Specific Recognition of a Stem-Loop RNA Structure by the Alphavirus Capsid Protein.

Alphaviruses are small enveloped viruses with positive-sense RNA genomes. During infection, the alphavirus capsid protein (Cp) selectively packages and assembles with the viral genomic RNA to form the nucleocapsid core, a process critical to the production of infectious virus. Prior studies of the alphavirus Semliki Forest virus (SFV) showed that packaging and assembly are promoted by Cp binding to multiple high affinity sites on the genomic RNA. Here, we developed an in vitro Cp binding assay based on fluorescently labeled RNA oligos. We used this assay to explore the RNA sequence and structure requirements for Cp binding to site #1, the top binding site identified on the genomic RNA during all stages of virus assembly. Our results identify a stem-loop structure that promotes specific binding of the SFV Cp to site #1 RNA. This structure is also recognized by the Cps of the related alphaviruses chikungunya virus and Ross River virus.

STING facilitates nuclear import of herpesvirus genome during infection.

Once inside the host cell, DNA viruses must overcome the physical barrier posed by the nuclear envelope to establish a successful infection. The mechanism underlying this process remains unclear. Here, we show that the herpesvirus exploits the immune adaptor stimulator of interferon genes (STING) to facilitate nuclear import of the viral genome. Following the entry of the viral capsid into the cell, STING binds the viral capsid, mediates capsid docking to the nuclear pore complex via physical interaction, and subsequently enables accumulation of the viral genome in the nucleus. Silencing STING in human cytomegalovirus (HCMV)-susceptible cells inhibited nuclear import of the viral genome and reduced the ensuing viral gene expression. Overexpressing STING increased the host cell's susceptibility to HCMV and herpes simplex virus 1 by improving the nuclear delivery of viral DNA at the early stage of infection. These observations suggest that the proviral activity of STING is conserved and exploited by the herpesvirus family. Intriguingly, in monocytes, which act as latent reservoirs of HCMV, STING deficiency negatively regulated the establishment of HCMV latency and reactivation. Our findings identify STING as a proviral host factor regulating latency and reactivation of herpesviruses.

Quantum Dots: A Promising Fluorescent Label for Probing Virus Trafficking.

Recent research has highlighted the immense potential of the quantum dot (QD)-based single-virus tracking (SVT) technique in virology. In these experiments, the infection behaviors of single viruses or viral components, labeled with QDs, could be tracked on time scales of milliseconds to hours in host cells. The trajectories of individual viruses are reconstructed with nanometer accuracy, and the underlying dynamic information on virus infection can be extracted to uncover the infection mechanisms of viruses. Therefore, QD-based single-virus tracking (QSVT) is an exquisitely selective and powerful approach to investigating how viruses are internalized in host cells dynamically to release their genome for viral replication and assembly that ensure the completion of viral life cycles.QDs are better candidates than organic dyes and fluorescent proteins for virus labeling and subsequent SVT due to the following considerations: (i) the high brightness of QDs makes it possible to label a virus with sufficient brightness using very few QDs or even just one QD; (ii) the extraordinary photostability of QDs allows one to track the infection process long term and quantify low probability events; (iii) the color-tunable emission property of QDs ensures multicolor labeling of various components of a virus simultaneously; and (iv) the abundant surface ligands of QDs facilitate the conjugation of a virus with a variety of labeling strategies. Therefore, the photoproperties of QDs make it possible to perform multicolor long-term SVT experiments quantitatively. Nowadays, the QD-based SVT (QSVT) technique has made prodigious achievements in unraveling the entry, trafficking, and uncoating mechanisms of viruses. This fascinating technique can provide spatiotemporal dynamic information on the viral journey in unprecedented detail and has revolutionized our understanding of virus infection.In this Account, we first introduce the advantages and the limitations of conventional SVT in virological research and the unique features of QDs as labels in the SVT field. We subsequently focus on the principles and related methods of QSVT and the current state of QD chemistry and QD-based virus labeling that resolves many issues associated with the tracking of individual viruses in live cells. Then we emphasize some new findings by this technique in the study of infection mechanisms. Finally, we will provide our insights into future challenges on this topic. With this Account, we hope to further stimulate the development of QSVT with a combined effort from different disciplines and, more importantly, to accelerate the applications of QSVT in virological research.

