Abhijeet Bakre

Respiratory syncytial virus modifies microRNAs regulating host genes that affect virus replication.

Respiratory syncytial virus (RSV) causes substantial morbidity and life-threatening lower respiratory tract disease in infants, young children and the elderly. Understanding the host response to RSV infection is critical for developing disease-intervention approaches. The role of microRNAs (miRNAs) in post-transcriptional regulation of host genes responding to RSV infection is not well understood. In this study, it was shown that RSV infection of a human alveolar epithelial cell line (A549) induced five miRNAs (let-7f, miR-24, miR-337-3p, miR-26b and miR-520a-5p) and repressed two miRNAs (miR-198 and miR-595), and showed that RSV G protein triggered let-7f expression. Luciferase–untranslated region reporters and miRNA mimics and inhibitors validated the predicted targets, which included cell-cycle genes (CCND1, DYRK2 and ELF4), a chemokine gene (CCL7) and the suppressor of cytokine signalling 3 gene (SOCS3). Modulating let-7 family miRNA levels with miRNA mimics and inhibitors affected RSV replication, indicating that RSV modulates host miRNA expression to affect the outcome of the antiviral host response, and this was mediated in part through RSV G protein expression.

MicroRNA Regulation of Human Protease Genes Essential for Influenza Virus Replication

Influenza A virus causes seasonal epidemics and periodic pandemics threatening the health of millions of people each year. Vaccination is an effective strategy for reducing morbidity and mortality, and in the absence of drug resistance, the efficacy of chemoprophylaxis is comparable to that of vaccines. However, the rapid emergence of drug resistance has emphasized the need for new drug targets. Knowledge of the host cell components required for influenza replication has been an area targeted for disease intervention. In this study, the human protease genes required for influenza virus replication were determined and validated using RNA interference approaches. The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-κB), cAMP/calcium signaling (CRE/CREB), and apoptosis. Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication. These findings identify unique host genes and microRNAs important for influenza replication providing potential new targets for disease intervention strategies.

Identification of Host Kinase Genes Required for Influenza Virus Replication and the Regulatory Role of MicroRNAs.

Human protein kinases (HPKs) have profound effects on cellular responses. To better understand the role of HPKs and the signaling networks that influence influenza virus replication, a small interfering RNA (siRNA) screen of 720 HPKs was performed. From the screen, 17 HPKs (NPR2, MAP3K1, DYRK3, EPHA6, TPK1, PDK2, EXOSC10, NEK8, PLK4, SGK3, NEK3, PANK4, ITPKB, CDC2L5 (CDK13), CALM2, PKN3, and HK2) were validated as essential for A/WSN/33 influenza virus replication, and 6 HPKs (CDK13, HK2, NEK8, PANK4, PLK4 and SGK3) were identified as vital for both A/WSN/33 and A/New Caledonia/20/99 influenza virus replication. These HPKs were found to affect multiple host pathways and regulated by miRNAs induced during infection. Using a panel of miRNA agonists and antagonists, miR-149* was found to regulate NEK8 expression, miR-548d-3p was found to regulate MAPK1 transcript expression, and miRs -1228 and -138 to regulate CDK13 expression. Up-regulation of miR-34c induced PLK4 transcript and protein expression and enhanced influenza virus replication, while miR-34c inhibition reduced viral replication. These findings identify HPKs important for influenza viral replication and show the miRNAs that govern their expression.

MicroRNA-555 has potent antiviral properties against poliovirus.

Vaccination with live-attenuated polio vaccine has been the primary reason for the drastic reduction of poliomyelitis worldwide. However, reversion of this attenuated poliovirus vaccine occasionally results in the emergence of vaccine-derived polioviruses that may cause poliomyelitis. Thus, the development of anti-poliovirus agents remains a priority for control and eradication of the disease. MicroRNAs (miRNAs) have been shown to regulate viral infection through targeting the viral genome or reducing host factors required for virus replication. However, the roles of miRNAs in poliovirus (PV) replication have not been fully elucidated. In this study, a library of 1200 miRNA mimics was used to identify miRNAs that govern PV replication. High-throughput screening revealed 29 miRNAs with antiviral properties against Sabin-2, which is one of the oral polio vaccine strains. In particular, miR-555 was found to have the most potent antiviral activity against three different oral polio attenuated vaccine strains tested. The results show that miR-555 reduced the level of heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP C) required for PV replication in the infected cells, which in turn resulted in reduction of PV positive-strand RNA synthesis and production of infectious progeny. These findings provide the first evidence for the role of miR-555 in PV replication and reveal that miR-555 could contribute to the development of antiviral therapeutic strategies against PV.

