Porcine reproductive and respiratory syndrome virus triggers Golgi apparatus fragmentation-mediated autophagy to facilitate viral self-replication.

Macroautophagy/autophagy is a cellular degradation and recycling process that maintains the homeostasis of organisms. A growing number of studies have reported that autophagy participates in infection by a variety of viruses. Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe financial losses to the global swine industry. Although much research has shown that PRRSV triggers autophagy for its own benefits, the exact molecular mechanisms involved in PRRSV-triggered autophagy remain to be fully elucidated. In the current study, we demonstrated that PRRSV infection significantly induced Golgi apparatus (GA) fragmentation, which promoted autophagy to facilitate viral self-replication. Mechanistically, PRRSV nonstructural protein 2 was identified to interact with and degrade the Golgi reassembly and stacking protein 65 dependent on its papain-like cysteine protease 2 activity, resulting in GA fragmentation. Upon GA fragmentation, GA-resident Ras-like protein in brain 2 was disassociated from Golgi matrix protein 130 and subsequently bound to unc-51 like autophagy activating kinase 1 (ULK1), which enhanced phosphorylation of ULK1 and promoted autophagy. Taken together, all these results expand the knowledge of PRRSV-triggered autophagy as well as PRRSV pathogenesis to support novel potential avenues for prevention and control of the virus. More importantly, these results provide the detailed mechanism of GA fragmentation-mediated autophagy, deepening the understanding of autophagic processes.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) infection results in a serious swine disease affecting pig farming worldwide. Despite that numerous studies have shown that PRRSV triggers autophagy for its self-replication, how PRRSV induces autophagy is incompletely understood. Here, we identify that PRRSV Nsp2 degrades GRASP65 to induce GA fragmentation, which dissociates RAB2 from GM130 and activates RAB2-ULK1-mediated autophagy to enhance viral replication. This work expands our understanding of PRRSV-induced autophagy and PRRSV replication, which is beneficial for anti-viral drug development.

Heat Shock Protein Member 8 (HSPA8) Is Involved in Porcine Reproductive and Respiratory Syndrome Virus Attachment and Internalization.

Porcine reproductive and respiratory syndrome virus (PRRSV), a porcine arterivirus, causes severe financial losses to global swine industry. Despite much research, the molecular mechanisms of PRRSV infection remains to be fully elucidated. In the current study, we uncovered the involvement of heat shock protein member 8 (HSPA8) in PRRSV attachment and internalization during infection for the first time. In detail, HSPA8 was identified to interact with PRRSV glycoprotein 4 (GP4), a major determinant for viral cellular tropism, dependent on its carboxy-terminal peptide-binding (PB) domain. Chemical inhibitors and specific small interference RNAs (siRNAs) targeting HSPA8 significantly suppressed PRRSV infection as indicated by decreased viral RNA abundance, infectivity, and titers. Especially, PRRSV attachment was inhibited by interference of its binding to HSPA8 with mouse anti-HSPA8 polyclonal antibodies (pAbs) and recombinant soluble HSPA8 protein. HSPA8 was further shown to participate in PRRSV internalization through clathrin-dependent endocytosis (CME). Collectively, these results demonstrate that HSPA8 is important for PRRSV attachment and internalization, which is a potential target to prevent and control the viral infection. IMPORTANCE PRRSV has caused huge economic losses to the pork industry around the world. Currently, safe and effective strategies are still urgently required to prevent and control PRRSV infection. As the first steps, PRRSV attachment and internalization are initiated by interactions between viral envelope proteins and host cell receptors/factors, which are not fully understood yet. Here, we identified the interaction between PRRSV GP4 and HSPA8, and demonstrated that HSPA8 was involved in PRRSV attachment and internalization. This work deepens our understanding of the molecular mechanisms involved in PRRSV infection, and provides novel insights for the development of antiviral drugs and vaccines against the virus.

Tumor Susceptibility Gene 101 (TSG101) Contributes to Virion Formation of Porcine Reproductive and Respiratory Syndrome Virus via Interaction with the Nucleocapsid (N) Protein along with the Early Secretory Pathway.

