Comparison of tissue tropism and host response to enteric and respiratory enteroviruses.

Enteroviruses (EVs) are among the most prevalent viruses worldwide. They are characterized by a high genetic and phenotypic diversity, being able to cause a plethora of symptoms. EV-D68, a respiratory EV, and EV-D94, an enteric EV, represent an interesting paradigm of EV tropism heterogeneity. They belong to the same species, but display distinct phenotypic characteristics and in vivo tropism. Here, we used these two viruses as well as relevant 3D respiratory, intestinal and neural tissue culture models, to highlight key distinctive features of enteric and respiratory EVs. We emphasize the critical role of temperature in restricting EV-D68 tissue tropism. Using transcriptomic analysis, we underscore fundamental differences between intestinal and respiratory tissues, both in the steady-state and in response to infection. Intestinal tissues present higher cell proliferation rate and are more immunotolerant than respiratory tissues. Importantly, we highlight the different strategies applied by EV-D94 and EV-D68 towards the host antiviral response of intestinal and respiratory tissues. EV-D68 strongly activates antiviral pathways while EV-D94, on the contrary, barely induces any host defense mechanisms. In summary, our study provides an insightful characterization of the differential pathogenesis of EV-D68 and EV-D94 and the interplay with their main target tissues.

A VP1 mutation acquired during an enterovirus 71 disseminated infection confers heparan sulfate binding ability and modulates ex vivo tropism.

Enterovirus 71 (EV71) causes hand, foot and mouth disease, a mild and self-limited illness that is sometimes associated with severe neurological complications. EV71 neurotropic determinants remain ill-defined to date. We previously identified a mutation in the VP1 capsid protein (L97R) that was acquired over the course of a disseminated infection in an immunocompromised host. The mutation was absent in the respiratory tract but was present in the gut (as a mixed population) and in blood and cerebrospinal fluid (as a dominant species). In this study, we demonstrated that this mutation does not alter the dependence of EV71 on the human scavenger receptor class B2 (SCARB2), while it enables the virus to bind to the heparan sulfate (HS) attachment receptor and modifies viral tropism in cell lines and in respiratory, intestinal and neural tissues. Variants with VP197L or VP197R were able to replicate to high levels in intestinal and neural tissues and, to a lesser extent, in respiratory tissues, but their preferred entry site (from the luminal or basal tissue side) differed in respiratory and intestinal tissues and correlated with HS expression levels. These data account for the viral populations sequenced from the patient's respiratory and intestinal samples and suggest that improved dissemination, resulting from an acquired ability to bind HS, rather than specific neurotropism determinants, enabled the virus to reach and infect the central nervous system. Finally, we showed that iota-carrageenan, a highly sulfated polysaccharide, efficiently blocks the replication of HS-dependent variants in cells and 2D neural cultures. Overall, the results of this study emphasize the importance of HS binding in EV71 pathogenesis and open new avenues for the development of antiviral molecules that may prevent this virus's dissemination.

Viral chimeras decrypt the role of enterovirus capsid proteins in viral tropism, acid sensitivity and optimal growth temperature.

Despite their genetic similarities, enteric and respiratory enteroviruses (EVs) have highly heterogeneous biophysical properties and cause a vast diversity of human pathologies. In vitro differences include acid sensitivity, optimal growth temperature and tissue tropism, which reflect a preferential in vivo replication in the respiratory or gastrointestinal tract and are thus key determinants of EV virulence. To investigate the underlying cause of these differences, we generated chimeras at the capsid-level between EV-D68 (a respiratory EV) and EV-D94 (an enteric EV). Although some chimeras were nonfunctional, EV-D94 with both the capsid and 2A protease or the capsid only of EV-D68 were both viable. Using this latter construct, we performed several functional assays, which indicated that capsid proteins determine acid sensitivity and tropism in cell lines and in respiratory, intestinal and neural tissues. Additionally, capsid genes were shown to also participate in determining the optimal growth temperature, since EV-D94 temperature adaptation relied on single mutations in VP1, while constructs with EV-D68 capsid could not adapt to higher temperatures. Finally, we demonstrate that EV-D68 maintains residual binding-capacity after acid-treatment despite a loss of infectivity. In contrast, non-structural rather than capsid proteins modulate the innate immune response in tissues. These unique biophysical insights expose another layer in the phenotypic diversity of one of world's most prevalent pathogens and could aid target selection for vaccine or antiviral development.

