Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important viral pathogens in the swine industry. Emerging evidence indicates that the host microRNAs (miRNAs) are involved in host-pathogen interactions. However, whether host miRNAs can target PRRSV and be used to inhibit PRRSV infection has not been reported. Recently, microRNA 181 (miR-181) has been identified as a positive regulator of immune response, and here we report that miR-181 can directly impair PRRSV infection. Our results showed that delivered miR-181 mimics can strongly inhibit PRRSV replication in vitro through specifically binding to a highly (over 96%) conserved region in the downstream of open reading frame 4 (ORF4) of the viral genomic RNA. The inhibition of PRRSV replication was specific and dose dependent. In PRRSV-infected Marc-145 cells, the viral mRNAs could compete with miR-181-targeted sequence in luciferase vector to interact with miR-181 and result in less inhibition of luciferase activity, further demonstrating the specific interactions between miR-181 and PRRSV RNAs. As expected, miR-181 and other potential PRRSV-targeting miRNAs (such as miR-206) are expressed much more abundantly in minimally permissive cells or tissues than in highly permissive cells or tissues. Importantly, highly pathogenic PRRSV (HP-PRRSV) strain-infected pigs treated with miR-181 mimics showed substantially decreased viral loads in blood and relief from PRRSV-induced fever compared to negative-control (NC)-treated controls. These results indicate the important role of host miRNAs in modulating PRRSV infection and viral pathogenesis and also support the idea that host miRNAs could be useful for RNA interference (RNAi)-mediated antiviral therapeutic strategies. P orcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important viral pathogens in pigs, leading to significant economic losses in the swine industry worldwide. PRRS is characterized by severe reproductive failure in sows and respiratory syndromes and persistent infection in young pigs. Atypical PRRS is characterized by high fever, high morbidity, and high mortality in pigs of all ages and emerged in China in 2006. The causative agent was confirmed to be a highly pathogenic PRRSV (HP-PRRSV) with a discontinuous deletion of 30 amino acids in nonstructural protein 2 (NSP2) (1). PRRSV is classified within the family Arteriviridae, order Nidovirales. It has a singlestranded, 5=-capped positive-sense RNA genome of approximately 15.4 kb, containing at least 9 opening reading frames (ORFs) (1, 2). MicroRNAs (miRNAs), which can be produced by hosts or viruses, are a class of small noncoding RNAs that can inhibit gene expression through base-pairing interactions between the loaded miRNA and its mRNA target. Recent studies have reported that cellular miRNAs can target viral RNAs during infections, resulting in inhibition of virus replication as a new antiviral defense (3-8) or a new pathway to alter the virus life cycle (9-11), whereas virusderive...

Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly infectious pathogen that causes severe diseases in pigs and great economic losses to the swine industry worldwide. Type I interferons (IFNs) play a crucial role in antiviral immunity. In the present study, we demonstrated that infection with the highly pathogenic PRRSV strain JXwn06 antagonized type I IFN expression induced by poly(I·C) in both porcine alveolar macrophages (PAMs) and blood monocyte-derived macrophages (BMo). Subsequently, we showed that the inhibition of poly(I·C)-induced IFN-␤ production by PRRSV was dependent on the blocking of NF-B signaling pathways. By screening PRRSV nonstructural and structural proteins, we demonstrated that nonstructural protein 4 (nsp4), a viral 3C-like serine protease, significantly suppressed IFN-␤ expression. Moreover, we verified that nsp4 inhibited NF-B activation induced by signaling molecules, including RIG-I, VISA, TRIF, and IKK␤. nsp4 was shown to target the NF-B essential modulator (NEMO) at the E349-S350 site to mediate its cleavage. Importantly, nsp4 mutants with defective protease activity abolished its ability to cleave NEMO and inhibit IFN-␤ production. These findings might have implications for our understanding of PRRSV pathogenesis and its mechanisms for evading the host immune response. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is a major agent of respiratory diseases in pigs.Like many other viruses, PRRSV has evolved a variety of strategies to evade host antiviral innate immunity for survival and propagation. In this study, we show that PRRSV nsp4 is a novel antagonist of the NF-B signaling pathway, which is responsible for regulating the expression of type I interferons and other crucial cytokines. We then investigated the underlying mechanism used by nsp4 to suppress NF-B-mediated IFN-␤ production. We found that nsp4 interfered with the NF-B signaling pathway through the cleavage of NEMO (a key regulator of NF-B signaling) at the E349-S350 site, leading to the downregulation of IFN-␤ production induced by poly(I·C). The data presented here may help us to better understand PRRSV pathogenesis.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression post-transcriptionally and play critical roles in intricate networks of host-pathogen interactions and innate immunity. Porcine reproductive and respiratory syndrome (PRRS) is one of the most important diseases affecting swine industry worldwide. Here, we demonstrated that miR-23, miR-378, and miR-505 were antiviral host factors against PRRS virus (PRRSV). Over-expression of the three miRNAs inhibited PRRSV infection in a dose-dependent manner, respectively. Blockage of the three endogenously expressed miRNAs significantly enhanced PRRSV replication. Different type 2 PRRSV strains harbored conserved miR-23, miR-378, and miR-505 target sites (TSs) that were sufficient to confer miRNA-mediated repression of PRRSV replication. Interestingly, miR-23 was capable of inducing type I interferon expression during PRRSV infection through IRF3/IRF7 activation, which might further lead to the inhibition of virus infection. These results suggest that miR-23, miR-378, and miR-505, especially miR-23, may have the potential to be used as antiviral therapy against PRRSV infection.

