Role of lipid rafts in porcine reproductive and respiratory syndrome virus infection in MARC-145 cells

Lipid rafts play an important role in the life cycle of many viruses. Cholesterol is a critical structural component of lipid rafts. Although the porcine reproductive and respiratory syndrome virus (PRRSV) has restricted cell tropism for cells of the monocyte/macrophage lineage, a non-macrophage cell MARC-145 was susceptible to PRRSV because of the expression of virus receptor CD163 on the cell surface, therefore MARC-145 cells is used as model cell for PRRSV studies. In order to determine if cholesterol is involved in PRRSV infection in MARC-145 cells, we used three pharmacological agents: methyl-β cyclodextrin (MβCD), mevinolin, and filipin complex to deplete cholesterol in MARC-145. Although these agents act by different mechanisms, they all significantly inhibited PRRSV infection. The inhibition could be prevented by addition of exogenous cholesterol. Cell membrane cholesterol depletion after virus infection had no effect on PRRSV production and cholesterol depletion pre-infection did not reduce the virus attachment, suggesting cholesterol is involved in virus entry. Further results showed that cholesterol depletion did not change expression levels of the PRRSV receptor CD163 in MARC-145, had no effect on clathrin-mediated endocytosis, but disturbed lipid-raft-dependent endocytosis. Collectively, these studies suggest that cholesterol is critical for PRRSV entry, which is likely to be mediated by a lipid-raft-dependent pathway.     

The design and recombinant protein expression of a consensus porcine interferon: CoPoIFN-α

CoPoIFN-α is a recombinant non-naturally occurring porcine interferon-α (IFN-α). It was designed by scanning 17 porcine IFN-α nonallelic subtypes and assigning the most frequently occurring amino acid in each position. We used a porcine IFN-α (PoIFN-α) derived from domestic pig as a control. Both porcine IFN-α genes were introduced into yeast expression vector PpICZα-A and expressed in Pichia pastoris. The antiviral unit of these two IFN-αs were assayed in MDBK, PK-15 and MARC-145 cells against vesicular stomatitis virus (VSV), and their inhibitory abilities on pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV) replication were also examined, respectively. We found the antiviral activity (units/mg) of CoPoIFN-α was 46.4, 63.6 and 53.5-fold higher than that of PoIFN-α for VSV inhibition in MDBK, PK-15 and MARC-145 cells, 4.8-fold higher for PRV inhibition in PK-15 cells, and 5-fold higher for PRRSV inhibition in MARC-145 cells. Our results also showed that the PRV and PRRSV-specific cytopathic effect (CPE) could be inhibited in the cells pretreated with CoPoIFN-α and PoIFN-α, and the virus titers in the cells pretreated with CoPoIFN-α were lower than those cells pretreated with PoIFN-α by 10-20-fold. The antiproliferative activity of CoPoIFN-α was significantly higher than that of PoIFN-α on a molar basis. The mRNA level of Mx1 and OAS1 genes in PK-15 cells induced by CoPoIFN-α were enhanced about 4.6-fold and 3.2-fold compared to that induced by PoIFN-α. Based on a homology model of CoPoIFN-α and IFNAR2, all of the different residues between native PoIFN-α and CoPoIFN-α were not involved in IFNAR1 binding site, and there is no direct interaction between these residues and IFNAR2, either. We speculate that the higher activity of CoPoIFN-α was likely due to the electrostatic potential introduced by residue Arg156 around the binding site or a structural perturbation caused by these different residues. This may enhance the overall binding affinity of CoPoIIFN-α and the receptors. Thus, CoPoIFN-α may have the potential to be used in therapy of porcine diseases.     

