Negative-strand Synthesis Research Articles

A highly discriminatory RNA strand-specific assay to facilitate analysis of the role of cis-acting elements in foot-and-mouth disease virus replication.

Foot-and-mouth-disease virus (FMDV), the aetiological agent responsible for foot-and-mouth disease (FMD), is a member of the genus Aphthovirus within the family Picornavirus. In common with all picornaviruses, replication of the single-stranded positive-sense RNA genome involves synthesis of a negative-sense complementary strand that serves as a template for the synthesis of multiple positive-sense progeny strands. We have previously employed FMDV replicons to examine viral RNA and protein elements essential to replication, but the factors affecting differential strand production remain unknown. Replicon-based systems require transfection of high levels of RNA, which can overload sensitive techniques such as quantitative PCR, preventing discrimination of specific strands. Here, we describe a method in which replicating RNA is labelled in vivo with 5-ethynyl uridine. The modified base is then linked to a biotin tag using click chemistry, facilitating purification of newly synthesised viral genomes or anti-genomes from input RNA. This selected RNA can then be amplified by strand-specific quantitative PCR, thus enabling investigation of the consequences of defined mutations on the relative synthesis of negative-sense intermediate and positive-strand progeny RNAs. We apply this new approach to investigate the consequence of mutation of viral cis-acting replication elements and provide direct evidence for their roles in negative-strand synthesis.

The strand-biased transcription of SARS-CoV-2 and unbalanced inhibition by remdesivir

SummarySevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive single-stranded RNA virus, causes the coronavirus disease 19 pandemic. During the viral replication and transcription, the RNA-dependent RNA polymerase “jumps” along the genome template, resulting in discontinuous negative-stranded transcripts. Although the sense-mRNA architectures of SARS-CoV-2 were reported, its negative strand was unexplored. Here, we deeply sequenced both strands of RNA and found SARS-CoV-2 transcription is strongly biased to form the sense strand with variable transcription efficiency for different genes. During negative strand synthesis, numerous non-canonical fusion transcripts are also formed, driven by 3-15 nt sequence homology scattered along the genome but more prone to be inhibited by SARS-CoV-2 RNA polymerase inhibitor remdesivir. The drug also represses more of the negative than the positive strand synthesis as supported by a mathematic simulation model and experimental quantifications. Overall, this study opens new sights into SARS-CoV-2 biogenesis and may facilitate the antiviral vaccine development and drug design.

Venezuelan Equine Encephalitis Virus nsP3 Phosphorylation Can Be Mediated by IKKβ Kinase Activity and Abrogation of Phosphorylation Inhibits Negative-Strand Synthesis.

Venezuelan equine encephalitis virus (VEEV), a mosquito transmitted alphavirus of the Togaviridae family, can cause a highly inflammatory and encephalitic disease upon infection. Although a category B select agent, no FDA-approved vaccines or therapeutics against VEEV currently exist. We previously demonstrated NF-κB activation and macromolecular reorganization of the IKK complex upon VEEV infection in vitro, with IKKβ inhibition reducing viral replication. Mass spectrometry and confocal microscopy revealed an interaction between IKKβ and VEEV non-structural protein 3 (nsP3). Here, using western blotting, a cell-free kinase activity assay, and mass spectrometry, we demonstrate that IKKβ kinase activity can directly phosphorylate VEEV nsP3 at sites 204/5, 142, and 134/5. Alanine substitution mutations at sites 204/5, 142, or 134/5 reduced VEEV replication by >30-100,000-fold corresponding to a severe decrease in negative-strand synthesis. Serial passaging rescued viral replication and negative-strand synthesis, and sequencing of revertant viruses revealed reversion to the wild-type TC-83 phosphorylation capable amino acid sequences at nsP3 sites 204/5, 142, and 135. Generation of phosphomimetic mutants using aspartic acid substitutions at site 204/5 resulted in rescue of both viral replication and negative-strand RNA production, whereas phosphomimetic mutant 134/5 rescued viral replication but failed to restore negative-strand RNA levels, and phosphomimetic mutant 142 did not rescue VEEV replication. Together, these data demonstrate that IKKβ can phosphorylate VEEV nsP3 at sites 204/5, 142, and 134/5, and suggest that phosphorylation is essential for negative-strand RNA synthesis at site 204/5, but may be important for infectious particle production at site 134/5.

