Nerea Irigoyen

Autoproteolytic Activity Derived from the Infectious Bursal Disease Virus Capsid Protein

Viral capsids are envisioned as vehicles to deliver the viral genome to the host cell. They are nonetheless dynamic protective shells, as they participate in numerous processes of the virus cycle such as assembly, genome packaging, binding to receptors, and uncoating among others. In so doing, they undergo large scale conformational changes. Capsid proteins with essential enzymatic activities are being described more frequently. Here we show that the precursor (pVP2) of the capsid protein VP2 of the infectious bursal disease virus (IBDV), an avian double-stranded RNA virus, has autoproteolytic activity. The pVP2 C-terminal region is first processed by the viral protease VP4. VP2 Asp-431, lying in a flexible loop preceding the C-terminal most α-helix, is responsible for the endopeptidase activity that cleaves the Ala-441—Phe-442 bond to generate the mature VP2 polypeptide. The D431N substitution abrogates the endopeptidase activity without introducing a significant conformational change, as deduced from the three-dimensional structure of the mutant protein at 3.1 Å resolution. Combinations of VP2 polypeptides containing mutations affecting either the cleavage or the catalytic site revealed that pVP2 proteolytic processing is the result of a monomolecular cis-cleavage reaction. The D431N mutation does not affect the assembly of the VP2 trimers that constitute the capsid building block. Although VP2 D431N trimers are capable of assembling both pentamers and hexamers, expression of a polyprotein gene harboring the D431N mutation does not result in the assembly of IBDV virus-like particles. Reverse genetics analyses demonstrate that pVP2 self-processing is essential for the assembly of an infectious IBDV progeny.

The use of duplex-specific nuclease in ribosome profiling and a user-friendly software package for Ribo-seq data analysis

Ribosome profiling is a technique that permits genome-wide, quantitative analysis of translation and has found broad application in recent years. Here we describe a modified profiling protocol and software package designed to benefit more broadly the translation community in terms of simplicity and utility. The protocol, applicable to diverse organisms, including organelles, is based largely on previously published profiling methodologies, but uses duplex-specific nuclease (DSN) as a convenient, species-independent way to reduce rRNA contamination. We show that DSN-based depletion compares favorably with other commonly used rRNA depletion strategies and introduces little bias. The profiling protocol typically produces high levels of triplet periodicity, facilitating the detection of coding sequences, including upstream, downstream, and overlapping open reading frames (ORFs) and an alternative ribosome conformation evident during termination of protein synthesis. In addition, we provide a software package that presents a set of methods for parsing ribosomal profiling data from multiple samples, aligning reads to coding sequences, inferring alternative ORFs, and plotting average and transcript-specific aspects of the data. Methods are also provided for extracting the data in a form suitable for differential analysis of translation and translational efficiency.

High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling

Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global “snap-shot” of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal frameshift site. To our knowledge this is the first application of ribosome profiling to an RNA virus.

Mechanical Stability and Reversible Fracture of Vault Particles

Vaults are the largest ribonucleoprotein particles found in eukaryotic cells, with an unclear cellular function and promising applications as vehicles for drug delivery. In this article, we examine the local stiffness of individual vaults and probe their structural stability with atomic force microscopy under physiological conditions. Our data show that the barrel, the central part of the vault, governs both the stiffness and mechanical strength of these particles. In addition, we induce single-protein fractures in the barrel shell and monitor their temporal evolution. Our high-resolution atomic force microscopy topographies show that these fractures occur along the contacts between two major vault proteins and disappear over time. This unprecedented systematic self-healing mechanism, which enables these particles to reversibly adapt to certain geometric constraints, might help vaults safely pass through the nuclear pore complex and potentiate their role as self-reparable nanocontainers.

Electrostatic Interactions between Capsid and Scaffolding Proteins Mediate the Structural Polymorphism of a Double-stranded RNA Virus

Capsid proteins that adopt distinct conformations constitute a paradigm of the structural polymorphism of macromolecular assemblies. We show the molecular basis of the flexibility mechanism of VP2, the capsid protein of the double-stranded RNA virus infectious bursal disease virus. The initial assembly, a procapsid-like structure, is built by the protein precursor pVP2 and requires VP3, the other infectious bursal disease virus major structural protein, which acts as a scaffold. The pVP2 C-terminal region, which is proteolyzed during virus maturation, contains an amphipathic α-helix that acts as a molecular switch. In the absence of VP3, efficient virus-like particle assembly occurs when the structural unit is a VP2-based chimeric protein with an N-terminal-fused His6 tag. The His tag has a positively charged N terminus and a negatively charged C terminus, both important for virion-like structure assembly. The charge distributions of the VP3 C terminus and His tag are similar. We tested whether the His tag emulates the role of VP3 and found that the presence of a VP3 C-terminal peptide in VP2-based chimeric proteins resulted in the assembly of virus-like particles. We analyzed the electrostatic interactions between these two charged morphogenetic peptides, in which a single residue was mutated to impede the predicted interaction, followed by a compensatory double mutation to rescue electrostatic interactions. The effects of these mutations were monitored by following the virus-like and/or virus-related assemblies. Our results suggest that the basic face of the pVP2 amphipathic α-helix interacts with the acidic region of the VP3 C terminus and that this interaction is essential for VP2 acquisition of competent conformations for capsid assembly.