Propagation of respiratory viruses in human airway epithelia reveals persistent virus-specific signatures.

BACKGROUND: The leading cause of acute illnesses, respiratory viruses, typically cause self-limited diseases, although severe complications can occur in fragile patients. Rhinoviruses (RVs), respiratory enteroviruses (EVs), influenza virus, respiratory syncytial viruses (RSVs), and coronaviruses are highly prevalent respiratory pathogens, but because of the lack of reliable animal models, their differential pathogenesis remains poorly characterized. OBJECTIVE: We sought to compare infections by respiratory viruses isolated from clinical specimens using reconstituted human airway epithelia. METHODS: Tissues were infected with RV-A55, RV-A49, RV-B48, RV-C8, and RV-C15; respiratory EV-D68; influenza virus H3N2; RSV-B; and human coronavirus (HCoV)-OC43. Replication kinetics, cell tropism, effect on tissue integrity, and cytokine secretion were compared. Viral adaptation and tissue response were assessed through RNA sequencing. RESULTS: RVs, RSV-B, and HCoV-OC43 infected ciliated cells and caused no major cell death, whereas H3N2 and EV-D68 induced ciliated cell loss and tissue integrity disruption. H3N2 was also detected in rare goblet and basal cells. All viruses, except RV-B48 and HCoV-OC43, altered cilia beating and mucociliary clearance. H3N2 was the strongest cytokine inducer, and HCoV-OC43 was the weakest. Persistent infection was observed in all cases. RNA sequencing highlighted perturbation of tissue metabolism and induction of a transient but important immune response at 4 days after infection. No majority mutations emerged in the viral population. CONCLUSION: Our results highlight the differential in vitro pathogenesis of respiratory viruses during the acute infection phase and their ability to persist under immune tolerance. These data help to appreciate the range of disease severity observed in vivo and the occurrence of chronic respiratory tract infections in immunocompromised hosts.

Chimeric rhinoviruses obtained via genetic engineering or artificially induced recombination are viable only if the polyprotein coding sequence derives from the same species.

UNLABELLED: Recombination is a widespread phenomenon that ensures both the stability and variation of RNA viruses. This phenomenon occurs with different frequencies within species of the Enterovirus genus. Intraspecies recombination is described frequently among non-rhinovirus enteroviruses but appears to be sporadic in rhinoviruses. Interspecies recombination is even rarer for rhinoviruses and mostly is related to ancient events which contributed to the speciation of these viruses. We reported that artificially engineered 5' untranslated region (UTR) interspecies rhinovirus/rhinovirus or rhinovirus/non-rhinovirus enterovirus recombinants are fully viable. Using a similar approach, we demonstrated in this study that exchanges of the P1-2A polyprotein region between members of the same rhinovirus species, but not between members of different species, give rise to competent chimeras. To further assess the rhinovirus intra- and interspecies recombination potential, we used artificially induced recombination by cotransfection of 5'-end-deleted and 3'-end-deleted and replication-deficient genomes. In this system, intraspecies recombination also resulted in viable viruses with high frequency, whereas no interspecies rhinovirus recombinants could be recovered. Mapping intraspecies recombination sites within the polyprotein highlighted recombinant hotspots in nonstructural genes and at gene boundaries. Notably, all recombinants occurring at gene junctions presented in-frame sequence duplications, whereas most intragenic recombinants were homologous. Taken together, our results suggest that only intraspecies recombination gives rise to viable rhinovirus chimeras in the polyprotein coding region and that recombination hotspots map to nonstructural genes with in-frame duplications at gene boundaries. These data provide new insights regarding the mechanism and limitations of rhinovirus recombination. IMPORTANCE: Recombination represents a means to ensure both the stability and the variation of RNA viruses. While intraspecies recombination is described frequently among non-rhinovirus enteroviruses, it seems to occur more rarely in rhinoviruses. Interspecies recombination is even rarer in this virus group and is mostly related to ancient events, which contributed to its speciation. We used engineered chimeric genomes and artificially induced RNA recombination to study experimentally the recombination potential of rhinoviruses and analyze recombination sites. Our results suggest that only intraspecies recombination gives rise to viable chimeras in the polyprotein coding region. Furthermore, characterization of intraspecies chimeras provides new insight into putative recombination hotspots within the polyprotein. In summary, we applied two powerful and complementary experimental approaches to improve current knowledge on rhinovirus recombination.