The Strand-Biased Transcription of SARS-CoV-2 And Unbalanced Inhibition by Remdesivir

SARS-CoV-2, a positive single-stranded RNA virus, caused the COVID-19 pandemic. During the viral replication and transcription, the RNA dependent RNA polymerase (RdRp) “jumps” along the genome template, resulting in discontinuous negative-stranded transcripts. In other coronaviruses, the negative strand RNA was found functionally relevant to the activation of host innate immune responses. Although the sense-mRNA architectures of SARS-CoV-2 were reported, its negative strand was unexplored. Here, we deeply sequenced both strands of RNA and found SARS-CoV-2 transcription is strongly biased to form the sense strand with also variable transcription efficiency for different protein-coding genes. During negative strand synthesis, apart from canonical sub-genomic ORFs, numerous non-canonical fusion transcripts are formed, driven by 3-15 nt sequence homology scattered along the genome but more prone to be inhibited by SARS-CoV-2 RNA polymerase inhibitor Remdesivir. The drug also represses more of the negative than the positive strand synthesis as supported by a mathematic simulation model and experimental quantifications. Overall, this study opens new sights into SARS-CoV-2 biogenesis and may facilitate the anti-viral vaccine development and drug design.

RNA Replication and Membrane Modification Require the Same Functions of Alphavirus Nonstructural Proteins.

The alphaviruses induce membrane invaginations known as spherules as their RNA replication sites. Here, we show that inactivation of any function (polymerase, helicase, protease, or membrane association) essential for RNA synthesis also prevents the generation of spherule structures in a Semliki Forest virus trans-replication system. Mutants capable of negative-strand synthesis, including those defective in RNA capping, gave rise to spherules. Recruitment of RNA to membranes in the absence of spherule formation was not detected.

Role of the Pepino mosaic virus 3′-untranslated region elements in negative-strand RNA synthesis in vitro

Pepino mosaic virus (PepMV) is a mechanically-transmitted positive-strand RNA potexvirus, with a 6410nt long single-stranded (ss) RNA genome flanked by a 5′-methylguanosine cap and a 3′ poly-A tail. Computer-assisted folding of the 64nt long PepMV 3′-untranslated region (UTR) resulted in the prediction of three stem-loop structures (hp1, hp2, and hp3 in the 3′–5′ direction). The importance of these structures and/or sequences for promotion of negative-strand RNA synthesis and binding to the RNA dependent RNA polymerase (RdRp) was tested in vitro using a specific RdRp assay. Hp1, which is highly variable among different PepMV isolates, appeared dispensable for negative-strand synthesis. Hp2, which is characterized by a large U-rich loop, tolerated base-pair changes in its stem as long as they maintained the stem integrity but was very sensitive to changes in the U-rich loop. Hp3, which harbours the conserved potexvirus ACUUAA hexamer motif, was essential for template activity. Template-RNA polymerase binding competition experiments showed that the ACUUAA sequence represents a high-affinity RdRp binding element.

The structural basis of transfer RNA mimicry and conformational plasticity by a viral RNA.

RNA is arguably the most functionally diverse biological macromolecule. In some cases a single discrete RNA sequence performs multiple roles and this can be conferred by a complex three-dimensional structure. This multifunctionality can also be driven or enhanced by the ability of a given RNA to assume different conformational (and therefore functional) states1. Despite its biological importance, a detailed structural understanding of the paradigm of RNA structure-driven multifunctionality is lacking. Examples to address this gap are found in single-stranded positive-sense RNA viruses, a prototype being the tRNA-like structure (TLS) found at the 3′ end of the Turnip Yellow Mosaic Virus (TYMV). This TLS not only acts like a tRNA to drive aminoacylation of the viral genomic RNA (gRNA)2-4, but also interacts with other structures in the gRNA's 3′ untranslated region5, contains the promoter for negative strand synthesis, and influences several infection-critical processes6. This TLS RNA can provide a glimpse into the structural basis of RNA multifunctionality and plasticity, but for decades its high-resolution structure has remained elusive. Here, we present the crystal structure of the complete TYMV TLS to 2.0 Å resolution. Globally, the RNA adopts a shape that mimics tRNA, but it uses a very different set of intramolecular interactions to achieve this shape. These interactions also allow the TLS to readily switch conformations. In addition, the TLS structure is ‘two-faced’: one ‘face’ closely mimics tRNA and drives aminoacylation, the other ‘face’ diverges from tRNA and enables additional functionality. The TLS is thus structured to perform several functions and interact with diverse binding partners, and we demonstrate its ability to specifically bind to ribosomes.

Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion

Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cells and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion.

Targeting Ribosome assembly on the HCV RNA using a small RNA molecule

Translation initiation of hepatitis C Virus (HCV) RNA is the initial obligatory step of the viral life cycle, mediated through the Internal Ribosome Entry Site (IRES) present in the 5′-untranslated region (UTR). Initiation on the HCV IRES is mediated by multiple structure-specific interactions between IRES RNA and host 40S ribosomal subunit. In the present study we demonstrate that the SLIIIef domain, in isolation from other structural elements of HCV IRES, retain the ability to interact with 40S ribosome subunit. A small RNA SLRef, mimicking the SLIIIef domain was found to interact specifically with human La protein and the ribosomal protein S5 and selectively inhibit HCV RNA translation. More importantly, SLRef RNA showed significant suppression of replication in HCV monocistronic replicon and decrease of negative strand synthesis in HCV cell culture system. Finally, using Sendai virus based virosome, the targeted delivery of SLRef RNA into mice liver succeeded in selectively inhibiting HCV IRES mediated translation in vivo.

Temperature and density dependent induction of a cytopathic effect following infection with non-cytopathic HAV strains

Hepatitis A virus infection and growth in cultured cells is protracted, cell-type restricted, and generally not accompanied by the appearance of a cytopathic effect, with the exception of some culture-adapted strains. We demonstrate that the non-cytopathic HAV strain HM175/clone 1 can be induced to exhibit a cytopathic phenotype in both persistently or acutely infected cells under co-dependent conditions of lower incubation temperature (<34°C) and reduced cell density in both monkey (FRhK-4) and human (A549) cells. This phenotype is not virus-strain restricted, as it was also observed in cells infected with HAV strains, HAS-15 and LSH/S. Cytopathic effect was accompanied by rRNA cleavage, indicating activation of the RNase L pathway, viral negative strand synthesis, caspase-3 activation, and apoptosis. The results indicate that a cytopathic phenotype may be present in some HAV strains that can be induced under appropriate conditions, suggesting the potential for development of a plaque assay for this virus.

Slow Conformational Dynamics in the Cystoviral RNA-Directed RNA Polymerase P2: Influence of Substrate Nucleotides and Template RNA

The RNA-directed RNA polymerase P2 from cystovirus ϕ6 catalyzes the de novo synthesis of positive and negative strands of the viral double-stranded RNA genome. P2 is mobile on the slow, microsecond to millisecond time scale with various motional modes, putatively assisting in RNA translocation and catalysis. Here we investigate the influence of the extreme 3'-end sequence of the single-stranded RNA templates and the nature of the substrate nucleotide triphosphates on these motional modes using multiple-quantum NMR spectroscopy. We find that P2, in the presence of templates bearing the proper genomic 3'-ends or the preferred initiation nucleotide, displays unique dynamic signatures that are different from those in the presence of nonphysiological templates or substrates. This suggests that dynamics may play a role in the fidelity of recognition of the correct substrates and template sequences to initiate RNA polymerization.

A Y-shaped RNA structure in the 3′ untranslated region together with the trans-activator and core promoter of Red clover necrotic mosaic virus RNA2 is required for its negative-strand RNA synthesis

Red clover necrotic mosaic virus (RCNMV) is a positive-strand RNA virus with a bipartite genome. RNA1 encodes N-terminally overlapping replication proteins, p27 and p88. RNA2 is replicated efficiently by the replication proteins supplied in trans, whereas RNA1 needs p88 preferentially in cis for its replication. cis-Acting elements required for RNA2 replication have been mapped to the 3′ terminal stem–loop structure conserved between RNA1 and RNA2, and to the protein-coding region including the trans-activator. Here, we have identified a Y-shaped RNA structure with three-way RNA junctions predicted in the 3′ untranslated region of RNA2 as a novel element required for negative-strand synthesis using an in vitro translation/replication system. We also show that, in addition to the 3′ terminal core promoter, several RNA elements including the trans-activator are also required for negative-strand synthesis. Functional roles and structural requirements of these cis-acting elements in RCNMV RNA replication are discussed.

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 (Delta49TD 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 Delta49TD RNA.