Establishment of a stable CHO cell line with high level expression of recombinant porcine IFN-β

A CHO cell clone (CHO-PoIFN-β) with stable porcine IFN-β expression under control of CMV promoter was selected under G418 pressure. In a 25cm(2) cell culture flask (5 ml culture medium), the cumulative protein yield of recombinant PoIFN-β reached 2.3×10(6) IU/ml. This cells clone maintained stable expression for at least 20 generations even in the absence of G418 selection pressure. The expressed recombinant PoIFN-β could induce the expression of porcine Mx protein in PK15 cells, and activate the chicken Mx promoter-controlled luciferase reporter gene expression, confirming that the recombinant PoIFN-β has the biological activity of natural porcine type-I interferon. In addition, the recombinant PoIFN-β fully protected PK15 cells against 1000 TCID(50) of porcine transmissible gastroenteritis virus and pseudo-rabies virus infection, demonstrating its high potential in therapeutic applications. This is the first report of establishing a mammalian cell line with stable expression of porcine IFN-β.     

Exostosin glycosyltransferase 1 reduces porcine reproductive and respiratory syndrome virus infection through proteasomal degradation of nsp3 and nsp5

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a serious threat to the swine industry worldwide. Exostosin glycosyltransferase 1 (EXT1), an enzyme involved in the biosynthesis of heparin sulfate, has also been reported to be a host factor essential for a wide variety of pathogens. However, the role of EXT1 in PRRSV infection remains uncharted. Here, we identified that PRRSV infection caused an increase of EXT1 expression. EXT1 knockdown promoted virus infection, whereas its overexpression inhibited virus infection, suggesting an inhibitory function of EXT1 to PRRSV infection. We found that EXT1 had no effects on the attachment, internalization, or release of PRRSV but did restrict viral RNA replication. EXT1 was determined to interact with viral nonstructural protein 3 (nsp3) and nsp5 via its N-terminal cytoplasmic tail and to enhance K48-linked polyubiquitination of these two nsps to promote their degradation. Furthermore, the C-terminal glycosyltransferase activity domain of EXT1 was necessary for nsp3 and nsp5 degradation. We also found that EXT2, a EXT1 homolog, interacted with EXT1 and inhibited PRRSV infection. Similarly, EXT1 effectively restricted porcine epidemic diarrhea virus and porcine enteric alphacoronavirus infection in Vero cells. Taken together, this study reveals that EXT1 may serve as a broad-spectrum host restriction factor and suggests a molecular basis for the potential development of therapeutics against PRRSV infection.     

CD163 expression confers susceptibility to porcine reproductive and respiratory syndrome viruses

Direct functional screening of a cDNA expression library derived from primary porcine alveolar macrophages (PAM) revealed that CD163 is capable of conferring a porcine reproductive and respiratory syndrome virus (PRRSV)-permissive phenotype when introduced into nonpermissive cells. Transient-transfection experiments showed that full-length CD163 cDNAs from PAM, human U937 cells (histiocytic lymphoma), African green monkey kidney cells (MARC-145 and Vero), primary mouse peritoneal macrophages, and canine DH82 (histocytosis) cells encode functional virus receptors. In contrast, CD163 splice variants without the C-terminal transmembrane anchor domain do not provide PRRSV receptor function. We established several stable cell lines expressing CD163 cDNAs from pig, human, and monkey, using porcine kidney (PK 032495), feline kidney (NLFK), or baby hamster kidney (BHK-21) as the parental cell lines. These stable cell lines were susceptible to PRRSV infection and yielded high titers of progeny virus. Cell lines were phenotypically stable over 80 cell passages, and PRRSV could be serially passed at least 60 times, yielding in excess of 10(5) 50% tissue culture infective doses/ml.     