Information Encoded by the Flavivirus Genomes beyond the Nucleotide Sequence.

The genus Flavivirus comprises numerous, small, single positive-stranded RNA viruses, many of which are important human pathogens. To store all the information required for their successful propagation, flaviviruses use discrete structural genomic RNA elements to code for functional information by the establishment of dynamic networks of long-range RNA-RNA interactions that promote specific folding. These structural elements behave as true cis-acting, non-coding RNAs (ncRNAs) and have essential regulatory roles in the viral cycle. These include the control of the formation of subgenomic RNAs, known as sfRNAs, via the prevention of the complete degradation of the RNA genome. These sfRNAs are important in ensuring viral fitness. This work summarizes our current knowledge of the functions performed by the genome conformations and the role of RNA-RNA interactions in these functions. It also reviews the role of RNA structure in the production of sfRNAs across the genus Flavivirus, and their existence in related viruses.

SARS-CoV-2 mutations: the biological trackway towards viral fitness.

The outbreak of pneumonia-like respiratory disorder at China and its rapid transmission world-wide resulted in public health emergency, which brought lineage B betacoronaviridae SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) into spotlight. The fairly high mutation rate, frequent recombination and interspecies transmission in betacoronaviridae are largely responsible for their temporal changes in infectivity and virulence. Investigation of global SARS-CoV-2 genotypes revealed considerable mutations in structural, non-structural, accessory proteins as well as untranslated regions. Among the various types of mutations, single-nucleotide substitutions are the predominant ones. In addition, insertion, deletion and frame-shift mutations are also reported, albeit at a lower frequency. Among the structural proteins, spike glycoprotein and nucleocapsid phosphoprotein accumulated a larger number of mutations whereas envelope and membrane proteins are mostly conserved. Spike protein and RNA-dependent RNA polymerase variants, D614G and P323L in combination became dominant world-wide. Divergent genetic variants created serious challenge towards the development of therapeutics and vaccines. This review will consolidate mutations in different SARS-CoV-2 proteins and their implications on viral fitness.

CNBP Binds and Unfolds In Vitro G-Quadruplexes Formed in the SARS-CoV-2 Positive and Negative Genome Strands.

The Coronavirus Disease 2019 (COVID-19) pandemic has become a global health emergency with no effective medical treatment and with incipient vaccines. It is caused by a new positive-sense RNA virus called severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). G-quadruplexes (G4s) are nucleic acid secondary structures involved in the control of a variety of biological processes including viral replication. Using several G4 prediction tools, we identified highly putative G4 sequences (PQSs) within the positive-sense (+gRNA) and negative-sense (-gRNA) RNA strands of SARS-CoV-2 conserved in related betacoronaviruses. By using multiple biophysical techniques, we confirmed the formation of two G4s in the +gRNA and provide the first evidence of G4 formation by two PQSs in the -gRNA of SARS-CoV-2. Finally, biophysical and molecular approaches were used to demonstrate for the first time that CNBP, the main human cellular protein bound to SARS-CoV-2 RNA genome, binds and promotes the unfolding of G4s formed by both strands of SARS-CoV-2 RNA genome. Our results suggest that G4s found in SARS-CoV-2 RNA genome and its negative-sense replicative intermediates, as well as the cellular proteins that interact with them, are relevant factors for viral genes expression and replication cycle, and may constitute interesting targets for antiviral drugs development.

Translatomic profiling reveals novel self-restricting virus-host interactions during HBV infection.

