Pseudorabies virus UL16 protein influences the inhibition of LRPPRC for the viral proliferation

Highlights

• PRV pUL16 localized to the mitochondria, nuclei and cytoplasm.

• PRV pUL16 weakened the inhibition of LRPPRC for the growth of PRV.

• LRPPRC influenced STING-mediated activation of NF-κB.

Abstract

Pseudorabies is caused by pseudorabies virus (PRV), a member of the Herpesvirus family, and has caused tremendous damage to the pig industry. Protein unique lone 16 (pUL16) is a conserved envelope protein in all herpesviruses, that is known to play an important role in several aspects, including virus diffusion in cells and virulence in mice. It has been shown that the pUL16 can interact with the virus proteins UL11, UL49, UL21, gD, and gE. However, the research to date on pUL16 has only focused on etiology, without discussing the possible cellular pathways involved in PRV infection. Leucine-rich PPR motif-containing protein (LRPPRC) is a multifunctional cellular protein that participates in various cellular processes, such as RNA processing, splicing, stabilization, editing, translation, and energy metabolism. This was the first caspase-independent apoptosis protein to be identified. In this study, immune precipitation and mass spectrometry was performed to define the function of the pUL16 in PRV infection to study the possible cellular pathways in which pUL16 may participate. It was found that LRPRRC could interact with PRV pUL16, which may indicate that UL16 is involved in a redox reaction or cellular apoptosis. This is the first study of the interaction between pUL16 and host proteins, which has positive significance to gain a further understanding of the pUL16.

Introduction

Pseudorabies, also known as Aujeszky's disease, is caused by pseudorabies virus (PRV). It is an acute, virulent, and reproductive disordered infectious disease affecting pigs. PRV can infect humans, cattle, cats, dogs, sheep, rabbits, rats, and wild animals. However, pigs are the definitive host of PRV. PRV belongs to the order Herpesvirales, family Herperviridae, subfamily alphaherpersvirinae, and genus Varicellovirus (http://tall.ictvonline.org/taxonomy/). Alphaherperviruses have the characteristics of a wide host spectrum, rapid replication, cytopathic effects, and latent infection in the nervous system. Based on the arrangement of repeated and unique sequences on the herpesvirus genome, it can be divided into A, B, C, D, E and F. PRV is similar to VZV (herpes zoster virus), it has Unique Short (US), Unique Long (UL), terminal repeat sequences (Schloegl et al., 2012), and an internal repeat sequence. According to GC sequence analysis, PRV can be divided into gene type I and gene type II. In particular, virus strains outside China consist of gene type I, including virus strains from Europe, America, Australia, and other Asian countries. The gene type II virus mainly comes from China. PRV is a linear double-stranded DNA virus with a genome of approximately 145 kb and an average GC content of about 73 %. PRV viral particles are composed of four different structures: DNA; icosahedral nucleocapsid; tegument composed of viral proteins; and an envelope composed of viral glycoproteins from the inside to outside. The PRV genome encodes 16 types of membrane proteins, among which 11 proteins are modified by N- and O- linked sugars and termed gB, gC, gD, gE, gG, gH, gI, gK, gL, gM, and gN (Mettenleiter, 2000). pUL16, a tegument protein, is encoded by the UL16 gene, which is in the subfamilies of α, β, and γ herpesviruses, including pUL16 of PRV, pUL16 of HSV-1, EBV BGLF2 (Baer et al., 1984; Chen et al., 1991), ORF 33 of KSHV (Guo et al., 2009), pUL16 of HSV-2 (Oshima et al., 1998), ORF 44 of VZV (https://pubmed.ncbi.nlm.nih.gov/3018124/), and pUL94 of HCMV (Wing et al., 1996). Except for infectious laryngotracheitis virus, the UL16 gene of all poultry and mammal herpesviruses is located in the PRV UL region. The UL16 gene has 987 nucleotides, encoding 328 amino acids, of approximately 34.8 kDa (Klupp et al., 2003). Among the cellular proteins interacting with pUL16, LRPPRC (leucine-rich PPR motif-containing protein), also known as LRP130 (130 kDa leucine-rich protein) is a protein rich in leucine pentapeptide repeat sequences. In pig cells, the gene is 4,185 bp in length and encodes 1,395 amino acids (Ghiso and Lennon, 1994; Hou et al., 1994). LRPPRC is a multifunctional protein located in the inner and outer nuclear membrane, nuclear matter, endoplasmic reticulum, cytoskeleton, and mitochondria (Liu et al., 2002; Tsuchiya et al., 2004). LRPPRC is found in various protein complexes of mammalian cells but has no direct activity on the function of the complexes. But it plays a different role through direct or indirect protein-protein interactions. The STRING database revealed that there were 25 proteins that could interact with LRPPRC, which were related to various cellular processes (e.g., RNA metabolism, acute reaction, activation of non-receptor tyrosine kinase, mitochondrial function, cell signaling pathway, plasma membrane transport, and protein degradation) (Snel et al., 2000; Szklarczyk et al., 2015, 2019; Szklarczyk et al., 2017).

At present, LRPPRC is related to human immunodeficiency virus-1 (HIV-1) nucleic acid during the early stage of viral infection (Nicoll et al., 2016). An LRPPRC knockout reduced HIV-1 replication in cell lines had no effect on virus production and RNA encapsulation (Schweitzer et al., 2012). LRPPRC is a non-structural protein 5A (NS5A) binding factor. Additionally, NS5A helps to suppress the innate immune response during HCV infection by utilizing the ability of LRPPRC to suppress the antiviral signal regulated by the mitochondrial antiviral signal protein (Refolo et al., 2019). In 2012, an RRV variant strain (JS-2012 strain) was isolated from a pig farm immunized with Bartha-K61 in Jiangsu Province. A PRV variant vaccine candidate strain, JS-2012-F120, was obtained after the purified JS-2012 strain continuously passaged in Vero cells at 39 ℃ for 120 generations (Liang et al., 2017; Ye et al., 2018).

In this study, the screening and identification of host proteins interacting with the PRV pUL16 found that LRPPRC could interact with the pUL16 of PRV, and thereby inhibit PRV proliferation. It has been reported that LRPPRC interacts with the HIV-1 MA protein to promote HIV-1 replication in host cells (Schweitzer et al., 2012). Alternatively, LRPPRC interacts with the NS5A of HCV, inhibits the MAVS-mediated innate immune pathway, and promotes HCV proliferation (Refolo et al., 2019). Previous studies have shown that LRPPRC promotes viral proliferation, while the results of this study showed that LRPPRC overexpression inhibited PRV proliferation more strongly on PRV lacking pUL16, whereas a knockdown of LRPPRC attenuated this inhibitory effect. Therefore, upon PRV infection, LRPPRC inhibited PRV proliferation. The pUL16 antagonized this inhibitory effect of LRPPRC on PRV proliferation. Moreover, the potential mechanism in this process was evaluated in this study.