Host-dependent roles of the viral 5′ untranslated region (UTR) in RNA stabilization and cap-independent translational enhancement mediated by the 3′ UTR of Red clover necrotic mosaic virus RNA1

The genome of Red clover necrotic mosaic virus (RCNMV) consists of RNA1 and RNA2, both lacking a cap structure and a poly(A)tail. RNA1 has a translational enhancer element (3′TE-DR1) in the 3′ untranslated region (UTR). In this study, we analyzed the roles of 5′ and 3′ UTRs of RNA1 in 3′TE-DR1-mediated cap-independent translation in cowpea and tobacco BY-2 protoplasts using a dual-luciferase (Luc) reporter assay system. Most mutations introduced into RNA1 5′ UTR in reporter Luc mRNA abolished or greatly reduced cap-independent translation in BY-2 protoplasts, whereas those mutations had no or much milder effects if any on translational activity in cowpea protoplasts. Our results suggest that a stem-loop structure predicted in the 5′ proximal region of RNA1 plays important roles in both translation and RNA stability. We also show that 3′TE-DR1-mediated cap-independent translation relies on a ribosome-scanning mechanism in both protoplasts.     

Functional role of the 5′ terminal cloverleaf in Coxsackievirus RNA replication

Using cell-free reactions, we investigated the role of the 5′ cloverleaf (5′CL) and associated C-rich sequence in Coxsackievirus B3 RNA replication. We showed that the binding of poly(C) binding protein (PCBP) to the C-rich sequence was the primary determinant of RNA stability. In addition, inhibition of negative-strand synthesis was only observed when PCBP binding to both stem-loop ‘b’ and the C-rich sequence was inhibited. Taken together, these findings suggest that PCBP binding to the C-rich sequence was sufficient to support RNA stability and negative-strand synthesis. Mutational analysis of the three conserved structural elements in stem-loop ‘d’ showed that they were required for efficient negative- and positive-strand synthesis. Finally, we showed an RNA with a 5′ terminal deletion (Δ49TD RNA), which was previously isolated from persistently infected cells, replicated at low but detectable levels in these reactions. Importantly, the critical replication elements identified in this study are still present in the Δ49TD RNA.     

Mutational analysis of three predicted 5'-proximal stem-loop structures in the genome of tick-borne encephalitis virus indicates different roles in RNA replication and translation

Flavivirus gene expression is modulated by RNA secondary structure elements at the terminal ends of the viral RNA molecule. For tick-borne encephalitis virus (TBEV), four stem-loop (SL) elements have been predicted in the first 180 nucleotides of the viral genome: 5′-SL1, 5′-SL2, 5′-SL3 and 5′-SL4. The last three of these appear to be unique to tick-borne flaviviruses. Here, we report their characterization by mutagenesis in a TBEV luciferase reporter system. By manipulating their thermodynamic properties, we found that an optimal stability of the 5′-SL2 is required for efficient RNA replication. 5′-SL3 formation is also important for viral RNA replication, but although it contains the viral start codon, its formation is dispensable for RNA translation. 5′-SL4 appears to facilitate both RNA translation and replication. Our data suggest that maintenance of the balanced thermodynamic stability of these SL elements is important for temporal regulation of its different functions.     

Classical swine fever virus NS5A protein interacts with 3'-untranslated region and regulates viral RNA synthesis

To investigate the function of classical swine fever virus (CSFV) NS5A protein, the experiments for viral RNA synthesis and viral replication were performed in the co-presence of NS5A and NS5B. Results showed that small concentrations of NS5A stimulated, large concentrations of NS5A inhibited, viral RNA synthesis and viral replication. Affinity chromatography experiments and UV-crosslinking assays revealed that CSFV NS5A and NS5B bound its cognate 3′UTR and that NS5A had higher affinity than NS5B protein in binding to 3′UTR. 200 ng of NS5A inhibited NS5B-3′UTR complex formation by about 95%. CSFV 3′UTR was found to contain two NS5A-binding sites, located in 3′UTRSL-1 (nt 161-231) and 3′UTRSL-2 (nt 90-160), respectively, a NS5B-binding site, also located in 3′UTRSL-1. The 3′UTRSL-1 is the common binding site for NS5A and NS5B. Furthermore, competitive electrophoretic mobility shift assays indicated that binding of CSFV NS5A to 3′UTRSL-1 is more efficiently than to 3′UTRSL-2. These results suggested that the different concentrations of NS5A, the different binding activities of NS5A and NS5B to 3′UTR and binding of NS5A to different regions of 3′UTR might contribute at least partially to modulation of CSFV replication.     