BACKGROUND & AIMS: HBV remains a global threat to human health. It remains incompletely understood how HBV self-restricts in the host during most adult infections. Thus, we performed multi-omics analyses to systematically interrogate HBV-host interactions and the life cycle of HBV. METHODS: RNA-sequencing and ribosome profiling were conducted with cell-based models for HBV replication and gene expression. The novel translational events or products hereby detected were then characterized, and functionally assessed in both cell and mouse models. Moreover, quasi-species analyses of HBV subpopulations were conducted with patients at immune tolerance or activation phases, using next- or third-generation sequencing. RESULTS: We identified EnhI-SL (Enhancer I-stem loop) as a new cis element in the HBV genome; mutations disrupting EnhI-SL were found to elevate viral polymerase expression. Furthermore, while re-discovering HpZ/P', a previously under-explored isoform of HBV polymerase, we also identified HBxZ, a novel short isoform of HBX. Having confirmed their existence, we functionally characterized them as potent suppressors of HBV gene expression and genome replication. Mechanistically, HpZ/P' was found to repress HBV gene expression partially by interacting with, and sequestering SUPV3L1. Activation of the host immune system seemed to reduce the abundance of HBV mutants deficient in HpZ/P' or with disruptions in EnhI-SL. Finally, SRSF2, a host RNA spliceosome protein that is downregulated by HBV, was found to promote the splicing of viral pre-genomic RNA and HpZ/P' biogenesis. CONCLUSION: This study has identified multiple self-restricting HBV-host interactions. In particular, SRSF2-HpZ/P' appeared to constitute another negative feedback mechanism in the HBV life cycle. Targeting host splicing machinery might thus represent a strategy to intervene in HBV-host interactions. LAY SUMMARY: There remain many unknowns about the natural history of HBV infection in adults. Herein, we identified new HBV-host mechanisms which could be responsible for self-restricting infections. Targeting these mechanisms could be a promising strategy for the treatment of HBV infections.

Real-Time Imaging of Polioviral RNA Translocation across a Membrane.

Genome transfer from a virus into a cell is a critical early step in viral replication. Enveloped viruses achieve the delivery of their genomes into the cytoplasm by merging the viral membrane with the cellular membrane via a conceptually simple mechanism called membrane fusion. In contrast, genome translocation mechanisms in nonenveloped viruses, which lack viral membranes, remain poorly understood. Although cellular assays provide useful information about cell entry and genome release, it is difficult to obtain detailed mechanistic insights due both to the inherent technical difficulties associated with direct visualization of these processes and to the prevalence of nonproductive events in cellular assays performed at a very high multiplicity of infection. To overcome these issues, we developed an in vitro single-particle fluorescence assay to characterize genome release from a nonenveloped virus (poliovirus) in real time using a tethered receptor-decorated liposome system. Our results suggest that poliovirus genome release is a complex process that consists of multiple rate-limiting steps. Interestingly, we found that the addition of exogenous wild-type capsid protein VP4, but not mutant VP4, enhanced the efficiency of genome translocation. These results, together with prior structural analysis, suggest that VP4 interacts with RNA directly and forms a protective, membrane-spanning channel during genome translocation. Furthermore, our data indicate that VP4 dynamically interacts with RNA, rather than forming a static tube for RNA translocation. This study provides new insights into poliovirus genome translocation and offers a cell-free assay that can be utilized broadly to investigate genome release processes in other nonenveloped viruses.IMPORTANCE The initial transfer of genomic material from a virus into a host cell is a key step in any viral infection. Consequently, understanding how viruses deliver their genomes into cells could reveal attractive therapeutic targets. Although conventional biochemical and cellular assays have provided useful information about cell entry, the mechanism used to deliver the viral genomes across the cellular membrane into the cytoplasm is not well characterized for nonenveloped viruses such as poliovirus. In this study, we developed a fluorescence imaging assay to visualize poliovirus genome release using a synthetic vesicle system. Our results not only provide new mechanistic insights into poliovirus genome translocation but also offer a cell-free assay to bridge gaps in understanding of this process in other nonenveloped viruses.

Packaging signal of influenza A virus.

Influenza A virus (IAV) contains a genome with eight single-stranded, negative-sense RNA segments that encode 17 proteins. During its assembly, all eight separate viral RNA (vRNA) segments are incorporated into virions in a selective manner. Evidence suggested that the highly selective genome packaging mechanism relies on RNA-RNA or protein-RNA interactions. The specific structures of each vRNA that contribute to mediating the packaging of the vRNA into virions have been described and identified as packaging signals. Abundant research indicated that sequences required for genome incorporation are not series and are varied among virus genotypes. The packaging signals play important roles in determining the virus replication, genome incorporation and genetic reassortment of influenza A virus. In this review, we discuss recent studies on influenza A virus packaging signals to provide an overview of their characteristics and functions.

