Mutational Analysis of the Influenza Virus cRNA Promoter and Identification of Nucleotides Critical for Replication

ABSTRACT Replication of the influenza A virus virion RNA (vRNA) requires the synthesis of full-length cRNA, which in turn is used as a template for the synthesis of more vRNA. A “corkscrew” secondary-structure model of the cRNA promoter has been proposed recently. However the data in support of that model were indirect, since they were derived from measurement, by use of a chloramphenicol acetyltransferase (CAT) reporter in 293T cells, of mRNA levels from a modified cRNA promoter rather than the authentic cRNA promoter found in influenza A viruses. Here we measured steady-state cRNA and vRNA levels from a CAT reporter in 293T cells, directly measuring the replication of the authentic influenza A virus wild-type cRNA promoter. We found that (i) base pairing between the 5′ and 3′ ends and (ii) base pairing in the stems of both the 5′ and 3′ hairpin loops of the cRNA promoter were required for in vivo replication. Moreover, nucleotides in the tetraloop at positions 4, 5, and 7 and nucleotides forming the 2-9 base pair of the 3′ hairpin loop were crucial for promoter activity in vivo. However, the 3′ hairpin loop was not required for polymerase binding in vitro. Overall, our results suggest that the corkscrew secondary-structure model is required for authentic cRNA promoter activity in vivo, although the precise role of the 3′ hairpin loop remains unknown.

Download Full-text

Secondary structure model of the naked segment 7 influenza A virus genomic RNA

Biochemical Journal ◽

10.1042/bcj20160651 ◽

2016 ◽

Vol 473 (23) ◽

pp. 4327-4348 ◽

Cited By ~ 12

Author(s):

Agnieszka Ruszkowska ◽

Elzbieta Lenartowicz ◽

Walter N. Moss ◽

Ryszard Kierzek ◽

Elzbieta Kierzek

Keyword(s):

Secondary Structure ◽

Influenza A Virus ◽

Rna Structure ◽

Influenza A ◽

Comparative Sequence Analysis ◽

Rnase H ◽

Structure Model ◽

Chemical Mapping ◽

Secondary Structure Model ◽

Negative Sense

The influenza A virus (IAV) genome comprises eight negative-sense viral (v)RNA segments. The seventh segment of the genome encodes two essential viral proteins and is specifically packaged alongside the other seven vRNAs. To gain insights into the possible roles of RNA structure both within and without virions, a secondary structure model of a naked (protein-free) segment 7 vRNA (vRNA7) has been determined using chemical mapping and thermodynamic energy minimization. The proposed structure model was validated using microarray mapping, RNase H cleavage and comparative sequence analysis. Additionally, the detailed structures of three vRNA7 fragment constructs — comprising independently folded subdomains — were determined. Much of the proposed vRNA7 structure is preserved between IAV strains, suggesting their importance in the influenza replication cycle. Possible structure rearrangements, which allow or preclude long-range RNA interactions, are also proposed.

Download Full-text

Conserved Structural Motifs of Two Distant IAV Subtypes in Genomic Segment 5 RNA

Viruses ◽

10.3390/v13030525 ◽

2021 ◽

Vol 13 (3) ◽

pp. 525

Author(s):

Paula Michalak ◽

Julita Piasecka ◽

Barbara Szutkowska ◽

Ryszard Kierzek ◽

Ewa Biala ◽

...

Keyword(s):