Virus infection of mammalian cells triggers host innate immune responses to restrict viral replication and induces adaptive immunity for viral elimination. In order to survive and propagate, viruses have evolved sophisticated mechanisms to subvert host defense system by encoding proteins that target key components of the immune signaling pathways. Porcine reproductive and respiratory syndrome virus (PRRSV), a RNA virus, impairs several processes of host immune responses including interfering with interferon production and signaling, modulating cytokine expression, manipulating apoptotic responses and regulating adaptive immunity. In this review, we highlight the molecular mechanisms of how PRRSV interferes with the different steps of initial antiviral host responses to establish persistent infection in pigs. Dissection of the PRRSV-host interaction is the key in understanding PRRSV pathogenesis and will provide a basis for the rational design of vaccines.

A major challenge in finding a cure for HIV-1/AIDS is the difficulty in identifying and eradicating persistent reservoirs of replication-competent provirus. Long noncoding RNAs (lncRNAs, >200 nucleotides) are increasingly recognized to play important roles in pathophysiology. Here, we report the first genome-wide expression analysis of lncRNAs in HIV-1-infected primary monocyte-derived macrophages (MDMs). We identified an lncRNA, which we named HIV-1-enhanced lncRNA (HEAL), that is upregulated by HIV-1 infection of MDMs, microglia, and T lymphocytes. Peripheral blood mononuclear cells of HIV-1-infected individuals show elevated levels of HEAL. Importantly, HEAL is a broad enhancer of multiple HIV-1 strains because depletion of HEAL inhibited X4, R5, and dual-tropic HIV replications and the inhibition was rescued by HEAL overexpression. HEAL forms a complex with the RNA-binding protein FUS, which facilitates HIV replication through at least two mechanisms: (i) HEAL-FUS complex binds the HIV promoter and enhances recruitment of the histone acetyltransferase p300, which positively regulates HIV transcription by increasing histone H3K27 acetylation and P-TEFb enrichment on the HIV promoter, and (ii) HEAL-FUS complex is enriched at the promoter of the cyclin-dependent kinase 2 gene, CDK2, to enhance CDK2 expression. Notably, HEAL knockdown and knockout mediated by RNA interference (RNAi) and CRISPR-Cas9, respectively, prevent HIV-1 recrudescence in T cells and microglia upon cessation of azidothymidine treatment in vitro. Our results suggest that silencing of HEAL or perturbation of the HEAL-FUS ribonucleoprotein complex could provide a new epigenetic silencing strategy to eradicate viral reservoirs and effect a cure for HIV-1/AIDS. IMPORTANCE Despite our increased understanding of the functions of lncRNAs, their potential to develop HIV/AIDS cure strategies remains unexplored. A genome-wide analysis of lncRNAs in HIV-1-infected primary monocyte-derived macrophages (MDMs) was performed, and 1,145 differentially expressed lncRNAs were identified. An lncRNA named HIV-1-enhanced lncRNA (HEAL) is upregulated by HIV-1 infection and promotes HIV replication in T cells and macrophages. HEAL forms a complex with the RNA-binding protein FUS to enhance transcriptional coactivator p300 recruitment to the HIV promoter. Furthermore, HEAL knockdown and knockout prevent HIV-1 recrudescence in T cells and microglia upon cessation of azidothymidine treatment, suggesting HEAL as a potential therapeutic target to cure HIV-1/AIDS.