The tRNA-like structure of Turnip yellow mosaic virus RNA is a 3'-translational enhancer

Many positive stand RNA viral genomes lack the poly(A) tail that is characteristic of cellular mRNAs and that promotes translation in cis. The 3′ untranslated regions (UTRs) of such genomes are expected to provide similar translation-enhancing properties as a poly(A) tail, yet the great variety of 3′ sequences suggests that this is accomplished in a range of ways. We have identified a translational enhancer present in the 3′ UTR of Turnip yellow mosaic virus (TYMV) RNA using luciferase reporter RNAs with generic 5′ sequences transfected into plant cells. The 3′ terminal 109 nucleotides comprising the tRNA-like structure (TLS) and an upstream pseudoknot (UPSK) act in synergy with a 5′-cap to enhance translation, with a minor contribution in stabilizing the RNA. Maximum enhancement requires that the RNA be capable of aminoacylation, but either the native valine or engineered methionine is acceptable. Mutations that decrease the affinity for translation elongation factor eEF1A (but also diminish aminoacylation efficiency) strongly decrease translational enhancement, suggesting that eEF1A is mechanistically involved. The UPSK seems to act as an important, though nonspecific, spacer element ensuring proper presentation of a functional TLS. Our studies have uncovered a novel type of translational enhancer and a new role for a plant viral TLS.     

Viral determinants in the NS3 helicase and 2K peptide that promote West Nile virus resistance to antiviral action of 2′,5′-oligoadenylate synthetase 1b

The interferon-inducible 2′,5′-oligoadenylate synthetase 1b (Oas1b) protein inhibits West Nile virus (WNV) infection by preventing viral RNA (vRNA) accumulation in infected cells. Serial passage of WNV in Oas1b-expressing mouse cells selected a virus variant with improved growth capacity. Two major amino acid substitutions were identified in this Oas1b-resistant WNV variant: NS3-S365G in the ATPase/helicase domain of NS3 and 2K-V9M in the C-terminal segment of NS4A. To assess their effect on antiviral activity of Oas1b, the NS3 and 2K mutations were engineered into an infectious WNV cDNA clone. The NS3 mutation alters requirement of ATP for ATPase activity and attenuates Oas1b-mediated suppression of vRNA accumulation. However, growth of NS3-mutant virus remains impaired in Oas1b-expressing cells. Only the 2K-V9M mutation efficiently rescued viral growth by promoting vRNA replication. Thus, WNV resistance to Oas1b antiviral action could be attributed to the 2K-V9M substitution with a potential role of NS3-S365G through rescue of vRNA accumulation.     

Cellular proteins mediate 5′-3′ end contacts of Norwalk virus genomic RNA

Long-range RNA-RNA interactions between the 5′ and 3′ ends are a common feature involved in the regulation of both the initiation of translation and the synthesis of the viral genomic RNAs. These interactions either take place by direct RNA-RNA contacts or can be mediated by proteins. By in silico analysis, we found three possible complementary sequences (CS) between the 5′ and the 3′ ends of the Norwalk virus genomic RNA. Co-precipitation assays demonstrated that physical contacts between the 5′ and the 3′ ends of the NV genomic RNA were stabilized by cellular proteins. Mutations and deletions within these regions, that altered the formation of the CS-1 motif disrupted the 5′-3′ end contacts, while mutations that restore complementarity of the CS-1 motif, recover the ability to form these contacts. These results suggest that the NV genomic 5′-3′ end contacts initially occur by RNA-RNA interactions but are further stabilized by cellular proteins.     