Experimental Data ◽

Secondary Structure ◽

Influenza A Virus ◽

Influenza A ◽

Chemical Mapping ◽

Rna Structures ◽

Structural Motifs ◽

2009 H1n1

The functionality of RNA is fully dependent on its structure. For the influenza A virus (IAV), there are confirmed structural motifs mediating processes which are important for the viral replication cycle, including genome assembly and viral packaging. Although the RNA of strains originating from distant IAV subtypes might fold differently, some structural motifs are conserved, and thus, are functionally important. Nowadays, NGS-based structure modeling is a source of new in vivo data helping to understand RNA biology. However, for accurate modeling of in vivo RNA structures, these high-throughput methods should be supported with other analyses facilitating data interpretation. In vitro RNA structural models complement such approaches and offer RNA structures based on experimental data obtained in a simplified environment, which are needed for proper optimization and analysis. Herein, we present the secondary structure of the influenza A virus segment 5 vRNA of A/California/04/2009 (H1N1) strain, based on experimental data from DMS chemical mapping and SHAPE using NMIA, supported by base-pairing probability calculations and bioinformatic analyses. A comparison of the available vRNA5 structures among distant IAV strains revealed that a number of motifs present in the A/California/04/2009 (H1N1) vRNA5 model are highly conserved despite sequence differences, located within previously identified packaging signals, and the formation of which in in virio conditions has been confirmed. These results support functional roles of the RNA secondary structure motifs, which may serve as candidates for universal RNA-targeting inhibitory methods.

Download Full-text

Genes for tRNA recycling are upregulated in response to infection with Theiler's mouse encephalitis virus

10.1101/2021.08.05.455008 ◽

2021 ◽

Author(s):

Mineaki Seki ◽

Akihiko Komuro ◽

Tatsuya Ishikawa ◽

Masayuki Takahashi ◽

Masayuki Nashimoto

Keyword(s):

Quality Control ◽

Viral Infection ◽

Influenza A Virus ◽

Hela Cells ◽

Influenza A ◽

Encephalitis Virus ◽

Encephalomyocarditis Virus ◽

Mrna Levels ◽

Recycling System

The concept of tRNA recycling has recently emerged from the studies of ribosome associated quality control. Therein tRNase ZS removes the 2', 3'>p from the ANKZF1-cleaved tRNA and the subsequent TRNT1 action re-generates the intact tRNA. To know the roles of the tRNA recycling in vivo, we investigated how viral infection affects the tRNA recycling system by analyzing the mRNA levels of tRNase ZS and TRNT1. We found that both genes in HeLa cells are upregulated in response to infection of Theiler's mouse encephalitis virus but not to that of an influenza A virus. Upregulation was also observed in cells infected with encephalomyocarditis virus with reduced efficiency. The levels of the IFN-β mRNA appeared to positively correlate with those of the tRNase ZS and TRNT1 mRNAs. The tRNase ZS gene may be regulated post-transcriptionally in the cells infected with Theiler's mouse encephalitis virus.

Download Full-text

Estrogen Up-Regulates Hepatic Expression of Suppressors of Cytokine Signaling-2 and -3 in Vivo and in Vitro

Endocrinology ◽

10.1210/en.2004-0061 ◽

2004 ◽

Vol 145 (12) ◽

pp. 5525-5531 ◽

Cited By ~ 50

Author(s):

Gary M. Leong ◽

Sofia Moverare ◽

Jesena Brce ◽

Nathan Doyle ◽

Klara Sjögren ◽

...

Keyword(s):

Promoter Activity ◽

Hepatoma Cells ◽

Cytokine Signaling ◽

Mrna Levels ◽

Dependent Manner ◽

Wild Type ◽

Hepatic Expression ◽

Suppressors Of Cytokine Signaling

Abstract Suppressors of cytokine signaling (SOCS) are important negative regulators of cytokine action. We recently reported that estrogen stimulates SOCS-2 expression and inhibits GH signaling in kidney cells. The effects of estrogen on SOCS expression in other tissues are unclear. The aim of this study was to investigate in vivo and in vitro whether estrogen affected SOCS expression in the liver, a major target organ of GH. The in vivo hepatic effects of estrogen on ovariectomized mice lacking estrogen receptor (ER)-α, ERβ, or both and their wild-type littermates were examined by DNA microarray analysis. In vitro, the effects of estrogen on SOCS expression in human hepatoma cells were examined by reverse transcription quantitative PCR. Long-term (3 wk) estrogen treatment induced a 2- to 3-fold increase in hepatic expression of SOCS-2 and -3 in wild-type and ERβ knockout mice but not in those lacking ERα or both ER subtypes. Short-term treatment (at 24 h) increased the mRNA level of SOCS-3 but not SOCS-2. In cultured hepatoma cells, estrogen increased SOCS-2 and -3 mRNA levels by 2-fold in a time- and dose-dependent manner (P < 0.05). Estrogen induced murine SOCS-3 promoter activity by 2-fold (P < 0.05) in constructs containing a region between nucleotides −1862 and −855. Moreover, estrogen and GH had additive effects on the SOCS-3 promoter activity. In summary, estrogen, via ERα, up-regulated hepatic expression of SOCS-2 and -3, probably through transcriptional activation. This indicates a novel mechanism of estrogen regulation of cytokine action.

