RESUMO
Endogenous retroviruses (ERVs) are subject to transcriptional repression in adult tissues, in part to prevent autoimmune responses. However, little is known about the epigenetic silencing of ERV expression. Here, we describe a new role for inhibitor of growth family member 3 (ING3), to add to an emerging group of ERV transcriptional regulators. Our results show that ING3 binds to several ERV promoters (for instance MER21C) and establishes an EZH2-mediated H3K27 trimethylation modification. Loss of ING3 leads to decreases of H3K27 trimethylation enrichment at ERVs, induction of MDA5-MAVS-interferon signaling, and functional inhibition of several virus infections. These data demonstrate an important new function of ING3 in ERV silencing and contributing to innate immune regulation in somatic cells.
Assuntos
Retrovirus Endógenos , Inativação Gênica , Proteínas de Homeodomínio/fisiologia , Imunidade Inata/genética , Proteínas Supressoras de Tumor/fisiologia , Sistemas CRISPR-Cas , Células HT29 , Células HeLa , Código das Histonas , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas Supressoras de Tumor/metabolismoRESUMO
Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-stimulated gene that shows broad antiviral activities against a wide range of enveloped viruses. Here, using an IFN-stimulated gene screen against vesicular stomatitis virus (VSV)-SARS-CoV and VSV-SARS-CoV-2 chimeric viruses, we identified CH25H and its enzymatic product 25-hydroxycholesterol (25HC) as potent inhibitors of SARS-CoV-2 replication. Internalized 25HC accumulates in the late endosomes and potentially restricts SARS-CoV-2 spike protein catalyzed membrane fusion via blockade of cholesterol export. Our results highlight one of the possible antiviral mechanisms of 25HC and provide the molecular basis for its therapeutic development.
Assuntos
Tratamento Farmacológico da COVID-19 , Endossomos/genética , Hidroxicolesteróis/farmacologia , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Antivirais/farmacologia , COVID-19/metabolismo , COVID-19/patologia , COVID-19/virologia , Endossomos/metabolismo , Humanos , Interferons/metabolismo , Fusão de Membrana/efeitos dos fármacos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/patogenicidade , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/genética , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
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.
Assuntos
Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Anticorpos de Domínio Único/farmacologia , Animais , Antivirais/farmacologia , Linhagem Celular , Células Cultivadas , Macrófagos Alveolares/virologia , Peptídeos , Síndrome Respiratória e Reprodutiva Suína/virologia , Ligação Proteica , Anticorpos de Domínio Único/metabolismo , Suínos/virologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) exhibits a highly restricted tropism for cells of the monocyte-macrophage lineage, utilizing porcine CD163 (pCD163) as an indispensable cellular receptor for infection. Transfection the gene of pCD163 into several non-permissive cell lines followed by protein expression confers susceptibility to PRRSV. A lack of specialized porcine antibody tools for use with existing porcine-derived primary cells and cell lines has hampered studies of both PRRSV pathogenesis and virus triggering of immune response cascades. Therefore, we constructed PRRSV-susceptible murine alveolar macrophage-derived MH-S and peritoneal macrophage-like RAW264.7 cell lines by achieving pCD163 cell surface expression in these cells. We then evaluated PRRSV susceptibility and cytokine expression patterns induced upon PRRSV infection of these pCD163-expressing cell lines. RESULTS: Growth of MH-SCD163 and RAW264.7CD163 cells was indistinguishable from growth of un-transfected parental cell lines. Meanwhile, various stages of the PRRSV replication cycle, including viral particle attachment, internalization, disassembly and infection were confirmed in both pCD163-transfected cell lines. Analysis of PRRSV replication using immunofluorescence staining of virus and viral titration of cell lysates demonstrated that both MH-SCD163 and RAW264.7CD163 cells supported replication of various genotype 2 PRRSV isolates. Moreover, PRRSV replication in MH-SCD163 cells was similar to that observed in porcine alveolar macrophages (PAMs) and was more efficient than in RAW264.7CD163 cells. However, peak virus titers in MH-SCD163 cells were attained at 60 h post-infection (pi) versus 48 hpi in PAMs. Analysis of cytokine