Altered cell function and increased replication of rhinoviruses and EV-D68 in airway epithelia of asthma patients.

Introduction: Rhinovirus (RV) infections constitute one of the main triggers of asthma exacerbations and an important burden in pediatric yard. However, the mechanisms underlying this association remain poorly understood. Methods: In the present study, we compared infections of in vitro reconstituted airway epithelia originating from asthmatic versus healthy donors with representative strains of RV-A major group and minor groups, RV-C, RV-B, and the respiratory enterovirus EV-D68. Results: We found that viral replication was higher in tissues derived from asthmatic donors for all tested viruses. Viral receptor expression was comparable in non-infected tissues from both groups. After infection, ICAM1 and LDLR were upregulated, while CDHR3 was downregulated. Overall, these variations were related to viral replication levels. The presence of the CDHR3 asthma susceptibility allele (rs6967330) was not associated with increased RV-C replication. Regarding the tissue response, a significantly higher interferon (IFN) induction was demonstrated in infected tissues derived from asthmatic donors, which excludes a defect in IFN-response. Unbiased transcriptomic comparison of asthmatic versus control tissues revealed significant modifications, such as alterations of cilia structure and motility, in both infected and non-infected tissues. These observations were supported by a reduced mucociliary clearance and increased mucus secretion in non-infected tissues from asthmatic donors. Discussion: Altogether, we demonstrated an increased permissiveness and susceptibility to RV and respiratory EV infections in HAE derived from asthmatic patients, which was associated with a global alteration in epithelial cell functions. These results unveil the mechanisms underlying the pathogenesis of asthma exacerbation and suggest interesting therapeutic targets.

Negative elongation factor NELF controls transcription of immediate early genes in a stimulus-specific manner.

The transcription rate of immediate early genes (IEGs) is controlled directly by transcription elongation factors at the transcription elongation step. Negative elongation factor (NELF) and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF) stall RNA polymerase II (pol II) soon after transcription initiation. Upon induction of IEG transcription, DSIF is converted into an accelerator for pol II elongation. To address whether and how NELF as well as DSIF controls overall IEG transcription, its expression was reduced using stable RNA interference in GH4C1 cells. NELF knock-down reduced thyrotropin-releasing hormone (TRH)-induced transcription of the IEGs c-fos, MKP-1, and junB. In contrast, epidermal growth factor (EGF)-induced transcription of these IEGs was unaltered or even slightly increased by NELF knock-down. Thus, stable knock-down of NELF affects IEG transcription stimulation-specifically. Conversely, DSIF knock-down reduced both TRH- and EGF-induced transcription of the three IEGs. Interestingly, TRH-induced activation of the MAP kinase pathway, a pathway essential for transcription of the three IEGs, was down-regulated by NELF knock-down. Thus, stable knock-down of NELF, by modulating intracellular signaling pathways, caused stimulation-specific loss of IEG transcription. These observations indicate that NELF controls overall IEG transcription via multiple mechanisms both directly and indirectly.

Gene-specific recruitment of positive and negative elongation factors during stimulated transcription of the MKP-1 gene in neuroendocrine cells.

MAP kinase phosphatase-1 (MKP-1) controls nuclear MAP kinase activity with important consequences on cell growth or apoptosis. MKP-1 transcription is initiated constitutively but elongation is blocked within exon 1. It is unclear how induction of MKP-1 is controlled. Here, we report that the transcriptional elongation factors P-TEFb, DSIF and NELF regulate MKP-1 transcription in the pituitary GH4C1 cell line. Prior to stimulation, DSIF, NELF and RNA polymerase II (pol II) associate with the promoter-proximal region of the MKP-1 gene upstream of the elongation block site. Thyrotropin-releasing hormone (TRH) leads to recruitment of P-TEFb along the whole gene and a marked increase of DSIF and pol II downstream of the elongation block site, whereas NELF remains confined to the promoter-proximal region. 5,6-Dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB) an inhibitor of P-TEFb eliminated TRH stimulation of MKP-1 transcription. DRB specifically inhibited TRH-induced recruitment of DSIF and P-TEFb to the MKP-1 gene. Furthermore, DRB treatment eliminated TRH-induced progression along the MKP-1 gene of pol II phosphorylated on Ser-2 of its CTD. These results indicate that P-TEFb is essential for gene-specific stimulated transcriptional elongation in mammalian cells via mechanisms which involve the activation of the DSIF-NELF complex and Ser-2 phosphorylation of pol II.