Download Full-text

Impact of Influenza A Virus Shutoff Proteins on Host Immune Responses

Vaccines ◽

10.3390/vaccines9060629 ◽

2021 ◽

Vol 9 (6) ◽

pp. 629

Author(s):

Megan M. Dunagan ◽

Kala Hardy ◽

Toru Takimoto

Keyword(s):

Immune Response ◽

Immune Responses ◽

Influenza A Virus ◽

Influenza A ◽

Human Populations ◽

Lymphocyte Migration ◽

Host Immune Responses ◽

Mhc Molecules ◽

The Impact

Influenza A virus (IAV) is a significant human pathogen that causes seasonal epidemics. Although various types of vaccines are available, IAVs still circulate among human populations, possibly due to their ability to circumvent host immune responses. IAV expresses two host shutoff proteins, PA-X and NS1, which antagonize the host innate immune response. By transcriptomic analysis, we previously showed that PA-X is a major contributor for general shutoff, while shutoff active NS1 specifically inhibits the expression of host cytokines, MHC molecules, and genes involved in innate immunity in cultured human cells. So far, the impact of these shutoff proteins in the acquired immune response in vivo has not been determined in detail. In this study, we analyzed the effects of PA-X and NS1 shutoff activities on immune response using recombinant influenza A/California/04/2009 viruses containing mutations affecting the expression of shutoff active PA-X and NS1 in a mouse model. Our data indicate that the virus without shutoff activities induced the strongest T and B cell responses. Both PA-X and NS1 reduced host immune responses, but shutoff active NS1 most effectively suppressed lymphocyte migration to the lungs, antibody production, and the generation of IAV specific CD4+ and CD8+ T cells. NS1 also prevented the generation of protective immunity against a heterologous virus challenge. These data indicate that shutoff active NS1 plays a major role in suppressing host immune responses against IAV infection.

Download Full-text

Phylogeny of Criconematina Siddiqi, 1980 (Nematoda: Tylenchida) based on morphology and D2-D3 expansion segments of the 28S-rRNA gene sequences with application of a secondary structure model

Nematology ◽

10.1163/156854105776186307 ◽

2005 ◽

Vol 7 (6) ◽

pp. 927-944 ◽

Cited By ~ 38

Author(s):

Renato Crozzoli ◽

Franco Lamberti ◽

Nicola Vovlas ◽

James Baldwin ◽

Sergei Subbotin ◽

...

Keyword(s):

Maximum Likelihood ◽

Secondary Structure ◽

Sequence Divergence ◽

Complex Model ◽

Rrna Gene ◽

28S Rrna ◽

Structure Model ◽

Secondary Structure Model ◽

Loop Region ◽

Unidentified Species

AbstractThe suborder Criconematina is a large group of ecto- and endoparasitic nematodes, including several species of major agricultural importance. The D2-D3 expansion segments of the 28S nuclear ribosomal RNA gene were amplified and sequenced from 23 nominal and six unidentified species from the genera Mesocriconema, Criconemoides, Ogma, Criconema, Xenocriconemella, Hemicriconemoides, Hemicycliophora, Paratylenchus, Tylenchulus, Trophonema and Sphaeronema, together with outgroup taxa from Tylenchidae (Aglenchus) and Atylenchidae (Eutylenchus). A sequence alignment optimised using the secondary structure model was analysed using maximum parsimony, maximum likelihood and Bayesian inference approaches under two models. All analyses yielded a similar topology with differences primarily in the position of poorly supported clades. Although some molecular trees differ from the previous morphologically based hypotheses of criconematid phylogeny, maximum likelihood tests did not yield statistically significant differences between some of the tested classical morphological and molecular topologies. DNA data support monophyly for the genera Mesocriconema, Hemicriconemoides and Criconema and reject the hypothesis of a single origin of criconematids with a cuticular sheath or 'double cuticle'. Application of the complex model of rRNA evolution, considering paired nucleotides for the stem and unpaired nucleotides for the loop region, resulted in a majority rule consensus Bayesian tree with unresolved relationships between main clades. This lack of resolution is expected by the low number of independently evolving nucleotides. Sequence divergence in this DNA segment between populations of Mesocriconema xenoplax, M. sphaerocephalum and Hemicriconemoides cocophillus suggest the presence of several sibling species under these taxa names.

