin substrate 1 (Rac1).

  • miR-142–3p blocks PRRSV entry into PAMs by downregulating Rac1 expression.

Abstract

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) has been recognized as one of the severest epidemics in pigs worldwide. microRNAs (miRNAs) play important roles in a variety of biological processes, including cell differentiation, proliferation and death, as well as viral infections and antiviral immune responses. In this study, we found that miR-142–3p was expressed lower in cells susceptible to PRRSV infection than in cells less or no permissive to PRRSV infection. Subsequently, we showed that overexpression of miR-142–3p remarkably inhibited PRRSV infection in PAMs, while blockage of endogenous miR-142–3p significantly enhanced PRRSV replication. Then, we demonstrated that miR-142–3p directly targeted Ras-related C3 botulinum toxin substrate 1 (Rac1), a member of Rho GTPases family, by using luciferase reporter assay and UV cross-linking and immunoprecipitation (CLIP) assay. Importantly, we verified that miR-142–3p inhibited PRRSV entry into PAMs and accordingly suppressed PRRSV infection by downregulating Rac1 expression. These findings reveal an important role of miR-142–3p in modulating PRRSV infection and provide us with some ideas for developing novel antiviral therapy against PRRSV infection.

Introduction

Porcine reproductive and respiratory syndrome (PRRS) is a disease widely prevalent in pigs and results in heavy economic losses annually (Zhang and Feng, 2021). The main characteristics of the disease are severe reproductive failures, late miscarriage in sows, and severe respiratory syndromes and high mortality rates in piglets (Kimman et al., 2009). PRRS was first reported in the United States in 1987 (Butler et al., 2014). The causative agent of PRRS was isolated in Lelystad in 1991 (Wensvoort et al., 1991), and another strain (called VR-2332) was isolated in the United States in the following year (Collins et al., 1992). PRRS virus (PRRSV) is an enveloped, single-stranded, and positive-sense RNA virus and belongs to the family Arteriviridae in the order Nidovirales. The viral genome is approximately 15.4 kb in length and contains 11 open reading frames (ORFs), encoding 8 structural proteins and at least 16 non-structure proteins (An et al., 2020). Due to the high mutation frequency of PRRSV, an atypical highly pathogenic PRRSV (HP-PRRSV) variant emerged in China in 2006, causing much severer PRRS characterized by high fever, high morbidity, and high mortality (Tian et al., 2007). Since then, HP-PRRSV has become the major circulating strain in China and Southeast Asia and posed a serious threat to pig farms. Despite the efforts made by scientists to develop diverse vaccines, there is still no satisfactory protection against variable strains.

PRRSV is highly restricted to mononuclear-macrophage lineage. Porcine alveolar macrophages (PAMs) have been identified as the predominately and consistently infected cells in vivo, while peripheral blood monocytes (BMo) and porcine peritoneal macrophages (PPMs) are refractory to PRRSV infection (Duan et al., 1997). Endocytosis is the main way for pathogens to enter eukaryotic cells. Previous studies have shown that PRRSV infects cells through low pH-dependent clathrin-mediated endocytosis (CME) (Nauwynck et al., 1999). And a few cellular molecules have been described as receptors for PRRSV infection, including heparan sulphate, CD163, porcine sialoadhesin (pSn), vimentin, CD151, CD209 (DC-SIGN), and non-muscle myosin heavy chain 9 (MYH9) (Ma et al., 2021). Moreover, an alternative pathway for PRRSV to infect host cells has been found recently (Wei et al., 2020). Phosphatidylserine (PS), a marker of apoptosis, is exposed by PRRSV on the envelope as viral apoptotic mimicry. Then, PS receptor TIM-1/4 recognizes PRRSV as apoptotic mimicry and subsequently induces macropinocytosis by the downstream Ras-related C3 botulinum toxin substrate 1 (Rac1), cell division control protein 42 (Cdc42), and p21-activated kinase 1 (Pak1).

microRNAs (miRNAs) represent small (~23nt), endogenous, and non-coding RNAs that play important roles in regulating gene expression by degrading mRNAs or suppressing translation (Bartel, 2009). miRNA “seed” region is very important for miRNAs to perform their functions. It has been shown that miRNAs are implicated in almost every biological process, including cell differentiation, proliferation, and cell death (Dong et al., 2013). In addition, miRNAs are also involved in a variety of cellular activities such as immune response and virus replication (Lodish et al., 2008). Emerging evidence has indicated that host miRNAs can modulate PRRSV infection mainly by targeting viral genome, regulating viral receptors, and regulating host antiviral response (Liu et al., 2017). For example, miR-181 not only inhibits viral replication by targeting viral genome, but also suppresses viral infection by targeting PRRSV receptor CD163 (Gao et al., 2013, Guo et al., 2013). In contrast, miR-30c upregulated by PRRSV infection promotes PRRSV replication via downregulating JAK1 and IFNAR2 expression (Liu et al., 2018, Zhang et al., 2016). Since more than 800 miRNAs have been identified in pigs (An et al., 2020), we want to know if there are other miRNAs that can regulate PRRSV replication.

In this study, we demonstrated that miR-142–3p played a role in inhibition of PRRSV infection. Our data verified that miR-142–3p inhibited PRRSV entry and infection via downregulating Rac1 expression. These findings not only help us understand PRRSV pathogenesis, but also provide a potential therapeutic target for us to control PRRS.

Section snippets

Cells and viruses

Porcine alveolar macrophages (PAMs), porcine peritoneal macrophages (PPMs) and peripheral blood monocytes (BMo) were obtained from 8-week-old specific-pathogen-free (SPF) pigs and were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS). CRL-2843 cells, a porcine alveolar macrophage cell line, were maintained in RPMI 1640 medium containing 10% FBS. All the cells were cultured at 37°C with 5% CO2.

The highly pathogenic PRRSV (HP-PRRSV) isolate HV (GenBank Accession No. JX317648) and

miR-142-3p inhibits PRRSV replication

Our previous studies have shown that miRNAs, which are expressed differently between cells susceptible to PRRSV (PAMs) and cells less or no permissive to PRRSV (BMo and PPMs), have potentials to regulate PRRSV replication (Guo et al., 2013, Zhang et al., 2016). Thus, we focused on miRNAs that have different expression in these cells. Through screening, we found that the expression of miR-142–3p in PPMs and BMo was about 5- and 10- fold higher than that in PAMs, respectively (Fig. 1A). It has

Discussion

In this study, we found that miR-142–3p had a potent antiviral effect on PRRSV infection. We demonstrated that miR-142–3p directly targeted Rac1, leading to the blockage of PRRSV entry and suppression of subsequent infections in PAMs (Fig. 6).

PRRSV has undoubtedly become a major financial problem affecting the global swine industry since its outbreak in the late 1980 s (Bloemraad et al., 1994). In order to control PRRSV, diverse vaccines have been developed. However, satisfactory PRRSV vaccines

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (Grant No. 31630076), China, and the National Major Special Project on New Varieties Cultivation for Transgenic Organisms (grant no. 2016ZX08009-003-006), China.

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