Stimulated initiation of mitogen-activated protein kinase phosphatase-1 (MKP-1) gene transcription involves the synergistic action of multiple cis-acting elements in the proximal promoter.

Mitogen-activated protein kinases (MAPKs) are inactivated by a dual specificity phosphatase, MAPK phosphatase-1 (MKP-1). MKP-1 is transcribed as an immediate early response gene (IEG) following various stimuli. In the pituitary cell line GH4C1, MKP-1 gene transcription is strongly induced by thyrotropin-releasing hormone (TRH) as well as by epidermal growth factor (EGF) as a consequence of activated MAPK/extracellular-signal-regulated kinase (ERK) signalling. Intriguingly, reporter gene analysis with the MKP-1 promoter showed strong basal transcription, but only limited induction by TRH and EGF. Site-directed mutagenesis of the reporter construct combined with band-shift and in vivo studies revealed that part of the constitutive activity of the MKP-1 promoter resides in two GC boxes bound by Sp1 and Sp3 transcription factors in the minimal promoter. Basal transcription of transiently transfected luciferase reporter can be initiated by either of the two GC boxes or also by either of the two cAMP/Ca(2+) responsive elements or by the E-box present in the proximal promoter. On the other hand, when analysed by stable transfection, the five responsive elements are acting in synergy to transactivate the MKP-1 proximal promoter. We show in this study that the MKP-1 promoter can function as a constitutive promoter or as a rapid and transient sensor for the activation state of MAPKs/ERKs. This dual mode of transcription initiation may have different consequences for the control of a block to elongation situated in the first exon of the MKP-1 gene, as described previously [Ryser, Tortola, van Haasteren, Muda, Li and Schlegel (2001) J. Biol. Chem. 276, 33319-33327].

Transcription elongation factors are involved in programming hormone production in pituitary neuroendocrine GH4C1 cells.

Transcription elongation of many eukaryotic genes is regulated. Two negative transcription elongation factors, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) are known to stall collaboratively RNA polymerase II promoter proximally. We discovered that DSIF and NELF are linked to hormone expression in rat pituitary GH4C1 cells. When NELF-E, a subunit of NELF or Spt5, a subunit of DSIF was stably knocked-down, prolactin (PRL) expression was increased both at the mRNA and protein levels. In contrast, stable knock-down of only Spt5 abolished growth hormone (GH) expression. Transient NELF-E knock-down increased coincidentally PRL expression and enhanced transcription of a PRL-promoter reporter gene. However, no direct interaction of NELF with the PRL gene could be demonstrated by chromatin immuno-precipitation. Thus, NELF suppressed PRL promoter activity indirectly. In conclusion, transcription regulation by NELF and DSIF is continuously involved in the control of hormone production and may contribute to neuroendocrine cell differentiation.

The transcription elongation factors NELF, DSIF and P-TEFb control constitutive transcription in a gene-specific manner.

We examined whether transcription elongation factors control constitutive transcription of the histone H1(0) and GAPDH genes. Chromatin immunoprecipitation demonstrated positive transcription elongation factor b (P-TEFb) and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF) present together with RNA polymerase II (pol II) throughout the histone H1(0) gene, whereas negative elongation factor (NELF) was confined to the 5' region. Contrarily, DSIF, NELF and pol II were confined to the 5' region on the GAPDH. Inhibition of those factors affected the constitutive transcription of the histone H1(0) gene but not the GAPDH gene. Thus, NELF, DSIF and P-TEFb control constitutive transcription in a gene-specific manner.