Dengue virus strain 2 capsid protein switches the annealing pathway and reduces intrinsic dynamics of the conserved 5' untranslated region.

The capsid protein of dengue virus strain 2 (DENV2C) promotes nucleic acid structural rearrangements using chaperone activity. However, the role of DENV2C during the interaction of RNA elements in the conserved 5' untranslated region (5'UTR) to the 3' untranslated region (3'UTR) is still unclear. Thus, we investigated the effect of DENV2C on the annealing mechanism of two RNA hairpin elements from the 5'UTR to their complementary sequences during (+)/(-) ds-RNAformation and (+) RNA circularization. DENV2C was found to switch the annealing pathway for RNA elements involved in (+)/(-) ds-RNA formation, but not for RNA elements related to (+) RNA circularization. In addition, we also determined that DENV2C modulates intrinsic dynamics and reduces kinetically trapped unfavourable conformations of the 5'UTR sequence. Thus, our results provide mechanistic insights by which DENV2C chaperones the interactions between RNA elements at the 5' and 3' ends during genome recombination, a prerequisite for DENV replication.

SARS-CoV-2: Targeted managements and vaccine development.

Infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) results in diverse outcomes. The symptoms appear to be more severe in males older than 65 and people with underlying health conditions; approximately one in five individuals could be at risk worldwide. The virus's sequence was rapidly established days after the first cases were reported and identified an RNA virus from the Coronaviridae family closely related to a Betacoronavirus virus found in bats in China. SARS-CoV-2 is the seventh coronavirus known to infect humans, and with the severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome (MERS), the only ones to cause severe diseases. Lessons from these two previous outbreaks guided the identification of critical therapeutic targets such as the spike viral proteins promoting the virus's cellular entry through the angiotensin-converting enzyme 2 (ACE2) receptor expressed on the surface of multiple types of eukaryotic cells. Although several therapeutic agents are currently evaluated, none seems to provide a clear path for a cure. Also, various types of vaccines are developed in record time to address the urgency of efficient SARS-CoV-2 prevention. Currently, 58 vaccines are evaluated in clinical trials, including 11 in phase III, and 3 of them reported efficacy above 90 %. The results so far from the clinical trials suggest the availability of multiple effective vaccines within months.

RNA secondary structure at the transcription start site influences EBOV transcription initiation and replication in a length- and stability-dependent manner.

Ebola virus (EBOV) RNA has the potential to form hairpin structures at the transcription start sequence (TSS) and reinitiation sites of internal genes, both on the genomic and antigenomic/mRNA level. Hairpin formation involving the TSS and the spacer sequence between promotor elements (PE) 1 and 2 was suggested to regulate viral transcription. Here, we provide evidence that such RNA structures form during RNA synthesis by the viral polymerase and affect its activity. This was analysed using monocistronic minigenomes carrying hairpin structure variants in the TSS-spacer region that differ in length and stability. Transcription and replication were measured via reporter activity and by qRT-PCR quantification of the distinct viral RNA species. We demonstrate that viral RNA synthesis is remarkably tolerant to spacer extensions of up to ~54 nt, but declines beyond this length limit (~25% residual activity for a 66-nt extension). Minor incremental stabilizations of hairpin structures in the TSS-spacer region and on the mRNA/antigenomic level were found to rapidly abolish viral polymerase activity, which may be exploited for antisense strategies to inhibit viral RNA synthesis. Finally, balanced viral transcription and replication can still occur when any RNA structure formation potential at the TSS is eliminated, provided that hexamer phasing in the promoter region is maintained. Altogether, the findings deepen and refine our insight into structure and length constraints within the EBOV transcription and replication promoter and suggest a remarkable flexibility of the viral polymerase in recognition of PE1 and PE2.

Transmembrane redox regulation of genome replication functions in positive-strand RNA viruses.