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused huge economic losses to global swine industry. As an intracellular obligate pathogen, PRRSV exploits host cellular machinery to establish infection. The endocytic sorting complex required for transport (ESCRT) system has been shown to participate in different life cycle stages of multiple viruses. In the present study, a systematic small interference RNA screening assay identified that certain ESCRT components contributed to PRRSV infection. Among them, tumor susceptibility gene 101 (TSG101) was demonstrated to be important for PRRSV infection by knockdown and overexpression assays. TSG101 was further revealed to be involved in virion formation rather than viral attachment, internalization, RNA replication and nucleocapsid (N) protein translation within the first round of PRRSV life cycle. In detail, TSG101 was determined to specially interact with PRRSV N protein and take effect on its subcellular localization along with the early secretory pathway. Taken together, these results provide evidence that TSG101 is a proviral cellular factor for PRRSV assembly, which will be a promising target to interfere with the viral infection. IMPORTANCE PRRSV infection results in a serious swine disease affecting pig farming in the world. However, efficient prevention and control of PRRSV is hindered by its complicated infection process. Until now, our understanding of PRRSV assembly during infection is especially limited. Here, we identified that TSG101, an ESCRT-I subunit, facilitated virion formation of PRRSV via interaction with the viral N protein along with the early secretory pathway. Our work actually expands the knowledge of PRRSV infection and provides a novel therapeutic target for prevention and control of the virus.

Structural comparison of CD163 SRCR5 from different species sheds some light on its involvement in porcine reproductive and respiratory syndrome virus-2 infection in vitro.

Porcine reproductive and respiratory syndrome (PRRS) is a serious disease burdening global swine industry. Infection by its etiological agent, PRRS virus (PRRSV), shows a highly restricted tropism of host cells and has been demonstrated to be mediated by an essential scavenger receptor (SR) CD163. CD163 fifth SR cysteine-rich domain (SRCR5) is further proven to play a crucial role during viral infection. Despite intense research, the involvement of CD163 SRCR5 in PRRSV infection remains to be elucidated. In the current study, we prepared recombinant monkey CD163 (moCD163) SRCR5 and human CD163-like homolog (hCD163L1) SRCR8, and determined their crystal structures. After comparison with the previously reported crystal structure of porcine CD163 (pCD163) SRCR5, these structures showed almost identical structural folds but significantly different surface electrostatic potentials. Based on these differences, we carried out mutational research to identify that the charged residue at position 534 in association with the one at position 561 were important for PRRSV-2 infection in vitro. Altogether the current work sheds some light on CD163-mediated PRRSV-2 infection and deepens our understanding of the viral pathogenesis, which will provide clues for prevention and control of PRRS.

Vesicular stomatitis virus glycoprotein suppresses nuclear factor kappa-B- and mitogen-activated protein kinase-mediated pro-inflammatory responses dependent on sialic acids.

Vesicular stomatitis (VS), characterized by vesicular lesions, produces significant economic losses in livestock industry. Infection by its causative agent, VS virus (VSV), has been previously shown to be mediated by the glycoprotein (G) during attachment, endocytosis and membrane fusion. In the current study, we revealed a novel role of VSV G protein in negative regulation of host cell pro-inflammatory responses. We determined that VSV G protein inhibited lipopolysaccharide (LPS)-induced pro-inflammatory responses as naïve VSV virions in murine peritoneal macrophage-like cell line RAW 264.7. Furthermore, we identified that VSV G protein suppressed nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK)-mediated pro-inflammatory pathways in a dose-dependent manner. Moreover, we demonstrated that α2-3-linked sialic acids on VSV G protein were involved in antagonizing NF-κB- and MAPK-mediated pro-inflammatory responses. All these results expand the knowledge of VSV pathogenesis and strengthen the importance of VSV G protein in host innate immunity, which support implications for the development of VSV-based vaccination and oncolysis.

Glycoprotein 5 Is Cleaved by Cathepsin E during Porcine Reproductive and Respiratory Syndrome Virus Membrane Fusion.