重组猪肺表面活性蛋白A在体外可抑制PRRSV感染宿主细胞

【目的】研究重组猪肺表面活性蛋白A(SP-A)在体外对猪繁殖与呼吸综合征病毒(PRRSV)感染的抑制作用。【方法】采用PCR方法从含有猪SP-A基因的质粒中扩增SP-A基因,并将其插入到含有人CD5信号肽序列的真核表达载体pcDNA3.1A-CD5中,构建成SP-A基因的真核分泌型表达载体pcDNA-CD5-SPA/MH。将重组表达载体通过磷酸钙介导转染HEK293T细胞进行瞬时表达,通过Western blot方法鉴定表达产物,采用Ni-NTA琼脂糖凝胶亲和层析法从培养基中分离和纯化重组SP-A蛋白,通过ELISA方法检测SP-A蛋白与PRRSV的结合活性。将SP-A蛋白与PRRSV孵育,然后感染MARC-145细胞和猪肺泡巨噬细胞,感染72 h后测定病毒滴度,分析重组SP-A蛋白对PRRSV感染的抑制作用。【结果】结果表明构建的真核表达载体能够介导SP-A基因在HEK293T细胞中进行分泌表达;表达的重组猪SP-A蛋白能够与PRRSV进行剂量依赖性结合;用重组猪SP-A蛋白与PRRSV进行孵育,然后感染MARC-145细胞和猪肺泡巨噬细胞,结果显示SP-A处理的PRRSV感染细胞后的病变程度明显低于对照组。感染72 h后,SP-A处理组的PRRSV在MARC-145细胞和猪肺泡巨噬细胞的滴度明显低于SP-A非处理组。【结论】重组猪SP-A在体外对PRRSV的感染有明显的抑制作用,揭示SP-A具有抗PRRSV的活性。     

Actively replicating West Nile virus is resistant to cytoplasmic delivery of siRNA

Background

Recent studies have shown that gamma interferon (IFN-γ) synergizes with the innate IFNs (IFN-α and IFN-β) to inhibit herpes simplex virus type 1 (HSV-1) replication in vitro. To determine whether this phenomenon is shared by other herpesviruses, we investigated the effects of IFNs on human cytomegalovirus (HCMV) replication.

Results

We have found that as with HSV-1, IFN-γ synergizes with the innate IFNs (IFN-α/β) to potently inhibit HCMV replication in vitro. While pre-treatment of human foreskin fibroblasts (HFFs) with IFN-α, IFN-β or IFN-γ alone inhibited HCMV plaque formation by ~30 to 40-fold, treatment with IFN-α and IFN-γ or IFN-β and IFN-γ inhibited HCMV plaque formation by 163- and 662-fold, respectively. The generation of isobole plots verified that the observed inhibition of HCMV plaque formation and replication in HFFs by IFN-α/β and IFN-γ was a synergistic interaction. Additionally, real-time PCR analyses of the HCMV immediate early (IE) genes (IE1 and IE2) revealed that IE mRNA expression was profoundly decreased in cells stimulated with IFN-α/β and IFN-γ (~5-11-fold) as compared to vehicle-treated cells. Furthermore, decreased IE mRNA expression was accompanied by a decrease in IE protein expression, as demonstrated by western blotting and immunofluorescence.

Conclusion

These findings suggest that IFN-α/β and IFN-γ synergistically inhibit HCMV replication through a mechanism that may involve the regulation of IE gene expression. We hypothesize that IFN-γ produced by activated cells of the adaptive immune response may potentially synergize with endogenous type I IFNs to inhibit HCMV dissemination in vivo.     

Tripartite motif-containing protein 38 negatively regulates TLR3/4- and RIG-I-mediated IFN-β production and antiviral response by targeting NAP1

Recognition of RNA virus through TLR and RIG-I-like receptor results in rapid expression of type I IFNs, which play an essential role in host antiviral responses. However, the mechanisms to terminate the production of type I IFNs are not well defined. In the current study, we identified a member of the tripartite motif (TRIM) family, TRIM38, as a negative regulator in TLR3/4- and RIG-I-mediated IFN-β signaling. Knockdown of TRIM38 expression by small interfering RNA resulted in augmented activation of IFN regulatory factor 3 and enhanced expression of IFN-β, whereas overexpression of TRIM38 had opposite effects. Coimmunoprecipitation and colocalization experiments demonstrated that TRIM38 interacted with NF-κB-activating kinase-associated protein 1 (NAP1), which is required for TLR-induced IFN regulatory factor 3 activation and IFN-β production. As an E3 ligase, TRIM38 promoted K48-linked polyubiquitination and proteasomal degradation of NAP1. Thus, knockdown of TRIM38 expression resulted in higher protein level of NAP1 in primary macrophages. Consistent with the inhibitory roles in TLR3/4- and RIG-I-mediated IFN-β signaling, knockdown of TRIM38 significantly inhibited the replication of vesicular stomatitis virus. Overexpression of TRIM38 resulted in enhanced replication of vesicular stomatitis virus. Therefore, our results demonstrate that TRIM38 is a negative regulator for TLR and RIG-I-mediated IFN-β production by targeting NAP1 for ubiquitination and subsequent proteasome-mediated degradation.     