expression showed that post-PRRSV infection, mRNA expression patterns of anti-inflammatory cytokines (IL-4 and IL-10) and pro-inflammatory cytokines (TNF-α and IFN-γ) in MH-SCD163 cells were more similar to those observed in PAMs versus levels in RAW264.7CD163 cells. CONCLUSIONS: MH-S and RAW264.7 cells were not susceptible to PRRSV infection until transfection and subsequent expression of pCD163 were achieved in these cell lines. The PRRSV-susceptible MH-SCD163 cell line efficiently supported viral replication of various genotype 2 PRRSV isolates and exhibited similar cytokine expression patterns as observed in PAMs. In conclusion, this work describes the development of new tools to further understand PRRSV pathogenesis and immune response mechanisms to PRRSV infection.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Macrófagos/citologia , Macrófagos/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/crescimento & desenvolvimento , Receptores de Superfície Celular/metabolismo , Cultura de Vírus/métodos , Animais , Linhagem Celular , Proliferação de Células , Macrófagos/metabolismo , Camundongos , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/metabolismo , Células RAW 264.7 , SuínosRESUMO
Rotaviruses (RVs) are one of the main causes of severe gastroenteritis, diarrhea, and death in children and young animals. While suckling mice prove to be highly useful small animal models of RV infection and pathogenesis, direct visualization tools are lacking to track the temporal dynamics of RV replication and transmissibility in vivo. Here, we report the generation of the first recombinant murine-like RV that encodes a Nano-Luciferase reporter (NLuc) using a newly optimized RV reverse genetics system. The NLuc-expressing RV was replication-competent in cell culture and both infectious and virulent in neonatal mice in vivo. Strong luciferase signals were detected in the proximal and distal small intestines, colon, and mesenteric lymph nodes. We showed, via a noninvasive in vivo imaging system, that RV intestinal replication peaked at days 2 to 5 post infection. Moreover, we successfully tracked RV transmission to uninoculated littermates as early as 3 days post infection, 1 day prior to clinically apparent diarrhea and 3 days prior to detectable fecal RV shedding in the uninoculated littermates. We also observed significantly increased viral replication in Stat1 knockout mice that lack the host interferon signaling. Our results suggest that the NLuc murine-like RV represents a non-lethal powerful tool for the studies of tissue tropism and host and viral factors that regulate RV replication and spread, as well as provides a new tool to facilitate the testing of prophylactic and therapeutic interventions in the future.
Assuntos
Infecções por Rotavirus , Rotavirus , Animais , Diarreia , Camundongos , Camundongos Knockout , Rotavirus/genética , Infecções por Rotavirus/genética , TropismoRESUMO
N6-methyladenosine (m6A) is an abundant mRNA modification and affects many biological processes. However, how m6A levels are regulated during physiological or pathological processes such as virus infections, and the in vivo function of m6A in the intestinal immune defense against virus infections are largely unknown. Here, we uncover a novel antiviral function of m6A modification during rotavirus (RV) infection in small bowel intestinal epithelial cells (IECs). We found that rotavirus infection induced global m6A modifications on mRNA transcripts by down-regulating the m6a eraser ALKBH5. Mice lacking the m6A writer enzymes METTL3 in IECs (Mettl3ΔIEC) were resistant to RV infection and showed increased expression of interferons (IFNs) and IFN-stimulated genes (ISGs). Using RNA-sequencing and m6A RNA immuno-precipitation (RIP)-sequencing, we identified IRF7, a master regulator of IFN responses, as one of the primary m6A targets during virus infection. In the absence of METTL3, IECs showed increased Irf7 mRNA stability and enhanced type I and III IFN expression. Deficiency in IRF7 attenuated the elevated expression of IFNs and ISGs and restored susceptibility to RV infection in Mettl3ΔIEC mice. Moreover, the global m6A modification on mRNA transcripts declined with age in mice, with a significant drop from 2 weeks to 3 weeks post birth, which likely has broad implications for the development of intestinal immune system against enteric viruses early in life. Collectively, we demonstrated a novel host m6A-IRF7-IFN antiviral signaling cascade that restricts rotavirus infection in vivo.