Porcine reproductive and respiratory syndrome (PRRS) is a serious viral disease affecting the global swine industry. Its causative agent, PRRS virus (PRRSV), is an enveloped virus, and therefore membrane fusion between its envelope and host cell target membrane is critical for viral infection. Though much research has focused on PRRSV infection, the detailed mechanisms involved in its membrane fusion remain to be elucidated. In the present study, we performed confocal microscopy in combination with a constitutively active (CA) or dominant negative (DN) mutant, specific inhibitors, and small interfering RNAs (siRNAs), as well as multiple other approaches, to explore PRRSV membrane fusion. We first observed that PRRSV membrane fusion occurred in Rab11-recycling endosomes during early infection using labeled virions and subcellular markers. We further demonstrated that low pH and cathepsin E in Rab11-recycling endosomes are critical for PRRSV membrane fusion. Moreover, PRRSV glycoprotein 5 (GP5) is identified as being cleaved by cathepsin E during this process. Taken together, our findings provide in-depth information regarding PRRSV pathogenesis, which support a novel basis for the development of antiviral drugs and vaccines.IMPORTANCE PRRS, caused by PRRSV, is an economically critical factor in pig farming worldwide. As PRRSV is a lipid membrane-wrapped virus, merging of the PRRSV envelope with the host cell membrane is indispensable for viral infection. However, there is a lack of knowledge on its membrane fusion. Here, we first explored when and where PRRSV membrane fusion occurs. Furthermore, we determined which host cell factors were involved in the process. Importantly, PRRSV GP5 is shown to be cleaved by cathepsin E during membrane fusion. Our work not only provides information on PRRSV membrane fusion for the first time but also deepens our understanding of the molecular mechanisms of PRRSV infection, which provides a foundation for future applications in the prevention and control of PRRS.

Prevalence and genetic characteristics of porcine reproductive and respiratory syndrome virus in central China during 2016-2017: NADC30-like PRRSVs are predominant.

NADC30-like strains of porcine reproductive and respiratory syndrome virus (PRRSV) were firstly reported in China in 2013. Since then, these strains have been epidemic in more than 13 provinces/regions. During 2016-2017, a total of 18 PRRSV isolates were obtained from 52 clinical samples in Henan province. Based on comparative and phylogenetic analyses of ORF5 and partial Nsp2 genes, 83.3% (15/18) isolates belonged to NADC30-like strains, and the ORF5 shared 87.4%-95.5% nucleotide identity with NADC30/JL580 and 84.2%-89.9% with JXA1/CH-1a, respectively. The genetic variation analysis showed that extensive amino acid substitutions happened in the significant regions of ORF5 including major linear antigenic epitopes (27-30aa, 37-45aa, 52-61aa) and the potential N-glycosylation sites (32-35aa). 16.7% (3/18) isolates were very close to HP-PRRSV derived attenuated strains. Moreover, these three isolates shared common residues at the positions 33D, 59 N, 164R, 196R in ORF5 and 303D, 399T, 575V, 598R, 604G in Nsp2, which were thought to be unique to modified live vaccines (MLVs) or their derivatives. Therefore, they were probably the revertants from MLVs. Our studies showed that the HP-PRRSV strains seemed to be gradually disappearing and NADC30-like strains had become the main causative agents of PRRS in central China. Comparing with HP-PRRSVs, the ORF5 of NADC30-like PRRSV strains displayed extensive amino acid mutations which may be related with immune evasion. Furthermore, the circulation of MLV derivatives in the fields made the diagnosis and control of PRRSV more complicated.

The CD163 long-range scavenger receptor cysteine-rich repeat: expression, purification and X-ray crystallographic characterization.

Scavenger receptors (SRs) play critical roles in various physiological and pathological pathways. One of them, CD163, is a multifunctional endocytic receptor and is characterized by a long-range scavenger receptor cysteine-rich (SRCR) repeat. However, the structural and functional details of this long-range SRCR repeat have not yet been elucidated. In this study, the CD163 long-range SRCR repeat was expressed in Drosophila Schneider 2 cells. The recombinant protein was homogeneous after purification by metal-affinity, cation-exchange and size-exclusion chromatography. Single crystals were obtained using 20% PEG 4000, 0.15 M potassium sodium tartrate tetrahydrate pH 8.5 and diffracted to 3.30 Šresolution. As the first view of a long-range SRCR repeat, this work lays the structural basis for a deep understanding of SRs and their multiple functions.

Identification of the RNA Pseudoknot within the 3' End of the Porcine Reproductive and Respiratory Syndrome Virus Genome as a Pathogen-Associated Molecular Pattern To Activate Antiviral Signaling via RIG-I and Toll-Like Receptor 3.