Involvement of sialoadhesin in entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV) shows a very restricted tropism for cells of the monocyte/macrophage lineage. It enters cells via receptor-mediated endocytosis. A monoclonal antibody (MAb) that is able to block PRRSV infection of porcine alveolar macrophages (PAM) and that recognizes a 210-kDa protein (p210) was described previously (MAb41D3) (X. Duan, H. Nauwynck, H. Favoreel, and M. Pensaert, J. Virol. 72:4520-4523, 1998). In the present study, the p210 protein was purified from PAM by immunoaffinity using MAb41D3 and was subjected to internal peptide sequencing after tryptic digestion. Amino acid sequence identities ranging from 56 to 91% with mouse sialoadhesin, a macrophage-restricted receptor, were obtained with four p210 peptides. Using these peptide data, the full p210 cDNA sequence (5,193 bp) was subsequently determined. It shared 69 and 78% amino acid identity, respectively, with mouse and human sialoadhesins. Swine (PK-15) cells resistant to viral entry were transfected with the cloned p210 cDNA and inoculated with European or American PRRSV strains. Internalized virus particles were detected only in PK-15 cells expressing the recombinant sialoadhesin, demonstrating that this glycoprotein mediated uptake of both types of strains. However, nucleocapsid disintegration, like that observed in infected Marc-145 cells as a result of virus uncoating after fusion of the virus with the endocytic vesicle membrane, was not observed, suggesting a block in the fusion process. The ability of porcine sialoadhesin to mediate endocytosis was demonstrated by specific internalization of MAb41D3 into PAM. Altogether, these results show that sialoadhesin is involved in the entry process of PRRSV in PAM.     

A vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multi-species type I interferon

Type I interferons (IFN) comprise a family of cytokines that signal through a common cellular receptor to induce a plethora of genes with antiviral and other activities. Recombinant IFNs are used for the treatment of hepatitis C virus infection, multiple sclerosis, and certain malignancies. The capability of type I IFN to suppress virus replication and resultant cytopathic effects is frequently used to measure their bioactivity. However, these assays are time-consuming and require appropriate biosafety containment. In this study, an improved IFN assay is presented which is based on a recombinant vesicular stomatitis virus (VSV) replicon encoding two reporter proteins, firefly luciferase and green fluorescent protein. The vector lacks the essential envelope glycoprotein (G) gene of VSV and is propagated on a G protein-expressing transgenic cell line. Several mammalian and avian cells turned out to be susceptible to infection with the complemented replicon particles. Infected cells readily expressed the reporter proteins at high levels five hours post infection. When human fibroblasts were treated with serial dilutions of human IFN-β prior to infection, reporter expression was accordingly suppressed. This method was more sensitive and faster than a classical IFN bioassay based on VSV cytopathic effects. In addition, the antiviral activity of human IFN-λ (interleukin-29), a type III IFN, was determined on Calu-3 cells. Both IFN-β and IFN-λ were acid-stable, but only IFN-β was resistant to alkaline treatment. The antiviral activities of canine, porcine, and avian type I IFN were analysed with cell lines derived from the corresponding species. This safe bioassay will be useful for the rapid and sensitive quantification of multi-species type I IFN and potentially other antiviral cytokines.     