5′ cis Elements Direct Nodavirus RNA1 Recruitment to Mitochondrial Sites of Replication Complex Formation

Positive-strand RNA viruses replicate their genomes on intracellular membranes, usually in conjunction with virus-induced membrane rearrangements. For the nodavirus flock house virus (FHV), we recently showed that multifunctional FHV replicase protein A induces viral RNA template recruitment to a membrane-associated state, but the site(s) and function of this recruitment were not determined. By tagging viral RNA with green fluorescent protein, we show here in Drosophila cells that protein A recruits FHV RNA specifically to the outer mitochondrial membrane sites of RNA replication complex formation. Using Drosophila cells and yeast cells, which also support FHV replication, we also defined the cis-acting regions that direct replication and template recruitment for FHV genomic RNA1. RNA1 nucleotides 68 to 205 were required for RNA replication and directed efficient protein A-mediated RNA recruitment in both cell types. RNA secondary structure prediction, structure probing, and phylogenetic comparisons in this region identified two stable, conserved stem-loops with nearly identical loop sequences. Further mutational analysis showed that both stem-loops and certain flanking sequences were required for RNA1 recruitment, negative-strand synthesis, and subsequent positive-strand amplification in yeast and Drosophila cells. Thus, we have shown that protein A recruits RNA1 templates to mitochondria, as expected for RNA replication, and identified a new RNA1 cis element that is necessary and sufficient for RNA1 template recognition and recruitment to these mitochondrial membranes for negative-strand RNA1 synthesis. These results establish RNA recruitment to the sites of replication complex formation as an essential, distinct, and selective early step in nodavirus replication.

Comparison and functional implications of the 3D architectures of viral tRNA-like structures

RNA viruses co-opt the host cell's biological machinery, and their infection strategies often depend on specific structures in the viral genomic RNA. Examples are tRNA-like structures (TLSs), found at the 3' end of certain plant viral RNAs, which can use the cell's aminoacyl tRNA-synthetases (AARSs) to drive addition of an amino acid to the 3' end of the viral RNA. TLSs are multifunctional RNAs involved in processes such as viral replication, translation, and viral RNA stability; these functions depend on their fold. Experimental result-based structural models of TLSs have been published. In this study, we further examine these structures using a combination of biophysical and biochemical approaches to explore the three-dimensional (3D) architectures of TLSs from the turnip yellow mosaic virus (TYMV), tobacco mosaic virus (TMV), and brome mosaic virus (BMV). We find that despite similar function, these RNAs are biophysically diverse: the TYMV TLS adopts a characteristic tRNA-like L shape, the BMV TLS has a large compact globular domain with several helical extensions, and the TMV TLS aggregates in solution. Both the TYMV and BMV TLS RNAs adopt structures with tight backbone packing and also with dynamic structural elements, suggesting complexities and subtleties that cannot be explained by simple tRNA mimicry. These results confirm some aspects of existing models and also indicate how these models can be improved. The biophysical characteristics of these TLSs show how these multifunctional RNAs might regulate various viral processes, including negative strand synthesis, and also allow comparison with other structured RNAs.

Evidence for alternate states of Cucumber mosaic virus replicase assembly in positive- and negative-strand RNA synthesis

Cucumber mosaic virus (CMV) encodes two viral replication proteins, 1a and 2a. Accumulating evidence implies that different aspects of 1a–2a interaction in replication complex assembly are involved in the regulation of virus replication. To further investigate CMV replicase assembly and to dissect the involvement of replicase activities in negative- and positive-strand synthesis, we transiently expressed CMV RNAs and/or proteins in Nicotiana benthamiana leaves using a DNA or RNA-mediated expression system. Surprisingly, we found that, even in the absence of 1a, 2a is capable of synthesizing positive-strand RNAs, while 1a and 2a are both required for negative-strand synthesis. We also report evidence that 1a capping activities function independently of 2a. Moreover, using 1a mutants, we show that capping activities of 1a are crucial for viral translation but not for RNA transcription. These results support the concept that two or more alternate states of replicase assembly are involved in CMV replication.