Once infected by viruses, cells can detect pathogen-associated molecular patterns (PAMPs) on viral nucleic acid by host pattern recognition receptors (PRRs) to initiate the antiviral response. Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of porcine reproductive and respiratory syndrome (PRRS), characterized by reproductive failure in sows and respiratory diseases in pigs of different ages. To date, the sensing mechanism of PRRSV has not been elucidated. Here, we reported that the pseudoknot region residing in the 3' untranslated regions (UTR) of the PRRSV genome, which has been proposed to regulate RNA synthesis and virus replication, was sensed as nonself by retinoic acid-inducible gene I (RIG-I) and Toll-like receptor 3 (TLR3) and strongly induced type I interferons (IFNs) and interferon-stimulated genes (ISGs) in porcine alveolar macrophages (PAMs). The interaction between the two stem-loops inside the pseudoknot structure was sufficient for IFN induction, since disruption of the pseudoknot interaction powerfully dampened the IFN induction. Furthermore, transfection of the 3' UTR pseudoknot transcripts in PAMs inhibited PRRSV replication in vitro Importantly, the predicted similar structures of other arterivirus members, including equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV), also displayed strong IFN induction activities. Together, in this work we identified an innate recognition mechanism by which the PRRSV 3' UTR pseudoknot region served as PAMPs of arteriviruses and activated innate immune signaling to produce IFNs that inhibit virus replication. All of these results provide novel insights into innate immune recognition during virus infection.IMPORTANCE PRRS is the most common viral disease in the pork industry. It is caused by PRRSV, a positive single-stranded RNA virus, whose infection often leads to persistent infection. To date, it is not yet clear how PRRSV is recognized by the host and what is the exact mechanism of IFN induction. Here, we investigated the nature of PAMPs on PRRSV and the associated PRRs. We found that the 3' UTR pseudoknot region of PRRSV, which has been proposed to regulate viral RNA synthesis, could act as PAMPs recognized by RIG-I and TLR3 to induce type I IFN production to suppress PRRSV infection. This report is the first detailed description of pattern recognition for PRRSV, which is important in understanding the antiviral response of arteriviruses, especially PRRSV, and extends our knowledge on virus recognition.

Molecular epidemiology of porcine reproductive and respiratory syndrome virus in Central China since 2014: The prevalence of NADC30-like PRRSVs.

Porcine reproductive and respiratory syndrome (PRRS), characterized by respiratory disorders in piglets and reproductive failure in sows, is still the great threat of swine industry. Recently, Emergence of the novel NADC30-like PRRS viruses (PRRSVs) has caused widespread outbreaks of PRRS. To investigate the epidemic characteristics of PRRSVs in Central China since 2014, 6372 clinical serum samples were tested by ELISA, 250 tissue samples were tested by RT-PCR, and among these, 30 ORF5 and 17 Nsp2 genes sequences were analyzed. Phylogenetic tree based on ORF5 revealed that, 17 isolates were clustered into subgroup 1, represented by the NADC30. And for the Nsp2, The strains which had a discontinuous 131-amino-acid deletion in Nsp2, called NADC30-like strains, were clustered into subgroup 2. Our data suggested that the NADC30-like PRRSV strains spread quickly and are now circulating and prevalent in Central China as well as the classical HP-PRRSV strains. In addition, amino acid variation analysis of GP5 revealed that the amino acid sequences of NADC30-like PRRSV strains underwent rapid evolution and contained extensive amino acid substitutions in important motifs, such as potential neutralization epitope and the N-glycosylation sites. In summary, our data would provide a large amount of detailed information on molecular variation and genetic diversity of PRRSV in central China.

The Crystal Structure of the Fifth Scavenger Receptor Cysteine-Rich Domain of Porcine CD163 Reveals an Important Residue Involved in Porcine Reproductive and Respiratory Syndrome Virus Infection.

Porcine reproductive and respiratory syndrome (PRRS) has become an economically critical factor in swine industry since its worldwide spread in the 1990s. Infection by its causative agent, PRRS virus (PRRSV), was proven to be mediated by an indispensable receptor, porcine CD163 (pCD163), and the fifth scavenger receptor cysteine-rich domain (SRCR5) is essential for virus infection. However, the structural details and specific residues of pCD163 SRCR5 involved in infection have not been defined yet. In this study, we prepared recombinant pCD163 SRCR5 in Drosophila melanogaster Schneider 2 (S2) cells and determined its crystal structure at a high resolution of 2.0 Å. This structure includes a markedly long loop region and shows a special electrostatic potential, and these are significantly different from those of other members of the scavenger receptor cysteine-rich superfamily (SRCR-SF). Subsequently, we carried out structure-based mutational studies to identify that the arginine residue at position 561 (Arg561) in the long loop region is important for PRRSV infection. Further, we showed Arg561 probably takes effect on the binding of pCD163 to PRRSV during virus invasion. Altogether the current work provides the first view of the CD163 SRCR domain, expands our knowledge of the invasion mechanism of PRRSV, and supports a molecular basis for prevention and control of the virus. IMPORTANCE: PRRS has caused huge economic losses to pig farming. The syndrome is caused by PRRSV, and PRRSV infection has been shown to be mediated by host cell surface receptors. One of them, pCD163, is especially indispensable, and its SRCR5 domain has been further demonstrated to play a significant role in virus infection. However, its structural details and the residues involved in infection are unknown. In this study, we determined the crystal structure of pCD163 SRCR5 and then carried out site-directed mutational studies based on the crystal structure to elucidate which residue is important. Our work not only provides structural information on the CD163 SRCR domain for the first time but also indicates the molecular mechanism of PRRSV infection and lays a foundation for future applications in prevention and control of PRRS.