Hepatitis C Virus Core-Derived Peptides Inhibit Genotype 1b Viral Genome Replication via Interaction with DDX3X

The protein DDX3X is a DEAD-box RNA helicase that is essential for the hepatitis C virus (HCV) life cycle. The HCV core protein has been shown to bind to DDX3X both in vitro and in vivo. However, the specific interactions between these two proteins and the functional importance of these interactions for the HCV viral life cycle remain unclear. We show that amino acids 16–36 near the N-terminus of the HCV core protein interact specifically with DDX3X both in vitro and in vivo. Replication of HCV replicon NNeo/C-5B RNA (genotype 1b) is significantly suppressed in HuH-7-derived cells expressing green fluorescent protein (GFP) fusions to HCV core protein residues 16–36, but not by GFP fusions to core protein residues 16–35 or 16–34. Notably, the inhibition of HCV replication due to expression of the GFP fusion to HCV core protein residues 16–36 can be reversed by overexpression of DDX3X. These results suggest that the protein interface on DDX3X that binds the HCV core protein is important for replicon maintenance. However, infection of HuH-7 cells by HCV viruses of genotype 2a (JFH1) was not affected by expression of the GFP fusion protein. These results suggest that the role of DDX3X in HCV infection involves aspects of the viral life cycle that vary in importance between HCV genotypes.     

The DEAD-Box RNA Helicase DDX1 Interacts with the Viral Protein 3D and Inhibits Foot-and-Mouth Disease Virus Replication

Foot-and-mouth disease virus(FMDV) can infect domestic and wild cloven-hoofed animals. The non-structural protein 3 D plays an important role in FMDV replication and pathogenesis. However, the interaction partners of 3 D, and the effects of those interactions on FMDV replication, remain incompletely elucidated. In the present study, using the yeast two-hybrid system, we identified a porcine cell protein, DEAD-box RNA helicase 1(DDX1), which interacted with FMDV 3 D. The DDX1-3 D interaction was further confirmed by co-immunoprecipitation experiments and an indirect immunofluorescence assay(IFA) in porcine kidney 15(PK-15) cells. DDX1 was reported to either inhibit or facilitate viral replication and regulate host innate immune responses. However, the roles of DDX1 during FMDV infection remain unclear. Our results revealed that DDX1 inhibited FMDV replication in an ATPase/helicase activity-dependent manner. In addition, DDX1 stimulated IFN-b activation in FMDV-infected cells. Together, our results expand the body of knowledge regarding the role of DDX1 in FMDV infection.     

Nonstructural protein 2 of porcine reproductive and respiratory syndrome virus inhibits the antiviral function of interferon-stimulated gene 15

Type I interferon (alpha/beta interferon [IFN-α/β]) stimulates the expression of interferon-stimulated gene 15 (ISG15), which encodes a ubiquitin-like protein, ISG15. Free ISG15 and ISG15 conjugates function in diverse cellular pathways, particularly regulation of antiviral innate immune responses. In this study, we demonstrate that ISG15 overexpression inhibits porcine reproductive and respiratory syndrome virus (PRRSV) replication in cell culture and that the antiviral activity of interferon is reduced by inhibition of ISG15 conjugation. PRRSV nonstructural protein 2 (nsp2) was previously identified as a potential antagonist of ISG15 production and conjugation. The protein contains a papain-like protease domain (PLP2) that plays a crucial role in the proteolytic cleavage of the PRRSV replicase polyproteins. PLP2 was also proposed to belong to the ovarian tumor domain-containing superfamily of deubiquitinating enzymes (DUBs), which is capable of inhibiting ISG15 production and counteracting ISG15 conjugation to cellular proteins. To determine whether this immune antagonist function could be selectively inactivated, we engineered a panel of mutants with deletions and/or mutations at the N-terminal border of the nsp2 PLP2-DUB domain. A 23-amino-acid deletion (amino acids 402 to 424 of the ORF1a-encoded protein) largely abolished the inhibitory effect of nsp2 on ISG15 production and conjugation, but no viable recombinant virus was recovered. A 19-amino-acid deletion (amino acids 402 to 420), in combination with a downstream point mutation (S465A), partially relieved the ISG15 antagonist function and yielded a viable recombinant virus. Taken together, our data demonstrate that ISG15 and ISGylation play an important role in the response to PRRSV infection and that nsp2 is a key factor in counteracting the antiviral function of ISG15.     

