Heme oxygenase-1 metabolite biliverdin, not iron, inhibits porcine reproductive and respiratory syndrome virus replication

Huang

et al. 2019

J Virol

Self Cite

Porcine reproductive and respiratory syndrome (PRRS) is of great concern to the swine industry due to pandemic outbreaks of the disease, current ineffective vaccinations, and a lack of efficient antiviral strategies. In our previous study, a PRRSV Nsp9-specific nanobody, Nb6, was successfully isolated, and the intracellularly expressed Nb6 could dramatically inhibit PRRSV replication in MARC-145 cells. However, despite its small size, the application of Nb6 protein in infected cells is greatly limited, as the protein itself cannot enter the cells physically. In this study, a trans-activating transduction (TAT) peptide was fused with Nb6 to promote protein entry into cells. TAT-Nb6 was expressed as an inclusion body in Escherichia coli, and indirect enzyme-linked immunosorbent assays and pulldown assays showed that E. coli-expressed TAT-Nb6 maintained the binding ability to E. coli-expressed or PRRSV-encoded Nsp9. We demonstrated that TAT delivered Nb6 into MARC-145 cells and porcine alveolar macrophages (PAMs) in a dose- and time-dependent manner, and TAT-Nb6 efficiently inhibited the replication of several PRRSV genotype 2 strains as well as a genotype 1 strain. Using a yeast two-hybrid assay, Nb6 recognition sites were identified in the C-terminal part of Nsp9 and spanned two discontinuous regions (Nsp9aa454–551 and Nsp9aa599–646). Taken together, these results suggest that TAT-Nb6 can be developed as an antiviral drug for the inhibition of PRRSV replication and controlling PRRS disease. IMPORTANCE The pandemic outbreak of PRRS, which is caused by PRRSV, has greatly affected the swine industry. We still lack an efficient vaccine, and it is an immense challenge to control its infection. An intracellularly expressed Nsp9-specific nanobody, Nb6, has been shown to be able to inhibit PRRSV replication in MARC-145 cells. However, its application is limited, because Nb6 cannot physically enter cells. Here, we demonstrated that the cell-penetrating peptide TAT could deliver Nb6 into cultured cells. In addition, TAT-Nb6 fusion protein could suppress the replication of various PRRSV strains in MARC-145 cells and PAMs. These findings may provide a new approach for drug development to control PRRS.

Section: Methodsmentioning

confidence: 99%

A Nanobody Targeting Viral Nonstructural Protein 9 Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication

Wang

Huang

et al. 2019

J Virol

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“…EGFP-PRRSV based on the genetic background of SD16 strain that expressed EGFP as an additional ORF (rHP-PRRSV/SD16/EGFP) was kindly provided by Professor En-min Zhou in Northwest A&F University (40). Viruses were propagated and titrated in Marc-145 cells by calculating the median tissue culture infective dose as previously described (41). SD16 PRRSV was used for the majority of the experiments, and thus indicated as PRRSV, unless otherwise specified.…”

Section: Cells Viruses and Reagentsmentioning

confidence: 99%

“…PRRSV has highly restricted cell tropism whereby it infects the monocyte-macrophage lineage cells including porcine alveolar macrophages (PAMs), the primary target of PRRSV infection in vivo (30,31). The African green monkey kidney cell line MA-104, and its sub-clone Marc-145 are also susceptible to PRRSV infection and have been frequently used in PRRSV studies in vitro (32,33). Although the mechanism of on October 30, 2020 by guest http://jvi.asm.org/ Downloaded from PRRSV particle entry into host cells remains to be fully understood, it has been shown that the envelope glycoproteins (GP3, GP4, GP5 and M) on the surface of the viral envelop regulate cell entry via binding to cell surface receptors, such as scavenger receptor (CD163), sialoadhesin (Sn) and heparan sulfate (HS) amongst others (34)(35)(36).…”

Section: Introductionmentioning

confidence: 99%

Interferon-Induced Transmembrane Protein 3 Is a Virus-Associated Protein Which Suppresses Porcine Reproductive and Respiratory Syndrome Virus Replication by Blocking Viral Membrane Fusion