The 5′-UTR of Turnip yellow mosaic virus does not include a critical encapsidation signal

Turnip yellow mosaic virus (TYMV) RNA has two hairpins in the 5′ untranslated region (UTR) with internal CC and CA mismatches that become protonated and are able to base pair at a pH near 5. The protonatable hairpins have previously been implicated as playing an important role in RNA encapsidation. We have examined the role of the 5′-UTR in the amplification and packaging of TYMV RNA using agroinfiltration of Chinese cabbage leaves to express various TYMV constructs with mutations affecting the 5′-UTR and the two hairpins. Mutations affecting the protonatable centers of the two hairpins, as well as deletion of one or both hairpins and deletion or mutation of the 17-nucleotide region upstream of the hairpins decreased viral amplification to varying extents (c. 10- to 1000-fold). However, in all these cases, the viral RNAs present in non-denaturing leaf extracts were predominantly ribonuclease resistant, indicative of encapsidation. These results show that, while the 5′ hairpins are necessary for efficient amplification of TYMV, there appears to be no essential role for the 5′-UTR or its protonatable hairpins in the packaging of TYMV RNA. In a second set of experiments, it was demonstrated that TYMV can efficiently amplify in plants held in the dark, and that the progeny RNAs are efficiently encapsidated. Together, these observations argue for a revision of the model for TYMV encapsidation in which packaging occurs in low pH conditions that are generated by proton gradients produced by photosynthetic activity in the light and RNA packaging is dependent on the protonatable 5′ hairpins.     

Structures required for poly(A) tail-independent translation overlap with, but are distinct from, cap-independent translation and RNA replication signals at the 3' end of Tobacco necrosis virus RNA

Tobacco necrosis necrovirus (TNV) RNA lacks both a 5' cap and a poly(A) tail but is translated efficiently, owing in part to a Barley yellow dwarf virus (BYDV)-like cap-independent translation element (BTE) in its 3' untranslated region (UTR). Here, we identify sequence downstream of the BTE that is necessary for poly(A) tail-independent translation in vivo by using RNA encoding a luciferase reporter gene flanked by viral UTRs. Deletions and point mutations caused loss of translation that was restored by adding a poly(A) tail, and not by adding a 5' cap. The two 3'-proximal stem-loops in the viral genome contribute to poly(A) tail-independent translation, as well as RNA replication. For all necroviruses, we predict a conserved 3' UTR secondary structure that includes the BTE at one end of a long helical axis and the stem-loops required for poly(A) tail-independent translation and RNA replication at the other end. This work shows that a viral genome can harbor distinct cap- and poly(A) tail-mimic sequences in the 3' UTR.     

Mutagen-mediated enhancement of HIV-1 replication in persistently infected cells

Lethal mutagenesis, a new antiviral strategy to extinguish virus through elevated mutation rates, was explored in H61-D cells an HIV-1 persistently infected lymphoid cell line. Three mutagenic agents: 5-hydroxy-2^′-deoxycytidine (5-OHdC), 5-fluorouracil (5-FU) and 2,2^′-difluoro-2^′-deoxycytidine (gemcitabine) were used. After 54 passages, treatments with 5-FU and gemcitabine reduced virus infectivity, p24 and RT activity. Treatment with the pyrimidine analog 5-OHdC resulted in increases of p24 production, RT activity and infectivity. Rise in viral replication by 5-OHdC during HIV-1 persistence is in contrast with its inhibitory effect in acute infections. Viral replication enhancement by 5-OHdC was associated with an increase in intracellular HIV-1 RNA mutations. Mechanisms of HIV-1 replication enhancement by 5-OHdC are unknown but some potential factors are discussed. Increase of HIV-1 replication by 5-OHdC cautions against the use, without previous analyses, of mutagenic nucleoside analogs for AIDS treatment.     