Download Full-text

Viral dosing of influenza A infection reveals involvement of RIPK3 and FADD, but not MLKL

Cell Death and Disease ◽

10.1038/s41419-021-03746-0 ◽

2021 ◽

Vol 12 (5) ◽

Author(s):

Teodora Oltean ◽

Emily Van San ◽

Tatyana Divert ◽

Tom Vanden Berghe ◽

Xavier Saelens ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Kinase Activity ◽

Dose Range ◽

Challenge Dose

AbstractRIPK3 was reported to play an important role in the protection against influenza A virus (IAV) in vivo. Here we show that the requirement of RIPK3 for protection against IAV infection in vivo is only apparent within a limited dose range of IAV challenge. We found that this protective outcome is independent from RIPK3 kinase activity and from MLKL. This shows that platform function of RIPK3 rather than its kinase activity is required for protection, suggesting that a RIPK3 function independent of necroptosis is implicated. In line with this finding, we show that FADD-dependent apoptosis has a crucial additional effect in protection against IAV infection. Altogether, we show that RIPK3 contributes to protection against IAV in a narrow challenge dose range by a mechanism that is independent of its kinase activity and its capacity to induce necroptosis.

Download Full-text

Antiviral activity of Ladania067, an extract from wild black currant leaves against influenza A virus in vitro and in vivo

Frontiers in Microbiology ◽

10.3389/fmicb.2014.00171 ◽

2014 ◽

Vol 5 ◽

Cited By ~ 12

Author(s):

Emanuel Haasbach ◽

Carmen Hartmayer ◽

Alice Hettler ◽

Alicja Sarnecka ◽

Ulrich Wulle ◽

...

Keyword(s):

Antiviral Activity ◽

Influenza A Virus ◽

Influenza A ◽

Black Currant

Download Full-text

Exacerbation of Influenza A Virus Disease Severity by Respiratory Syncytial Virus Co-Infection in a Mouse Model

Viruses ◽

10.3390/v13081630 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1630 ◽

Cited By ~ 1

Author(s):

Junu A. George ◽

Shaikha H. AlShamsi ◽

Maryam H. Alhammadi ◽

Ahmed R. Alsuwaidi

Keyword(s):

T Cells ◽

Respiratory Syncytial Virus ◽

Influenza A Virus ◽

Influenza A ◽

Immune Cell ◽

Virus Disease ◽

Viral Loads ◽

Syncytial Virus

Influenza A virus (IAV) and respiratory syncytial virus (RSV) are leading causes of childhood infections. RSV and influenza are competitive in vitro. In this study, the in vivo effects of RSV and IAV co-infection were investigated. Mice were intranasally inoculated with RSV, with IAV, or with both viruses (RSV+IAV and IAV+RSV) administered sequentially, 24 h apart. On days 3 and 7 post-infection, lung tissues were processed for viral loads and immune cell populations. Lung functions were also evaluated. Mortality was observed only in the IAV+RSV group (50% of mice did not survive beyond 7 days). On day 3, the viral loads in single-infected and co-infected mice were not significantly different. However, on day 7, the IAV titer was much higher in the IAV+RSV group, and the RSV viral load was reduced. CD4 T cells were reduced in all groups on day 7 except in single-infected mice. CD8 T cells were higher in all experimental groups except the RSV-alone group. Increased airway resistance and reduced thoracic compliance were demonstrated in both co-infected groups. This model indicates that, among all the infection types we studied, infection with IAV followed by RSV is associated with the highest IAV viral loads and the most morbidity and mortality.

Download Full-text