Duan

Zhao

et al. 2020

J Virol

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Porcine reproductive and respiratory syndrome virus (PRRSV) infection eliminates production of type I interferons (IFNs) in host cells, which triggers an antiviral immune response through the induction of downstream IFN stimulated genes (ISGs), thus escaping the fate of host-mediated clearance. The IFN-induced transmembrane 3 (IFITM3) has recently been identified as an ISG and plays a pivotal role against enveloped RNA viruses by restricting cell entry. However, the role of IFITM3 in PRRSV replication is unknown. The present study demonstrated that overexpression of IFITM3 suppresses PRRSV replication, while silencing of endogenous IFITM3 prominently promoted PRRSV replication. Additionally, it was shown that IFITM3 undergoes S-palmitoylation and ubiquitination modification, and both posttranslational modifications contribute to the anti-PRRSV activity of IFITM3. Further study showed that PRRSV particles are transported into endosomes and then into lysosomes during the early stages of infection, and confocal microscopy results revealed that PRRSV particles are transported to IFITM3 positive cellular vesicles. By using a single virus particle fluorescent labeling technique, we confirmed that IFITM3 can restrict PRRSV membrane fusion by inducing accumulation of cholesterol in cellular vesicles. Additionally, we found that both endogenous and exogenous IFITM3 are incorporated into newly producing PRRS virions and diminish viral intrinsic infectivity. By using cell co-culture systems, we found that IFITM3 effectively restricted PRRSV intercellular transmission, which may have been caused by disrupted membrane fusion and reduced viral infectivity. In conclusion, our results demonstrate, for the first time, that swine IFITM3 interferes with the life cycle of PRRSV, and possibly other enveloped Arteritis viruses, at multiple steps. IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS), which is caused by PRRS virus (PRRSV), is of great economic significance to the swine industry. Due to the complicated immune escape mechanisms of PRRSV, there are no effective vaccines or therapeutic drugs currently available against PRRS. Identification of cellular factors and underlying mechanisms that establish an effective antiviral state against PRRSV can provide unique strategies for developing antiviral vaccines or drugs. As an IFN stimulated gene, the role of IFN-induced transmembrane 3 (IFITM3) in PRRSV infection has not been reported as of yet. In the present study, it was shown that IFITM3 can exert a potent anti-PRRSV effect, and PRRS virions are trafficked to IFITM3-containing cell vesicles, where viral membrane fusion is impaired by cholesterol accumulation that is induced by IFITM3. Additionally, both endogenous and exogenous IFITM3 are incorporated into newly assembled progeny virions and this decreased their intrinsic infectivity.

“…Viral replication was determined by titration as described previously [40] with the following modifications. MARC-145 cells were seeded in a 96-well plate 16 h before viral infection.…”

Section: Virus Titrationmentioning

confidence: 99%

Cellular microRNA miR-c89 inhibits replication of porcine reproductive and respiratory syndrome virus by targeting the host factor porcine retinoid X receptor β

Journal of General Virology

Feng

Yan

et al. 2019

Self Cite

MicroRNAs (miRNAs) play critical roles in the complex networks of virus–host interactions. Our previous research showed that porcine reproductive and respiratory syndrome virus (PRRSV) infection markedly upregulates miR-c89 expression, suggesting that miR-c89 may play an important role in PRRSV infection. The present study sought to determine the function of miR-c89 and its molecular mechanism during PRRSV infection. Using quantitative reverse transcription PCR (RT-qPCR) verification, we demonstrated that both highly pathogenic PRRSV and low-pathogenic PRRSV infection induced miR-c89 expression. The overexpression of miR-c89 significantly suppressed the replication of a variety of PRRSV strains, regardless of the timing of infection. Further, miR-c89 can directly target the 3′UTR of porcine retinoid X receptor β (RXRB) mRNA in a sequence-specific manner. Knockdown affected RXRB expression, as siRNA can suppress the replication of a variety of PRRSV strains. This work not only provides new insights into PRRSV–cell interactions, but also highlights the potential for the use of miR-c89 in the development of new antiviral strategies to combat PRRSV infection.