Depurination within the intergenic region of Brome mosaic virus RNA3 inhibits viral replication in vitro and in vivo

Pokeweed antiviral protein (PAP) is a glycosidase of plant origin that has been shown to depurinate some viral RNAs in vitro. We have demonstrated previously that treatment of Brome mosaic virus (BMV) RNAs with PAP inhibited their translation in a cell-free system and decreased their accumulation in barley protoplasts. In the current study, we map the depurination sites on BMV RNA3 and describe the mechanism by which replication of the viral RNA is inhibited by depurination. Specifically, we demonstrate that the viral replicase exhibited reduced affinity for depurinated positive-strand RNA3 compared with intact RNA3, resulting in less negative-strand product. This decrease was due to depurination within the intergenic region of RNA3, between ORF3 and 4, and distant from the 3′ terminal core promoter required for initiation of negative-strand RNA synthesis. Depurination within the intergenic region alone inhibited the binding of the replicase to full-length RNA3, whereas depurination outside the intergenic region permitted the replicase to initiate negative-strand synthesis; however, elongation of the RNA product was stalled at the abasic nucleotide. These results support a role of the intergenic region in controlling negative-strand RNA synthesis and contribute new insight into the effect of depurination by PAP on BMV replication.

Isolation of RNA aptamers specific for the 3' X tail of HCV

The 3' end of the HCV genome, designated as the 3' X tail, comprises an almost invariant 98-nucleotide sequence containing three highly conserved stem-loop structures (3' SL1, 3' SL2, and 3' SL3). Since these sequences are all critical for the initiation of negative-strand synthesis and essential for viral replication, they are attractive targets for novel anti-HCV drugs. To obtain effective RNA aptamers specific for the 3' X tail, and with the aim of developing novel inhibitors of HCV replication, we performed in vitro selection of aptamers with specificity for the 3' X tail. In vitro selection, namely SELEX (systematic evolution of ligands by exponential enrichment) is a useful strategy for isolating nucleic acid sequences from a randomized oligonucleotide pool that have a high affinity for a target molecule. After four selection cycles, a pool of the 3' X tail-specific RNA aptamers were obtained. This RNA pool included 39 clones that could be divided into three main classes (cSL1, cSL2, and cSL3) which harbor complementary sequences to the apical loops of 3' SL1, 3' SL2, and 3' SL3, respectively. Biochemical analyses are in progress to evaluate whether these RNA aptamers have the potential to block HCV replication.

Aichi Virus 2A Protein Is Involved in Viral RNA Replication

The Aichi virus 2A protein is not a protease, unlike many other picornavirus 2A proteins, and it is related to a cellular protein, H-rev107. Here, we examined the replication properties of two 2A mutants in Vero cells and a cell-free translation/replication system. In one mutant, amino acids 36 to 126 were replaced with an unrelated amino acid sequence. In the other mutant, the NC motif conserved in the H-rev107 family of proteins was changed to alanine residues. The two mutations abolished virus replication in cells. The mutations affected both negative- and positive-strand synthesis, the defect in positive-strand synthesis being more severe than that in negative-strand synthesis.

Interaction between Polypeptide 3ABC and the 5′-Terminal Structural Elements of the Genome of Aichi Virus: Implication for Negative-Strand RNA Synthesis

Secondary structural elements at the 5' end of picornavirus genomic RNA function as cis-acting replication elements and are known to interact specifically with viral P3 proteins in several picornaviruses. In poliovirus, ribonucleoprotein complex formation at the 5' end of the genome is required for negative-strand synthesis. We have previously shown that the 5'-end 115 nucleotides of the Aichi virus genome, which are predicted to fold into two stem-loops (SL-A and SL-C) and one pseudoknot (PK-B), act as a cis-acting replication element and that correct folding of these structures is required for negative-strand synthesis. In this study, we investigated the interaction between the 5'-terminal 120 nucleotides of the genome and the P3 proteins, 3AB, 3ABC, 3C, and 3CD, by gel shift assay and Northwestern analysis. The results showed that 3ABC and 3CD bound to the 5'-terminal region specifically. The binding of 3ABC was observed on both assays, while that of 3CD was detected only on Northwestern analysis. No binding of 3AB or 3C was observed. Binding assays using mutant RNAs demonstrated that disruption of the base pairings of the stem of SL-A and one of the two stem segments of PK-B (stem-B1) abolished the 3ABC binding. In addition, the specific nucleotide sequence of stem-B1 was responsible for the efficient 3ABC binding. These results suggest that the interaction of 3ABC with the 5'-terminal region of the genome is involved in negative-strand synthesis. On the other hand, the ability of 3CD to interact with the 5'-terminal region did not correlate with the RNA replication ability.