Analysis of the effects of alterations in the tick-borne encephalitis virus 3'-noncoding region on translation and RNA replication using reporter replicons.

The 3'-noncoding region (3'-NCR) of the flavivirus genome includes a variable region that tolerates the insertion of heterologous genetic information. Natural isolates of tick-borne encephalitis virus (TBEV) have particularly long variable regions, which, for some strains, include an internal poly(A) tract. We constructed luciferase reporter replicons of TBEV to analyze the impact of various manipulations of the 3'-NCR on viral RNA translation and replication. The choice of the reporter gene, its position and processing within the viral polyprotein, and the choice of standards were found to be important for obtaining a sensitive and reliable test system. We observed that truncation or complete removal of the internal poly(A) tract, or even the entire variable region, had no significant impact on translation and replication of the RNA in mammalian cell culture. Substitution of the variable region with foreign genetic elements impaired RNA replication to various degrees but generally had no influence on viral translation. Expression cassettes driven by an IRES element inhibited RNA replication more strongly than did repetitive protein-binding elements derived from a bacteriophage, even when the ligand that binds these elements was co-expressed in the cells. Previously identified mutations in the IRES partially relieved this inhibition when introduced into the reporter replicon but provided no evidence for intramolecular competition for translation factors. Impairment of replication appeared to depend more on the type of foreign insert than on its length. These results provide a rational basis for the construction of TBEV-based vectors or vaccines as well as molecular tools for studying flavivirus replication.

The hepatitis C virus RNA 3'-untranslated region strongly enhances translation directed by the internal ribosome entry site.

The positive-strand RNA genome of the hepatitis C virus (HCV) is flanked by 5'- and 3'-untranslated regions (UTRs). Translation of the viral RNA is directed by the internal ribosome entry site (IRES) in the 5'-UTR, and subsequent viral RNA replication requires sequences in the 3'-UTR and in the 5'-UTR. Addressing previous conflicting reports on a possible function of the 3'-UTR for RNA translation in this study, we found that reporter construct design is an important parameter in experiments testing 3'-UTR function. A translation enhancer function of the HCV 3'-UTR was detected only after transfection of monocistronic reporter RNAs or complete RNA genomes having a 3'-UTR with a precise 3' terminus. The 3'-UTR strongly stimulates HCV IRES-dependent translation in human hepatoma cell lines but only weakly in nonliver cell lines. The variable region, the poly(U . C) tract, and the most 3' terminal stem-loop 1 of the highly conserved 3' X region contribute significantly to translation enhancement, whereas stem-loops 2 and 3 of the 3' X region are involved only to a minor extent. Thus, the signals for translation enhancement and for the initiation of RNA minus-strand synthesis in the HCV 3'-UTR partially overlap, supporting the idea that these sequences along with viral and possibly also cellular factors may be involved in an RNA 3'-5' end interaction and a switch between translation and RNA replication.

3' untranslated region in a light neurofilament (NF-L) mRNA triggers aggregation of NF-L and mutant superoxide dismutase 1 proteins in neuronal cells.

The pathogenesis of neurodegenerative diseases is believed to involve abnormal aggregation of proteins, but the mechanisms initiating protein aggregation are unclear. Here we report a novel phenomenon that could be instrumental in triggering protein aggregation in neurodegenerative diseases. We show that the 3' untranslated region (3'UTR) of a light neurofilament (NF-L) transcript enhances the reactivity of its own translated product and leads to loss of solubility and aggregation of NF-L protein and to coaggregation of mutant superoxide dismutase 1 (SOD1) protein. Full-length mouse NF-L cDNAs, with and without NF-L 3'UTR, were fused to the C terminus of a green fluorescent protein (GFP) reporter gene, and the GFP-tagged NF-L proteins were examined in transfected Neuro2a cells. The GFP-tagged NF-L protein expressed from the transgene containing NF-L 3'UTR, but not from the transgene lacking NF-L 3'UTR, colocalizes with endogenous heavy neurofilament protein and, at high-level expression, leads to loss of solubility and aggregation of GFP-tagged NF-L protein. Aggregation of GFP-tagged NF-L protein triggers coaggregation and loss of solubility of coexpressed DsRed-tagged mutant (G93A) SOD1 protein but not wild-type SOD1 protein. Deletional mutagenesis maps the RNA sequence causing aggregation of GFP-tagged NF-L protein to the proximal 45 nucleotides of NF-L 3'UTR. This is the site of a major destabilizing element in NF-L RNA and binding site for RNA-binding proteins. Our findings support a working model whereby NF-L RNA, or cognate RNA-binding factors, enhances the reactivity of NF-L protein and provides a triggering mechanism leading to aggregation of NF-L and other proteins in neurodegenerative diseases.

The 3'-untranslated regions of cytoskeletal muscle mRNAs inhibit translation by activating the double-stranded RNA-dependent protein kinase PKR.

Cytoskeletal proteins are associated with actin in the microfilaments and have a major role in microfilament assembly and function. The expression of some of these proteins has been implicated in cell growth and transformation. Specifically, the 3'-untranslated regions (3'-UTRs) of tropomyosin, troponin and cardiac actin can induce muscle cell differentiation and appear to function as tumor suppressors. These RNA sequences are predicted to fold to form secondary structures with extended stretches of duplex. We show that the 3'-UTRs of the cytoskeletal mRNAs interact with the RNA-binding domain of the RNA-activated protein kinase PKR. Correspondingly, these RNAs activate PKR in vitro and inhibit globin translation in the rabbit reticulocyte lysate translation system. These data are consistent with a mechanism whereby PKR mediates the differentiation- and tumor-related actions of the cytoskeletal 3'-UTR sequences.

Distinct regions in the 3' untranslated region are responsible for targeting and stabilizing utrophin transcripts in skeletal muscle cells.

In this study, we have sought to determine whether utrophin transcripts are targeted to a distinct subcellular compartment in skeletal muscle cells, and have examined the role of the 3' untranslated region (UTR) in regulating the stability and localization of utrophin transcripts. Our results show that utrophin transcripts associate preferentially with cytoskeleton-bound polysomes via actin microfilaments. Because this association is not evident in myoblasts, our findings also indicate that the localization of utrophin transcripts with cytoskeleton-bound polysomes is under developmental influences. Transfection of LacZ reporter constructs containing the utrophin 3'UTR showed that this region is critical for targeting chimeric mRNAs to cytoskeleton-bound polysomes and controlling transcript stability. Deletion studies resulted in the identification of distinct regions within the 3'UTR responsible for targeting and stabilizing utrophin mRNAs. Together, these results illustrate the contribution of posttranscriptional events in the regulation of utrophin in skeletal muscle. Accordingly, these findings provide novel targets, in addition to transcriptional events, for which pharmacological interventions may be envisaged to ultimately increase the endogenous levels of utrophin in skeletal muscle fibers from Duchenne muscular dystrophy (DMD) patients.