Antibody-dependent enhancement of PRRSV infection down-modulates TNF-α and IFN-ß transcription in macrophages.

Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease, resulting in important economic losses in pig farming. Previous studies have shown that Fcγ receptor (FcγR)-mediated entry of infectious PRRSV immune complexes into macrophages plays a pivotal role in the pathogenesis of the disease. This study demonstrates that PRRSV was able to suppress the transcription of key antiviral genes tumor necrosis factor-α (TNF-α) and interferon-ß (IFN-ß), when infection was via the ADE pathway. Investigation of this infection pathway found that PRRSV suppresses the antiviral genes by disrupting the transcription of the genes coding for the associated transcription factors interferon regulatory factor-1 (IRF-1), interferon regulatory factor-3 (IRF-3) and nuclear factor kappa B (NF-κB). The ADE pathway of infection allows PRRSV to specifically target antiviral genes and alters the innate intracellular immune responses in macrophages. The ADE mechanism described in this study furthers our understanding of pathogenesis following PRRSV infection and is of general relevance to virally induced disease and in relation to antiviral vaccination strategies.

Impairment of the antibody-dependent phagocytic function of PMNs through regulation of the FcγRs expression after porcine reproductive and respiratory syndrome virus infection.

Porcine reproductive and respiratory syndrome (PRRS) is identified as one of the most important etiological agents in multifactorial respiratory disease of swine and can predispose pigs to secondary infections by other pathogens, usually bacteria. To understand the mechanism for an increased susceptibility to secondary bacterial infections, we investigated the antibody-dependent phagocytosis behaviour and killing ability of PMNs after infection by PRRSV strains BJ-4 or HN07-1. PMN's antibody-dependent phagocytosis and their ability to kill E.coli were both noticeably decreased following PRRSV infection, in particular with the highly pathogenic strain HN07-1. As the change in this function of the PMNs may reflect a variation in the expression of FcγRs, the expression profiles of the activating and the inhibitory FcγRs were examined. We found that RNA expression of the inhibitory receptor FcγRIIB was up-regulated post-infection, and this was greater after infection with the more virulent PRRSV strain HN07-1. The activating receptor FcγRIIIA RNA expression was on the other hand inhibited to the same extent by both PRRSV strains. Neutralizing antibody titers post-infection by PRRSV strains BJ-4 or HN07-1 were also detected. All of the pigs in infection groups showed viraemia by the end of the study (56 DPI). These observations may help to understand the mechanism of increased susceptibility to secondary bacterial infections following PRRSV infection.

Porcine reproductive and respiratory syndrome virus and bacterial endotoxin act in synergy to amplify the inflammatory response of infected macrophages.

In 2006 China experienced outbreaks of a severe form of porcine reproductive and respiratory syndrome (PRRS) characterized by high fever, morbidity and mortality in swine irrespective of age. It is thought that secondary bacterial infections may contribute to the generation of this severe form of the disease. To determine the mechanisms by which a highly pathogenic PRRSV strain causes high fever we used an in vitro model to investigate the production of the pro-inflammatory cytokines IL-1ß and TNF-α by macrophages in response to inoculation with PRRSV with or without LPS. Firstly we demonstrated, through an animal inoculation trial, that the isolate HN07-1 was a highly pathogenic strain and sequencing showed that the virus had the same genomic characteristics as previously described isolates. Porcine alveolar macrophage (PAM) cultures infected with PRRSV strains showed increased cytokine secretion and this was greater in the more virulent strain. Addition of LPS further increased cytokine secretion and again the effect was greater with the more virulent strain. Incubation of PAMs with PRRSV strain HN07-1 resulted in a significant increase in surface CD14 expression. This may explain the synergistic action between PRRSV and LPS in the induction of inflammatory cytokine secretion seen in the PAMs and so offer an explanation for the high fever that is characteristic of infections by the highly pathogenic PRRSV.