Targeting enteroviral 2A protease by a 16-mer synthetic peptide: Inhibition of 2A-induced apoptosis in a stable Tet-on HeLa cell line

Enteroviridae such as coxsackievirus are important infectious agents causing viral heart diseases. Viral protease 2A (2A^pro) initiates the virus life cycle, and is an excellent target for developing antiviral drugs. Here, to evaluate the validity of the 2A^pro as a proper therapeutic target, and based on the existing information and molecular dynamics, a 16-mer peptide was designed to specifically target the active site of protease 2A^pro in order to block the activity of CVB3 2A^pro. We showed that the peptide could compete with endogenous substrate in a concentration-dependent manner. Further, we established a HeLa cell line that expressed 2A^pro. Expression of 2A^pro resulted in significant morphological alteration and eventual cell death. Western blot and viability assay showed that the 16-mer peptide (200 μg/ml) could significantly block 2A^pro activity and its cytotoxic effect. Future modification of the 16-mer peptide can improve its affinity for 2A^pro and therefore develop effective antiviral drug.     

Phosphorylation of enteroviral 2Apro at Ser/Thr125 benefits its proteolytic activity and viral pathogenesis

Enteroviral 2A proteinase (2A^pro), a well-established and important viral functional protein, plays a key role in shutting down cellular cap-dependent translation, mainly via its proteolytic activity, and creating optimal conditions for Enterovirus survival. Accumulated data show that viruses take advantage of various signaling cascades for their life cycle; studies performed by us and others have demonstrated that the extracellular signal-regulated kinase (ERK) pathway is essential for enterovirus A71 (EV-A71) and other viruses replication. We recently showed that ERK1/2 is required for the proteolytic activity of viral 2A^pro; however, the mechanism underlying the regulation of 2A^pro remains unknown. Here, we demonstrated that the 125th residue Ser125 of EV-A71 2A^pro or Thr125 of coxsackievirus B3 2A^pro, which is highly conserved in the Enterovirus, was phosphorylated by ERK1/2. Importantly, 2A^pro with phosphor-Ser/Thr125 had much stronger proteolytic activity toward eukaryotic initiation factor 4GI and rendered the virus more efficient for multiplication and pathogenesis in hSCARB2 knock-in mice than that in nonphospho-Ser/Thr125A (S/T125A) mutants. Notably, phosphorylation-mimic mutations caused deleterious changes in 2A^pro catalytic function (S/T125D/E) and in viral propagation (S125D). Crystal structure simulation analysis showed that Ser125 phosphorylation in EV-A71 2A^pro enabled catalytic Cys to adopt an optimal conformation in the catalytic triad His-Asp-Cys, which enhances 2A^pro proteolysis. Therefore, we are the first to report Ser/Thr125 phosphorylation of 2A^pro increases enteroviral adaptation to the host to ensure enteroviral multiplication, causing pathogenicity. Additionally, weakened viruses containing a S/T125A mutation could be a general strategy to develop attenuated Enterovirus vaccines.     

Structural plasticity of Barley yellow dwarf virus-like cap-independent translation elements in four genera of plant viral RNAs

The 3′ untranslated regions (UTRs) of many plant viral RNAs contain cap-independent translation elements (3′ CITEs). Among the 3′ CITEs, the Barley yellow dwarf virus (BYDV)-like translation elements (BTEs) form a structurally variable and widely distributed group. Viruses in three genera were known to harbor 3′ BTEs, defined by the presence of a 17-nt consensus sequence. To understand BTE function, knowledge of phylogenetically conserved structure is essential, yet the secondary structure has been determined only for the BYDV BTE. Here we show that Rose spring dwarf-associated luteovirus, and two viruses in a fourth genus, Umbravirus, contain functional BTEs, despite deviating in the 17 nt consensus sequence. Structure probing by selective 2′-hydroxyl acylation and primer extension (SHAPE) revealed conserved and highly variable structures in BTEs in all four genera. We conclude that BTEs tolerate striking evolutionary plasticity in structure, while retaining the ability to stimulate cap-independent translation.     

