ABSTRACT
The small messenger RNA (SmRNA) of the Andes orthohantavirus (ANDV), a rodent-borne member of the Hantaviridae family of viruses of the Bunyavirales order, encodes a multifunctional nucleocapsid (N) protein and for a nonstructural (NSs) protein of unknown function. We have previously shown the expression of the ANDV-NSs, but only in infected cell cultures. In this study, we extend our early findings by confirming the expression of the ANDV-NSs protein in the lungs of experimentally infected golden Syrian hamsters. Next, we show, using a virus-free system, that the ANDV-NSs protein antagonizes the type I interferon (IFN) induction pathway by suppressing signals downstream of the melanoma differentiation-associated protein 5 (MDA5) and the retinoic acid-inducible gene 1 (RIG-I) and upstream of TBK1. Consistent with this observation, the ANDV-NSs protein antagonized mitochondrial antiviral-signaling protein (MAVS)-induced IFN-ß, NF-κB, IFN-regulatory factor 3 (IRF3), and IFN-sensitive response element (ISRE) promoter activity. Results demonstrate that ANDV-NSs binds to MAVS in cells without disrupting the MAVS-TBK-1 interaction. However, in the presence of the ANDV-NSs ubiquitination of MAVS is reduced. In summary, this study provides evidence showing that the ANDV-NSs protein acts as an antagonist of the cellular innate immune system by suppressing MAVS downstream signaling by a yet not fully understand mechanism. Our findings reveal new insights into the molecular regulation of the hosts' innate immune response by the Andes orthohantavirus.IMPORTANCEAndes orthohantavirus (ANDV) is endemic in Argentina and Chile and is the primary etiological agent of hantavirus cardiopulmonary syndrome (HCPS) in South America. ANDV is distinguished from other hantaviruses by its unique ability to spread from person to person. In a previous report, we identified a novel ANDV protein, ANDV-NSs. Until now, ANDV-NSs had no known function. In this new study, we established that ANDV-NSs acts as an antagonist of cellular innate immunity, the first line of defense against invading pathogens, hindering the cellular antiviral response during infection. This study provides novel insights into the mechanisms used by ANDV to establish its infection.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Orthohantavirus/genetics , Viral Nonstructural Proteins/genetics , Animals , Cell Line , Chlorocebus aethiops , HEK293 Cells , Hantavirus Infections/genetics , Host-Pathogen Interactions/immunology , Humans , Immunity, Innate/immunology , Interferon Regulatory Factor-3/metabolism , Interferon Type I/metabolism , Interferon-beta/genetics , NF-kappa B/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction/immunology , Vero Cells , Viral Nonstructural Proteins/metabolismABSTRACT
Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL). The HTLV-1 basic leucine zipper protein (HBZ) is expressed in all cases of ATL and is directly associated with virus pathogenicity. The two isoforms of the HBZ protein are synthesized from antisense messenger RNAs (mRNAs) that are either spliced (sHBZ) or unspliced (usHBZ) versions of the HBZ transcript. The sHBZ and usHBZ mRNAs have entirely different 5'untranslated regions (5'UTR) and are differentially expressed in cells, with the sHBZ protein being more abundant. Here, we show that differential expression of the HBZ isoforms is regulated at the translational level. Translation initiation of the usHBZ mRNA relies on a cap-dependent mechanism, while the sHBZ mRNA uses internal initiation. Based on the structural data for the sHBZ 5'UTR generated by SHAPE in combination with 5' and 3' deletion mutants, the minimal region harboring IRES activity was mapped to the 5'end of the sHBZ mRNA. In addition, the sHBZ IRES recruited the 40S ribosomal subunit upstream of the initiation codon, and IRES activity was found to be dependent on the ribosomal protein eS25 and eIF5A.
Subject(s)
Basic-Leucine Zipper Transcription Factors/genetics , Human T-lymphotropic virus 1/genetics , Peptide Chain Initiation, Translational , RNA, Messenger/genetics , RNA, Viral/genetics , Retroviridae Proteins/genetics , 5' Untranslated Regions/genetics , Animals , Basic-Leucine Zipper Transcription Factors/metabolism , COS Cells , Chlorocebus aethiops , Gene Expression Regulation, Viral , HEK293 Cells , HeLa Cells , Human T-lymphotropic virus 1/metabolism , Humans , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA Splicing , RNA, Messenger/metabolism , RNA, Viral/metabolism , Retroviridae Proteins/metabolismABSTRACT
The 5' untranslated region (UTR) of the full-length mRNA of the mouse mammary tumor virus (MMTV) harbors an internal ribosomal entry site (IRES). In this study, we show that the polypyrimidine tract-binding protein (PTB), an RNA-binding protein with four RNA recognition motifs (RRMs), binds to the MMTV 5' UTR stimulating its IRES activity. There are three isoforms of PTB: PTB1, PTB2, and PTB4. Results show that PTB1 and PTB4, but not PTB2, stimulate MMTV-IRES activity. PTB1 promotes MMTV-IRES-mediated initiation more strongly than PTB4. When expressed in combination, PTB1 further enhanced PTB4 stimulation of the MMTV-IRES, while PTB2 fully abrogates PTB4-induced stimulation. PTB1-induced stimulation of MMTV-IRES was not altered in the presence of PTB4 or PTB2. Mutational analysis reveals that stimulation of MMTV-IRES activity is abrogated when PTB1 is mutated either in RRM1/RRM2 or RRM3/RRM4. In contrast, a PTB4 RRM1/RRM2 mutant has reduced effect over MMTV-IRES activity, while stimulation of the MMTV-IRES activity is still observed when the PTB4 RRM3/RMM4 mutant is used. Therefore, PTB1 and PTB4 differentially stimulate the IRES activity. In contrast, PTB2 acts as a negative modulator of PTB4-induced stimulation of MMTV-IRES. We conclude that PTB1 and PTB4 act as IRES trans-acting factors of the MMTV-IRES.
Subject(s)
5' Untranslated Regions , Mammary Tumor Virus, Mouse/genetics , Polypyrimidine Tract-Binding Protein/metabolism , RNA Caps , RNA, Messenger/genetics , Binding Sites , Gene Knockdown Techniques , Genes, Viral , HEK293 Cells , Humans , Internal Ribosome Entry Sites , Polypyrimidine Tract-Binding Protein/geneticsABSTRACT
In this study, we demonstrate the identification of an internal ribosome entry site (IRES) within the 5'-untranslated region (5'-UTR) of the mouse mammary tumor virus (MMTV). The 5'-UTR of the full-length mRNA derived from the infectious, complete MMTV genome was cloned into a dual luciferase reporter construct containing an upstream Renilla luciferase gene (RLuc) and a downstream firefly luciferase gene (FLuc). In rabbit reticulocyte lysate, the MMTV 5'-UTR was capable of driving translation of the second cistron. In vitro translational activity from the MMTV 5'-UTR was resistant to the addition of m(7)GpppG cap-analog and cleavage of eIF4G by foot-and-mouth disease virus (FMDV) L-protease. IRES activity was also demonstrated in the Xenopus laevis oocyte by micro-injection of capped and polyadenylated bicistronic RNAs harboring the MMTV-5'-UTR. Finally, transfection assays showed that the MMTV-IRES exhibits cell type-dependent translational activity, suggesting a requirement for as yet unidentified cellular factors for its optimal function.