Assuntos
Intestinos/imunologia , Infecções por Rotavirus/imunologia , Rotavirus/classificação , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Animais , Linhagem Celular , Testes Genéticos , Humanos , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Carga ViralRESUMO
Pathogenic coronaviruses are a major threat to global public health. Here, using a recombinant reporter virus-based compound screening approach, we identified small-molecule inhibitors that potently block the replication of severe acute respiratory syndrome virus 2 (SARS-CoV-2). Among them, JIB-04 inhibited SARS-CoV-2 replication in Vero E6 cells with a 50% effective concentration of 695 nM, with a specificity index of greater than 1,000. JIB-04 showed in vitro antiviral activity in multiple cell types, including primary human bronchial epithelial cells, against several DNA and RNA viruses, including porcine coronavirus transmissible gastroenteritis virus. In an in vivo porcine model of coronavirus infection, administration of JIB-04 reduced virus infection and associated tissue pathology, which resulted in improved weight gain and survival. These results highlight the potential utility of JIB-04 as an antiviral agent against SARS-CoV-2 and other viral pathogens. IMPORTANCE The coronavirus disease 2019 (COVID-19), the disease caused by SARS-CoV-2 infection, is an ongoing public health disaster worldwide. Although several vaccines are available as a preventive measure and the FDA approval of an orally bioavailable drug is on the horizon, there remains a need for developing antivirals against SARS-CoV-2 that could work on the early course of infection. By using infectious reporter viruses, we screened small-molecule inhibitors for antiviral activity against SARS-CoV-2. Among the top hits was JIB-04, a compound previously studied for its anticancer activity. Here, we showed that JIB-04 inhibits the replication of SARS-CoV-2 as well as different DNA and RNA viruses. Furthermore, JIB-04 conferred protection in a porcine model of coronavirus infection, although to a lesser extent when given as therapeutic rather than prophylactic doses. Our findings indicate a limited but still promising utility of JIB-04 as an antiviral agent in the combat against COVID-19 and potentially other viral diseases.
Assuntos
COVID-19 , SARS-CoV-2 , Chlorocebus aethiops , Humanos , Animais , Suínos , Antivirais/farmacologia , COVID-19/metabolismo , Replicação Viral , Células VeroRESUMO
Rotavirus (RV)-encoded nonstructural protein 1 (NSP1), the product of gene segment 5, effectively antagonizes host interferon (IFN) signaling via multiple mechanisms. Recent studies with the newly established RV reverse genetics system indicate that NSP1 is not essential for the replication of the simian RV SA11 strain in cell culture. However, the role of NSP1 in RV infection in vivo remains poorly characterized due to the limited replication of heterologous simian RVs in the suckling mouse model. Here, we used an optimized reverse genetics system and successfully recovered recombinant murine RVs with or without NSP1 expression. While the NSP1-null virus replicated comparably with the parental murine RV in IFN-deficient and IFN-competent cell lines in vitro, it was highly attenuated in 5-day-old wild-type suckling pups in both the 129sv and C57BL/6 backgrounds. In the absence of NSP1 expression, murine RV had significantly reduced replication in the ileum, systemic spread to mesenteric lymph nodes, fecal shedding, diarrhea occurrence, and transmission to uninoculated littermates. The defective replication of the NSP1-null RV in small intestinal tissues occurred as early as 1 day postinfection. Of interest, the replication and pathogenesis defects of NSP1-null RV were only minimally rescued in Stat1 knockout pups, suggesting that NSP1 facilitates RV replication in an IFN-independent manner. Our findings highlight a pivotal function of NSP1 during homologous RV infections in vivo and identify NSP1 as an ideal viral protein for targeted attenuation for future vaccine development. IMPORTANCE Rotavirus remains one of the most important causes of severe diarrhea and dehydration in young children worldwide. Although NSP1 is dispensable for rotavirus replication in cell culture, its exact role in virus infection in vivo remains unclear. In this study, we demonstrate, for the first time in a pathologically valid homologous small animal model, that in the context of a fully replication-competent, pathogenic, and transmissible murine rotavirus, loss of NSP1 expression substantially attenuated virus replication in the gastrointestinal tract, diarrheal disease, and virus transmission. Notably, the NSP1-deficient murine rotavirus also replicated poorly in mice lacking host interferon or inflammasome signaling. Our data provide the first piece of evidence that NSP1 is essential for murine rotavirus replication in vivo, making it an attractive target for developing improved next-generation rotavirus vaccines better suited for socioeconomically disadvantaged and immunocompromised individuals.
Assuntos
Intestinos/virologia , Infecções por Rotavirus/virologia , Rotavirus/fisiologia , Rotavirus/patogenicidade , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Animais , Humanos , Interferons/genética , Interferons/metabolismo , Intestinos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Rotavirus/genética , Infecções por Rotavirus/genética , Infecções por Rotavirus/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Proteínas não Estruturais Virais/genéticaRESUMO
Pathogenic coronaviruses represent a major threat to global public health. Here, using a recombinant reporter virus-based compound screening approach, we identified several small-molecule inhibitors that potently block the replication of the newly emerged severe acute respiratory syndrome virus 2 (SARS-CoV-2). Among them, JIB-04 inhibited SARS-CoV-2 replication in Vero E6 cells with an EC50 of 695 nM, with a specificity index of greater than 1,000. JIB-04 showed in vitro antiviral activity in multiple cell types against several DNA and RNA viruses, including porcine coronavirus transmissible gastroenteritis virus. In an in vivo porcine model of coronavirus infection, administration of JIB-04 reduced virus infection and associated tissue pathology, which resulted in improved weight gain and survival. These results highlight the potential utility of JIB-04 as an antiviral agent against SARS-CoV-2 and other viral pathogens.
RESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) has a highly restricted tropism for cells of the monocyte-macrophage lineage, including porcine alveolar macrophages (PAMs). PRRSV entry into permissive cells involves several mediators in addition to two required host cell receptors, CD163 and MYH9. It is unknown whether CD163 directly interacts and/or cooperates with MYH9 to facilitate PRRSV infection. In this study, CD163 and MYH9 were co-immunoprecipitated from PAMs regardless of PRRSV infection status. Further truncation analysis indicated that the CD163 N-terminal region, containing scavenger receptor cysteine-rich domains 1 to 4 (SRCR1-4), directly interacts with the MYH9 C-terminal domain region without involvement of other adaptor proteins. Meanwhile, non-permissive HEK293T cells that stably expressed truncated swine CD163 SRCR1-4 domain did not support virus attachment. However, virus attachment to cells stably expressing SRCR5-CT domain was demonstrated to occur without appreciable virus internalization. The involvement of the SRCR1-4 domain in virus internalization was further demonstrated by the fact that incubation of recombinant SRCR1-4 protein with PAMs abolished subsequent virus internalization by permissive cells. These results demonstrated that CD163 SRCR1-4 interacts with the MYH9 C-terminal domain to facilitate PRRSV virion internalization in permissive cells, thus expanding our understanding of PRRSV cell-invasion mechanisms.
RESUMO
Prevention and control of infection by porcine reproductive and respiratory syndrome virus (PRRSV) remains a challenge, due to our limited understanding of the PRRSV invasion mechanism. Our previous study has shown that PRRSV glycoprotein GP5 interacts with MYH9 C-terminal domain protein (PRA). Here we defined that the first ectodomain of GP5 (GP5-ecto-1) directly interacted with PRA and this interaction triggered PRA and endogenous MYH9 to form filament assembly. More importantly, MYH9 filament assembly was also formed in GP5-ecto-1-transfected MARC-145 cells. Notably, PRRSV infection of MARC-145 cells and porcine alveolar macrophages also induced endogenous MYH9 aggregation and polymerization that were required for subsequent PRRSV internalization. Moreover, overexpression of S100A4, a MYH9-specific disassembly inducer, in MARC-145 cells significantly resulted in diminished MYH9 aggregation and marked inhibition of subsequent virion internalization and infection by both PRRSV-1 and PRRSV-2 isolates. The collective results of this work reveal a novel molecular mechanism employed by MYH9 that helps PRRSV gain entry into permissive cells.
RESUMO
MYH9 has been identified as an indispensable cellular protein for porcine reproductive and respiratory syndrome virus (PRRSV) entry into permissive cells using the monoclonal anti-idiotypic antibody (Mab2-5G2) recognizing an antibody that specifically interacts with PRRSV glycoprotein 5 (GP5). More recently, we found that Mab2-5G2 interacted with the MYH9 C-terminal domain, designated PRA, which is required for PRRSV internalization. In this study, we demonstrate that blocking of MYH9 with Mab2-5G2 significantly diminished PRRSV internalization by porcine alveolar macrophage (PAM) via interruption of direct interaction between GP5 and MYH9, and thus remarkably inhibited subsequent infection of PAMs by PRRSV-2 isolates. Moreover, the three-dimensional structure of the Mab2-5G2 Fab-PRA complex determined via homology modeling predicted potential docking sites required for PRRSV internalization. Further analysis of Mab2-5G2-binding sites within PRA highlighted that the amino acids E1670, K1673, E1679, and I1683 in PRA are the key Mab2-5G2-binding residues. Notably, recombinant PRA protein blocked the interaction between PRRSV GP5 and cellular MYH9 by preventing translocation of MYH9 from the cytoplasm to the cell membrane, an essential step for PRRSV virion internalization. Meanwhile, porcine cell line permissive for PRRSV bearing point mutation of E1670A in MYH9 demonstrated reduced susceptibility for PRRSV infection. In conclusion, this work increases understanding of both PRRSV pathogenesis and the mechanistic role played by MYH9 in PRRSV infection.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/virologia , Cadeias Pesadas de Miosina/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Proteínas do Envelope Viral/imunologia , Internalização do Vírus , Aminoácidos/química , Animais , Anticorpos Monoclonais/imunologia , Sítios de Ligação de Anticorpos , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , SuínosRESUMO
Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important infectious diseases impacting the swine industry worldwide. Prevention and control of PRRS have been problematic, as vaccination has achieved little success. MYH9 (encoded by the gene MYH9) is an essential cellular factor for PRRS virus (PRRSV) infection. The MYH9 C-terminal domain (designated PRA) interacts with viral glycoprotein 5 (GP5), a major PRRSV envelope protein. In this study, we investigated whether soluble PRA could serve as a novel blocking agent of PRRSV infection. Our data showed that preincubation of PRRSV with PRA inhibited virus infection of susceptible cells in a dose-dependent manner. Notably, PRA also exhibited broad-spectrum ability to inhibit infection with diverse strains of both PRRSV genotype 1 and 2. Analysis of the interaction between PRA and PRRSV GP5 revealed that PRA is able to capture PRRSV virions. In conclusion, our data suggest that PRA could serve as a novel broad-spectrum inhibitor of infection by heterogeneous PRRSV strains in vivo.
Assuntos
Antivirais/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Proteínas Recombinantes/metabolismo , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Animais , Antivirais/isolamento & purificação , Células Cultivadas , Macrófagos Alveolares/virologia , Cadeias Pesadas de Miosina/genética , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , SuínosRESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important viral pathogens in the swine industry. Current antiviral strategies do not effectively prevent and control PRRSV. Recent reports show that microRNAs (miRNAs) play vital roles in viral infections by post transcriptionally regulating the expression of viral or host genes. Our previous research showed that non-muscle myosin heavy chain 9 (MYH9) is an essential factor for PRRSV infection. Using bioinformatic prediction and experimental verification, we demonstrate that MYH9 expression is regulated by the miRNA let-7f-5p, which binds to the MYH9 mRNA 3'UTR and may play an important role during PRRSV infection. To understand how let-7f-5p regulates PRRSV infection, we analyzed the expression pattern of both let-7f-5p and MYH9 in porcine alveolar macrophages (PAMs) after infection with either highly pathogenic PRRSV (HP-PRRSV) or classical type PRRSV (N-PRRSV) using a deep sequencing approach with quantitative real-time PCR validation. Our results showed that both HP-PRRSV and N-PRRSV infection reduced let-7f-5p expression while also inducing MYH9 expression. Furthermore, let-7f-5p significantly inhibited PRRSV replication through suppression of MYH9 expression. These findings not only provide new insights into the pathogenesis of PRRSV, but also suggest potential new antiviral strategies against PRRSV infection.
RESUMO
Many viruses encode microRNAs (miRNAs) that are small non-coding single-stranded RNAs which play critical roles in virus-host interactions. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically impactful viruses in the swine industry. The present study sought to determine whether PRRSV encodes miRNAs that could regulate PRRSV replication. Four viral small RNAs (vsRNAs) were mapped to the stem-loop structures in the ORF1a, ORF1b and GP2a regions of the PRRSV genome by bioinformatics prediction and experimental verification. Of these, the structures with the lowest minimum free energy (MFE) values predicted for PRRSV-vsRNA1 corresponded to typical stem-loop, hairpin structures. Inhibition of PRRSV-vsRNA1 function led to significant increases in viral replication. Transfection with PRRSV-vsRNA1 mimics significantly inhibited PRRSV replication in primary porcine alveolar macrophages (PAMs). The time-dependent increase in the abundance of PRRSV-vsRNA1 mirrored the gradual upregulation of PRRSV RNA expression. Knockdown of proteins associated with cellular miRNA biogenesis demonstrated that Drosha and Argonaute (Ago2) are involved in PRRSV-vsRNA1 biogenesis. Moreover, PRRSV-vsRNA1 bound specifically to the nonstructural protein 2 (NSP2)-coding sequence of PRRSV genome RNA. Collectively, the results reveal that PRRSV encodes a functional PRRSV-vsRNA1 which auto-regulates PRRSV replication by directly targeting and suppressing viral NSP2 gene expression. These findings not only provide new insights into the mechanism of the pathogenesis of PRRSV, but also explore a potential avenue for controlling PRRSV infection using viral small RNAs.