RIG-I/MDA5/MAVS are required to signal a protective IFN response in rotavirus-infected intestinal epithelium

Rotavirus is a dsRNA virus that infects epithelial cells that line the surface of the small intestine. It causes severe diarrheal illness in children and ～500,000 deaths per year worldwide. We studied the mechanisms by which intestinal epithelial cells (IECs) sense rotavirus infection and signal IFN-β production, and investigated the importance of IFN-β production by IECs for controlling rotavirus production by intestinal epithelium and virus excretion in the feces. In contrast with most RNA viruses, which interact with either retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated gene 5 (MDA5) inside cells, rotavirus was sensed by both RIG-I and MDA5, alone and in combination. Rotavirus did not signal IFN-β through either of the dsRNA sensors TLR3 or dsRNA-activated protein kinase (PKR). Silencing RIG-I or MDA5, or their common adaptor protein mitochondrial antiviral signaling protein (MAVS), significantly decreased IFN-β production and increased rotavirus titers in infected IECs. Overexpression of laboratory of genetics and physiology 2, a RIG-I-like receptor that interacts with viral RNA but lacks the caspase activation and recruitment domains required for signaling through MAVS, significantly decreased IFN-β production and increased rotavirus titers in infected IECs. Rotavirus-infected mice lacking MAVS, but not those lacking TLR3, TRIF, or PKR, produced significantly less IFN-β and increased amounts of virus in the intestinal epithelium, and shed increased quantities of virus in the feces. We conclude that RIG-I or MDA5 signaling through MAVS is required for the activation of IFN-β production by rotavirus-infected IECs and has a functionally important role in determining the magnitude of rotavirus replication in the intestinal epithelium.     

Characterization of the biochemical properties and identification of amino acids forming the catalytic center of 3C-like proteinase of porcine reproductive and respiratory syndrome virus

Purpose of work  

The non-structural protein 4 (Nsp4) of porcine reproductive and respiratory syndrome virus (PRRSV) functions as a 3C-like proteinase (3CLpro) and plays a pivotal role in gene expression and replication. We have examined the biochemical properties of PRRSV 3CLpro and identified those amino acid residues involved in its catalytic activity as a prelude to developing anti-PRRSV strategies.     

Suppression of Autophagy Enhanced Growth Inhibition and Apoptosis of Interferon-β in Human Glioma Cells

Interferon-beta (IFN-β) is a cytokine with anti-viral, anti-proliferative, and immunomodulatory effects. In this study, we investigated the effects of IFN-β on the induction of autophagy and the relationships among autophagy, growth inhibition, and apoptosis induced by IFN-β in human glioma cells. We found that IFN-β induced autophagosome formation and conversion of microtubule associated protein 1 light chain 3 (LC3) protein, whereas it inhibited cell growth through caspase-dependent cell apoptosis. The Akt/mTOR signaling pathway was involved in autophagy induced by IFN-β. A dose- and time-dependent increase of p-ERK 1/2 expression was also observed in human glioma cells treated with IFN-β. Autophagy induced by IFN-β was suppressed when p-ERK1/2 was impaired by treatment with U0126. We also demonstrated that suppression of autophagy significantly enhanced growth inhibition and cell apoptosis induced by IFN-β, whereas inhibition of caspase-dependent cell apoptosis impaired autophagy induced by IFN-β. Collectively, these findings indicated that autophagy induced by IFN-β was associated with the Akt/mTOR and ERK 1/2 signaling pathways, and inhibition of autophagy could enhance the growth inhibitory effects of IFN-β and increase apoptosis in human glioma cells. Together, these findings support the possibility that autophagy inhibitors may improve IFN-β therapy for gliomas.