Positive-strand RNA virus genome replication takes place on intracellular membranes that separate the reduced cytosol from the oxidized extracellular/luminal milieu. Ongoing studies of these membrane-bounded genome replication complexes have revealed underlying common principles in their structure, assembly and functionalization, including transmembrane features and redox dependencies. Among these, members of the alphavirus, flavivirus, and picornavirus supergroups all encode membrane-permeabilizing viroporins required for efficient RNA replication. For flaviviruses and particularly alphavirus supergroup members, these viroporins are linked to activating viral RNA capping and potentially other later-stage RNA replication functions, and to local transmembrane release of oxidizing potential to trigger these changes in cytoplasmic RNA replication complexes. Further exploration of these emerging shared principles could spur development of broad-spectrum antivirals.

The Short- and Long-Range RNA-RNA Interactome of SARS-CoV-2.

The Coronaviridae is a family of positive-strand RNA viruses that includes SARS-CoV-2, the etiologic agent of the COVID-19 pandemic. Bearing the largest single-stranded RNA genomes in nature, coronaviruses are critically dependent on long-distance RNA-RNA interactions to regulate the viral transcription and replication pathways. Here we experimentally mapped the in vivo RNA-RNA interactome of the full-length SARS-CoV-2 genome and subgenomic mRNAs. We uncovered a network of RNA-RNA interactions spanning tens of thousands of nucleotides. These interactions reveal that the viral genome and subgenomes adopt alternative topologies inside cells and engage in different interactions with host RNAs. Notably, we discovered a long-range RNA-RNA interaction, the FSE-arch, that encircles the programmed ribosomal frameshifting element. The FSE-arch is conserved in the related MERS-CoV and is under purifying selection. Our findings illuminate RNA structure-based mechanisms governing replication, discontinuous transcription, and translation of coronaviruses and will aid future efforts to develop antiviral strategies.

Genomic Epidemiology and its importance in the study of the COVID-19 pandemic.

Not available.

The Role of the RNA-RNA Interactome in the Hepatitis C Virus Life Cycle.

RNA virus genomes are multifunctional entities endowed with conserved structural elements that control translation, replication and encapsidation, among other processes. The preservation of these structural RNA elements constraints the genomic sequence variability. The hepatitis C virus (HCV) genome is a positive, single-stranded RNA molecule with numerous conserved structural elements that manage different steps during the infection cycle. Their function is ensured by the association of protein factors, but also by the establishment of complex, active, long-range RNA-RNA interaction networks-the so-called HCV RNA interactome. This review describes the RNA genome functions mediated via RNA-RNA contacts, and revisits some canonical ideas regarding the role of functional high-order structures during the HCV infective cycle. By outlining the roles of long-range RNA-RNA interactions from translation to virion budding, and the functional domains involved, this work provides an overview of the HCV genome as a dynamic device that manages the course of viral infection.

Codon usage pattern and its influencing factors in different genomes of hepadnaviruses.

Codon usage bias (CUB) arises from the preference for a codon over codons for the same amino acid. The major factors contributing to CUB are evolutionary forces, compositional properties, gene expression, and protein properties. The present analysis was performed to investigate the compositional properties and the extent of CUB across the genomes of members of the family Hepadnaviridae, as previously no work using bioinformatic tools has been reported. The viral genes were found to be AT rich with low CUB. Analysis of relative synonymous codon usage (RSCU) was used to identify overrepresented and underrepresented codons for each amino acid. Correlation analysis of overall nucleotide composition and its composition at the third codon position suggested that mutation pressure might influence the CUB. A highly significant correlation was observed between GC12 and GC3 (r = 0.910, p < 0.01), indicating that directional mutation affected all three codon positions across the genome. Translational selection (P2) and mutational responsive index (MRI) values of genes suggested that mutation plays a more important role than translational selection in members of the family Hepadnaviridae.

Short Direct Repeats in the 3' Untranslated Region Are Involved in Subgenomic Flaviviral RNA Production.