siRNA Genome Screening Approaches to Therapeutic Drug Repositioning

Bridging high-throughput screening (HTS) with RNA interference (RNAi) has allowed for rapid discovery of the molecular basis of many diseases, and identification of potential pathways for developing safe and effective treatments. These features have identified new host gene targets for existing drugs paving the pathway for therapeutic drug repositioning. Using RNAi to discover and help validate new drug targets has also provided a means to filter and prioritize promising therapeutics. This review summarizes these approaches across a spectrum of methods and targets in the host response to pathogens. Particular attention is given to the utility of drug repurposing utilizing the promiscuous nature of some drugs that affect multiple molecules or pathways, and how these biological pathways can be targeted to regulate disease outcome.

MicroRNA screening identifies miR-134 as a regulator of poliovirus and enterovirus 71 infection

MicroRNAs (miRNAs) regulate virus replication through multiple mechanisms. Poliovirus causes a highly debilitating disease and though global efforts to eradicate polio have sharply decreased polio incidence, unfortunately three countries (Afghanistan, Nigeria and Pakistan) remain polio-endemic. We hypothesize that understanding the host factors involved in polio replication will identify novel prophylactic and therapeutic targets against polio and related viruses. In this data set, employing genome wide screens of miRNA mimics and inhibitors, we identified miRNAs which significantly suppressed polio replication. Specifically, miR-134 regulates poliovirus replication via modulation of ras-related nuclear protein (RAN), an important component of the nuclear transport system. MiR-134 also inhibited other Picornaviridae viruses including EV71, a growing concern and a high priority for vaccination in Asian countries like China. These findings demonstrate a novel mechanism for miRNA regulation of poliovirus and other Picornaviridae viruses in host cells, and thereby may provide a novel approach in combating infection and a potential approach for the development of anti-Picornaviridae strategies.

The Central Conserved Region (CCR) of Respiratory Syncytial Virus (RSV) G Protein Modulates Host miRNA Expression and Alters the Cellular Response to Infection

Respiratory Syncytial Virus (RSV) infects respiratory epithelial cells and deregulates host gene expression by many mechanisms including expression of RSV G protein (RSV G). RSV G protein encodes a central conserved region (CCR) containing a CX3C motif that functions as a fractalkine mimic. Disruption of the CX3C motif (a.a. 182–186) located in the CCR of the G protein has been shown to affect G protein function in vitro and the severity of RSV disease pathogenesis in vivo. We show that infection of polarized Calu3 respiratory cells with recombinant RSV having point mutations in Cys173 and 176 (C173/176S) (rA2-GC12), or Cys186 (C186S) (rA2-GC4) is associated with a decline in the integrity of polarized Calu-3 cultures and decreased virus production. This is accompanied with downregulation of miRNAs let-7f and miR-24 and upregulation of interferon lambda (IFNλ), a primary antiviral cytokine for RSV in rA2-GC12/rA2-GC4 infected cells. These results suggest that residues in the cysteine noose region of RSV G protein can modulate IFN λ expression accompanied by downregulation of miRNAs, and are important for RSV G protein function and targeting.

A universal mammalian vaccine cell line substrate

Using genome-wide small interfering RNA (siRNA) screens for poliovirus, influenza A virus and rotavirus, we validated the top 6 gene hits PV, RV or IAV to search for host genes that when knocked-down (KD) enhanced virus permissiveness and replication over wild type Vero cells or HEp-2 cells. The enhanced virus replication was tested for 12 viruses and ranged from 2-fold to >1000-fold. There were variations in virus-specific replication (strain differences) across the cell lines examined. Some host genes (CNTD2, COQ9, GCGR, NDUFA9, NEU2, PYCR1, SEC16G, SVOPL, ZFYVE9, and ZNF205) showed that KD resulted in enhanced virus replication. These findings advance platform-enabling vaccine technology, the creation of diagnostic cells substrates, and are informative about the host mechanisms that affect virus replication in mammalian cells.

Roles of Non-coding RNAs in Respiratory Syncytial Virus (RSV) Infection.

Analysis of host gene expression profiles following viral infections of target cells/tissues can reveal crucial insights into the host: virus interaction and enables the development of novel therapeutics and prophylactics. Regions of the host genome that do not code for protein, encode structural, and functional non-coding RNAs that are important not only in regulation of host gene expression but also may impact viral replication. This review summarizes the role of host non-coding RNAs during replication of multiple respiratory viruses with a focus on Respiratory Syncytial Virus (RSV), an important pediatric pathogen. This review highlights the current state of knowledge and understanding regarding the function(s) of ncRNAs for respiratory viral infection and host immunity in general.