Up-regulation of P-TEFb by the MEK1-extracellular signal-regulated kinase signaling pathway contributes to stimulated transcription elongation of immediate early genes in neuroendocrine cells.

The positive elongation factor P-TEFb appears to function as a crucial C-terminal-domain (CTD) kinase for RNA polymerase II (Pol II) transcribing immediate early genes (IEGs) in neuroendocrine GH4C1 cells. Chromatin immunoprecipitation indicated that in resting cells Pol II occupied the promoter-proximal regions of the c-fos and junB genes, together with the negative elongation factors DSIF and NELF. Thyrotropin-releasing hormone (TRH)-induced recruitment of positive transcription elongation factor b (P-TEFb) abolished the pausing of Pol II and enhanced phosphorylation of CTD serine 2, resulting in transcription elongation. In addition, P-TEFb was essential for splicing and 3'-end processing of IEG transcripts. Importantly, the MEK1-extracellular signal-regulated kinase (ERK) signaling pathway activated by TRH up-regulated nuclear CDK9 and CDK9/cyclinT1 dimers (i.e., P-TEFb), facilitating the recruitment of P-TEFb to c-fos and other IEGs. Thus, in addition to established gene transcription control via promoter response elements, the MEK1-ERK signaling pathway controls transcription elongation by Pol II via the up-regulation of nuclear CDK9 integrated into P-TEFb.

The rate of c-fos transcription in vivo is continuously regulated at the level of elongation by dynamic stimulus-coupled recruitment of positive transcription elongation factor b.

In mammalian cells, multiple stimuli induce the expression of the immediate early gene c-fos. The specificity of c-fos transcriptional response depends on the activation of signaling protein kinases, transcription factors, and chromatin-modifying complexes but also on a regulated block to elongation in the first intron. Here we show by chromatin immunoprecipitation that finely tuned control of c-fos gene expression by distinct stimuli is associated with a dynamic regulation of transcription elongation and differential phosphorylation of the C-terminal domain of RNA polymerase II. Comparison of two stimuli of c-fos expression in the pituitary cell line GH4C1, namely the thyrotropin-releasing hormone versus depolarizing KCl, shows that both stimuli increase initiation, but only thyrotropin-releasing hormone is efficient to stimulate elongation and thus produce high transcription rates. To control elongation, the elongation factor P-TEFb is recruited to the 5'-end of the gene in a stimuli and time-dependent manner. Transition from initiation to elongation depends also on the dynamic recruitment of the initiation factors TFIIB and TFIIE but not TFIID, which remains constitutively bound on the promoter. It thus appears that tight coupling of signaling input to transcriptional output rate is achieved by c-fos gene-specific mechanisms, which control post-initiation steps rather than pre-initiation complex assembly.

Spontaneous calcium oscillations control c-fos transcription via the serum response element in neuroendocrine cells.

In excitable cells the localization of Ca2+ signals plays a central role in the cellular response, especially in the control of gene transcription. To study the effect of localized Ca2+ signals on the transcriptional activation of the c-fos oncogene, we stably expressed various c-fos beta-lactamase reporter constructs in pituitary AtT20 cells. A significant, but heterogenous expression of c-fos beta-lactamase was observed in unstimulated cells, and a further increase was observed using KCl depolarization, epidermal growth factor (EGF), pituitary adenylate cyclase-activating polypeptide (PACAP), and serum. The KCl response was almost abolished by a nuclear Ca2+ clamp, indicating that a rise in nuclear Ca2+ is required. In contrast, the basal expression was not affected by the nuclear Ca2+ clamp, but it was strongly reduced by nifedipine, a specific antagonist of l-type Ca2+ channels. Spontaneous Ca2+ oscillations, blocked by nifedipine, were observed in the cytosol but did not propagate to the nucleus, suggesting that a rise in cytosolic Ca2+ is sufficient for basal c-fos expression. Inactivation of the c-fos promoter cAMP/Ca2+ response element (CRE) had no effect on basal or stimulated expression, whereas inactivation of the serum response element (SRE) had the same marked inhibitory effect as nifedipine. These experiments suggest that in AtT20 cells spontaneous Ca2+ oscillations maintain a basal c-fos transcription through the serum response element. Further induction of c-fos expression by depolarization requires a nuclear Ca2+ increase.