Mosquito-borne flaviviruses consist of a positive-sense genome RNA flanked by the untranslated regions (UTRs). There is a panel of highly complex RNA structures in the UTRs with critical functions. For instance, Xrn1-resistant RNAs (xrRNAs) halt Xrn1 digestion, leading to the production of subgenomic flaviviral RNA (sfRNA). Conserved short direct repeats (DRs), also known as conserved sequences (CS) and repeated conserved sequences (RCS), have been identified as being among the RNA elements locating downstream of xrRNAs, but their biological function remains unknown. In this study, we revealed that the specific DRs are involved in the production of specific sfRNAs in both mammalian and mosquito cells. Biochemical assays and structural remodeling demonstrate that the base pairings in the stem of these DRs control sfRNA formation by maintaining the binding affinity of the corresponding xrRNAs to Xrn1. On the basis of these findings, we propose that DRs functions like a bracket holding the Xrn1-xrRNA complex for sfRNA formation.IMPORTANCE Flaviviruses include many important human pathogens. The production of subgenomic flaviviral RNAs (sfRNAs) is important for viral pathogenicity as a common feature of flaviviruses. sfRNAs are formed through the incomplete degradation of viral genomic RNA by the cytoplasmic 5'-3' exoribonuclease Xrn1 halted at the Xrn1-resistant RNA (xrRNA) structures within the 3'-UTR. The 3'-UTRs of the flavivirus genome also contain distinct short direct repeats (DRs), such as RCS3, CS3, RCS2, and CS2. However, the biological functions of these ancient primary DR sequences remain largely unknown. Here, we found that DR sequences are involved in sfRNA formation and viral virulence and provide novel targets for the rational design of live attenuated flavivirus vaccine.

Finally, a Role Befitting Astar: Strongly Conserved, Unessential Microvirus A* Proteins Ensure the Product Fidelity of Packaging Reactions.

In microviruses, 60 copies of the positively charged DNA binding protein J guide the single-stranded DNA genome into the icosahedral capsid. Consequently, ∼12% of the genome is icosahedrally ordered within virions. Although the internal volume of the ϕX174, G4, and α3 capsids are nearly identical, their genome lengths vary widely from 5,386 (ϕX174) to 6,067 (α3) nucleotides. As the genome size increases, the J protein's length and charge decreases. The ϕX174 J protein is 37 amino acids long and has a charge of +12, whereas the 23-residue G4 and α3 proteins have respective +6 and +8 charges. While the large ϕX174 J protein can substitute for the smaller ones, the converse is not true. Thus, the smallest genome, ϕX174, requires the more stringent J protein packaging guide. To investigate this further, a chimeric virus (ϕXG4J) was generated by replacing the indigenous ϕX174 J gene with that of G4. The resulting mutant, ϕXG4J, was not viable on the level of plaque formation without ϕX174 J gene complementation. During uncomplemented infections, capsids dissociated during packaging or quickly thereafter. Those that survived were significantly less stable and infectious than the wild type. Complementation-independent ϕXG4J variants were isolated. They contained duplications that increased genome size by as much as 3.8%. Each duplication started at nucleotide 991, creating an additional DNA substrate for the unessential but highly conserved A* protein. Accordingly, ϕXG4J viability and infectivity was also restored by the exogenous expression of a cloned A* gene.IMPORTANCE Double-stranded DNA viruses typically package their genomes into a preformed capsid. In contrast, single-stranded RNA viruses assemble their coat proteins around their genomes via extensive nucleotide-protein interactions. Single-stranded DNA (ssDNA) viruses appear to blend both strategies, using nucleotide-protein interactions to organize their genomes into preformed shells, likely by a controlled process. Chaotic genome-capsid associations could inhibit packaging or genome release during the subsequent infection. This process appears to be partially controlled by the unessential A* protein, a shorter version of the essential A protein that mediates rolling-circle DNA replication. Protein A* may elevate fitness by ensuring the product fidelity of packaging reactions. This phenomenon may be widespread in ssDNA viruses that simultaneously synthesize and package DNA with rolling circle and rolling circle-like DNA replication proteins. Many of these viruses encode smaller, unessential, and/or functionally undefined in-frame versions of A/A*-like proteins.