An alternative -1/+2 open reading frame exists within viral N(pro)(1-19) region of bovine viral diarrhea virus SD-1

We previously reported the engineering of an N^pro-disrupted bovine viral diarrhea virus (BVDV), BSD1-N^pro/eGFP2A (Fan and Bird, 2008a). Here, we report that BSD1-N^pro/eGFP2A survives a single nucleotide missing in its C-terminal eGFP region. By using our established reverse genetics system for BVDV, we confirm that the viral mutant is rescued through a −1/+2 ORF initiated in the N^pro(1-19)/eGFP region of the mutant viral genome. We furthermore uncover that this event occurs in the N^pro(1-19) region of BVDV strain SD-1. The rescued viral mutant showed dramatic reductions in levels of both viral RNA and viral protein in host cells. Although the mutant is similar to the native strain in viral kinetics, the peak yield of the mutant is decreased dramatically. These findings reveal the existence of an alternative −1/+2 ORF in the N^pro(1-19) region during the replication of BVDV and open a new avenue to understand the life cycle and pathogenesis of pestiviruses.     

Fig mosaic virus mRNAs show generation by cap-snatching

Fig mosaic virus (FMV), a member of the newly described genus Emaravirus, has four negative-sense single-stranded genomic RNAs, and each codes for a single protein in the viral complementary RNA (vcRNA). In this study we show that FMV mRNAs for genome segments 2 and 3 contain short (12-18 nucleotides) heterogeneous nucleotide leader sequences at their 5′ termini. Furthermore, by using the high affinity cap binding protein eIF4E_K119A, we also determined that a 5′ cap is present on a population of the FMV positive-sense RNAs, presumably as a result of cap-snatching. Northern hybridization results showed that the 5′ capped RNA3 segments are slightly smaller than the homologous vcRNA3 and are not polyadenylated. These data suggest that FMV generates 5′ capped mRNAs via cap-snatching, similar to strategies used by other negative-sense multipartite ssRNA viruses.     

A fluorescence resonance energy transfer-based fluorometer assay for screening anti-coxsackievirus B3 compounds

In view of the need to develop a simple and rapid method to screen for antiviral therapeutic agents, a fluorescence resonance energy transfer (FRET)-based reporter system consisting of engineered mammalian cells expressing a cyan fluorescent protein-yellow fluorescent protein (CFP-YFP) pair linked by a short peptide containing the cleavage site of viral protease 2A (2A^pro) was developed. By detecting the 2A^pro produced early during the virus infection cycle, the CFP-YFP pair effectively identifies infectious coxsackievirus B3 (CVB3), a picornavirus that causes viral myocarditis in humans. The reporter system was used to screen a library of 2000 drugs and natural products for potential antiviral compounds. The reporter cells were treated with the test compounds, challenged with CVB3, and then examined using a fluorometer at 24 h post-infection. Sixty-four compounds, mostly therapeutic drugs, antimicrobial compounds and compounds with unknown functions, caused at least 50% inhibition of 2A^pro activity. Three known antiviral compounds, cosmosiin, ribavirin and baicalein, were also identified in the screening. The developed method is an effective strategy for rapid screening, and identifies compounds that inhibit CVB3 2A^pro. This method should be a valuable aid in the antiviral drug discovery effort.     

The nuclear protein Sam68 is cleaved by the FMDV 3C protease redistributing Sam68 to the cytoplasm during FMDV infection of host cells

Picornavirus infection can lead to disruption of nuclear pore traffic, shut-off of cell translation machinery, and cleavage of proteins involved in cellular signal transduction and the innate response to infection. Here, we demonstrated that the FMDV 3C^pro induced the cleavage of nuclear RNA-binding protein Sam68 C-terminus containing the nuclear localization sequence (NLS). Consequently, it stimulated the redistribution of Sam68 to the cytoplasm. The siRNA knockdown of Sam68 resulted in a 1000-fold reduction in viral titers, which prompted us to study the effect of Sam68 on FMDV post-entry events. Interestingly, Sam68 interacts with the internal ribosomal entry site within the 5′ non-translated region of the FMDV genome, and Sam68 knockdown decreased FMDV IRES-driven activity in vitro suggesting that it could modulate translation of the viral genome. The results uncover a novel role for Sam68 in the context of picornaviruses and the proteolysis of a new cellular target of the FMDV 3C^pro.