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.

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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.

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.

Mobile genetic elements, by virtue of their ability to move to new chromosomal locations, are considered important in shaping the evolutionary course of the genome. They are widespread in the biological kingdom. Among the protozoan parasites several types of transposable elements are encountered. The largest variety is seen in the trypanosomatids-Trypanosoma brucei, Trypanosoma cruzi and Crithidia fasciculata. They contain elements that insert site-specifically in the spliced-leader RNA genes, and others that are dispersed in a variety of genomic locations. Giardia lamblia contains three families of transposable elements. Two of these are subtleomeric in location while one is chromosome-internal. Entamoeba histolytica has an abundant retrotransposon dispersed in the genome. Nucleotide sequence analysis of all the elements shows that they are all retrotransposons, and, with the exception of one class of elements in T. cruzi, all of them are non-long-terminal-repeat retrotransposons. Although most copies have accumulated mutations, they can potentially encode reverse transcriptase, endonuclease and nucleic-acid-binding activities. Functionally and phylogenetically they do not belong to a single lineage, showing that retrotransposons were acquired early in the evolution of protozoan parasites. Many of the potentially autonomous elements that encode their own transposition functions have nonautonomous counterparts that probably utilize the functions in trans. In this respect these elements are similar to the mammalian LINEs and SINEs (long and short interspersed DNA elements), showing a common theme in the evolution of retrotransposons. So far there is no report of a DNA transposon in any protozoan parasite. The genome projects that are under way for most of these organisms will help understand the evolution and possible function of these genetic elements.

Influenza virus causes epidemics and sporadic pandemics resulting in morbidity, mortality and economic losses. Influenza viruses require host genes to replicate. RNA interference (RNAi) screens can identify host genes coopted by influenza for replication. Targeting these pro-influenza genes can provide therapeutic strategies to reduce virus replication. Using human lung (A549) cells, 19 pro-influenza GPCR and 13 pro-influenza ion channel genes were identified using small inferring RNAs (siRNA). These pro-influenza genes were authenticated by testing A/WSN/33, A/CA/04/09, and B/Yamagata/16/1988-infected A549 cells resulting in 16 pro-influenza GPCR and 5 pro-influenza ion channel genes being validated. These findings showed that several GPCR and ion channel genes are needed for production of infectious influenza virus. These data provide potential targets for the development of host-directed therapeutic strategies to impede the influenza productive cycle to limit infection. IMPORTANCE: Influenza epidemics result in morbidity and mortality each year. Vaccines are the most effective preventive measure but require annual reformulation as mismatch of vaccine strains can result in vaccine failure. Antiviral measures are desirable particularly when vaccines fail. In this study, we used RNAi screening to identify several GPCR and ion channel genes needed for influenza virus replication. Understanding the host genes usurped by influenza during viral replication can help identify host genes that can be targeted for drug repurposing or for the development of antiviral drugs. Targeting host genes is also refractory to drug resistance generated by viral mutations, as well as provides a platform for the development of broad spectrum anti-viral drugs.

Human respiratory syncytial virus (RSV) is a major health challenge in the young and elderly owing to the lack of a safe and effective vaccine and proven antiviral drugs. Understanding the mechanisms by which viral genes and proteins modulate the host response to infection is critical for identifying novel disease intervention strategies. In this study, the RSV non-structural protein NS1 was shown to suppress miR-24 expression during infection. Lack of NS1 was linked to increased expression of miR-24, whilst NS1 overexpression suppressed miR-24 expression. NS1 was found to induce Kruppel-like factor 6 (KLF6), a transcription factor that positively regulates the transforming growth factor (TGF)-b pathway to induce cell cycle arrest. Silencing of KLF6 led to increased miR-24 expression via downregulation of TGF-b. Treatment with exogenous TGF-b suppressed miR-24 expression and induced KLF6. Confocal microscopy showed co-localization of KLF6 and RSV NS1. These findings indicated that RSV NS1 interacts with KLF6 and modulates miR-24 expression and TGF-b, which facilitates RSV replication. Received 23 March 2015Accepted 6 August 2015 INTRODUCTIONHuman respiratory syncytial virus (RSV) is a ubiquitous negative-sense ssRNA virus that can cause severe lung disease following infection (CDC, 2013;Hall et al., 2009;Nair et al., 2010). RSV is a member of the genus Pneumovirus, family Paramyxoviridae with a non-segmented genome that encodes 10 genes and 11 proteins (NS1, NS2, N, P, M, SH, G, F, M2-1, M2-2 and L). The two non-structural proteins (NS1 and NS2), which are not part of the intact virion, are transcribed and translated during infection (Moore et al., 2008).RSV infection rates are high and by age 2 years the majority of young children have experienced at least one infection (CDC, 2008(CDC, , 2013Hall et al., 2009;Mori et al., 2014;Nair et al., 2010;Stockman et al., 2012; Zhou et al., 2012). RSV infection in high-risk individuals, such as infants, young children, immunocompromised adults and the elderly, can manifest as serious pulmonary inflammatory disease including bronchiolitis and pneumonia (Hoffman et al., 2004;Moore et al., 2013;Openshaw & Chiu, 2013;Oshansky et al., 2009b;Psarras et al., 2004;Vicencio, 2010). There is also substantial evidence that early RSV infection can mediate airway remodelling, a feature that predisposes individuals to asthma development and exacerbation (Fong et al., 2000;Foronjy et al., 2014;Hirakawa et al., 2013;Hotard et al., 2015;Liesman et al., 2014;Meng et al., 2014;Piedimonte, 2002Piedimonte, , 2003Tan et al., 2008;Wu et al., 2011). Transforming growth factor (TGF)-b is expressed during RSV infection of lung epithelial cells (Gibbs et al., 2009;McCann & Imani, 2007;Mgbemena et al., 2011) and several studies indicate that it plays an important role in asthma development (de Faria et al., 2008;Fong et al., 2000;Gagliardo et al., 2013;Hoshino et al., 1998;Howell & McAnulty, 2006; Pelaia et al., 2007;Sharma et al., 2009). TGF-b also regulates aspects of the inflammatory cytokine r...

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.