Host proteolytic activity is necessary for infectious bursal disease virus capsid protein assembly

Background: Virus capsid assembly and maturation are regulated by viral and host molecular factors. Results: The puromycin-sensitive aminopeptidase cleaves the capsid protein precursor of a double-stranded RNA virus (IBDV). Conclusion: Puromycin-sensitive aminopeptidase activity is essential for IBDV replication. Significance: Understanding crucial host factors for virus assembly is important for selecting efficient heterologous expression systems and for determining potential antiviral targets. In many viruses, a precursor particle, or procapsid, is assembled and undergoes massive chemical and physical modification to produce the infectious capsid. Capsid assembly and maturation are finely tuned processes in which viral and host factors participate. We show that the precursor of the VP2 capsid protein (pVP2) of the infectious bursal disease virus (IBDV), a double-stranded RNA virus, is processed at the C-terminal domain (CTD) by a host protease, the puromycin-sensitive aminopeptidase (PurSA). The pVP2 CTD (71 residues) has an important role in determining the various conformations of VP2 (441 residues) that build the T = 13 complex capsid. pVP2 CTD activity is controlled by co- and posttranslational proteolytic modifications of different targets by the VP4 viral protease and by VP2 itself to yield the mature VP2-441 species. Puromycin-sensitive aminopeptidase is responsible for the peptidase activity that cleaves the Arg-452-Arg-453 bond to generate the intermediate pVP2-452 polypeptide. A pVP2 R453A substitution abrogates PurSA activity. We used a baculovirus-based system to express the IBDV polyprotein in insect cells and found inefficient formation of virus-like particles similar to IBDV virions, which correlates with the absence of puromycin-sensitive aminopeptidase in these cells. Virus-like particle assembly was nonetheless rescued efficiently by coexpression of chicken PurSA or pVP2-452 protein. Silencing or pharmacological inhibition of puromycin-sensitive aminopeptidase activity in cell lines permissive for IBDV replication caused a major blockade in assembly and/or maturation of infectious IBDV particles, as virus yields were reduced markedly. PurSA activity is thus essential for IBDV replication.

Modulation of Ribosomal Frameshifting Frequency and Its Effect on the Replication of Rous Sarcoma Virus

ABSTRACT Programmed −1 ribosomal frameshifting is widely used in the expression of RNA virus replicases and represents a potential target for antiviral intervention. There is interest in determining the extent to which frameshifting efficiency can be modulated before virus replication is compromised, and we have addressed this question using the alpharetrovirus Rous sarcoma virus (RSV) as a model system. In RSV, frameshifting is essential in the production of the Gag-Pol polyprotein from the overlapping gag and pol coding sequences. The frameshift signal is composed of two elements, a heptanucleotide slippery sequence and, just downstream, a stimulatory RNA structure that has been proposed to be an RNA pseudoknot. Point mutations were introduced into the frameshift signal of an infectious RSV clone, and virus replication was monitored following transfection and subsequent infection of susceptible cells. The introduced mutations were designed to generate a range of frameshifting efficiencies, yet with minimal impact on encoded amino acids. Our results reveal that point mutations leading to a 3-fold decrease in frameshifting efficiency noticeably reduce virus replication and that further reduction is severely inhibitory. In contrast, a 3-fold stimulation of frameshifting is well tolerated. These observations suggest that small-molecule inhibitors of frameshifting are likely to have potential as agents for antiviral intervention. During the course of this work, we were able to confirm, for the first time in vivo, that the RSV stimulatory RNA is indeed an RNA pseudoknot but that the pseudoknot per se is not absolutely required for virus viability.

Modulation of Stop Codon Read-Through Efficiency and Its Effect on the Replication of Murine Leukemia Virus

ABSTRACT Translational readthrough—suppression of termination at a stop codon—is exploited in the replication cycles of several viruses and represents a potential target for antiviral intervention. In the gammaretroviruses, typified by Moloney murine leukemia virus (MuLV), gag and pol are in the same reading frame, separated by a UAG stop codon, and termination codon readthrough is required for expression of the viral Gag-Pol fusion protein. Here, we investigated the effect on MuLV replication of modulating readthrough efficiency. We began by manipulating the readthrough signal in the context of an infectious viral clone to generate a series of MuLV variants in which readthrough was stimulated or reduced. In carefully controlled infectivity assays, it was found that reducing the MuLV readthrough efficiency only 4-fold led to a marked defect and that a 10-fold reduction essentially abolished replication. However, up to an ∼8.5-fold stimulation of readthrough (up to 60% readthrough) was well tolerated by the virus. These high levels of readthrough were achieved using a two-plasmid system, with Gag and Gag-Pol expressed from separate infectious clones. We also modulated readthrough by silencing expression of eukaryotic release factors 1 and 3 (eRF1 and eRF3) or by introducing aminoglycosides into the cells. The data obtained indicate that gammaretroviruses tolerate a substantial excess of viral Gag-Pol synthesis but are very sensitive to a reduction in levels of this polyprotein. Thus, as is also the case for ribosomal frameshifting, antiviral therapies targeting readthrough with inhibitory agents are likely to be the most beneficial. IMPORTANCE Many pathogenic RNA viruses and retroviruses use ribosomal frameshifting or stop codon readthrough to regulate expression of their replicase enzymes. These translational “recoding” processes are potential targets for antiviral intervention, but we have only a limited understanding of the consequences to virus replication of modulating the efficiency of recoding, particularly for those viruses employing readthrough. In this paper, we describe the first systematic analysis of the effect of increasing or decreasing readthrough efficiency on virus replication using the gammaretrovirus MuLV as a model system. We find unexpectedly that MuLV replication is only slightly inhibited by substantial increases in readthrough frequency, but as with other viruses that use recoding strategies, replication is quite sensitive to even modest reductions. These studies provide insights into both the readthrough process and MuLV replication and have implications for the selection of antivirals against gammaretroviruses.

Activation of the unfolded protein response and inhibition of translation initiation during coronavirus infection

Coronaviruses (CoV), such as severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), are of medical importance with high mortality rates and significant zoonotic and pandemic potential. Here, we apply ribosome profiling and parallel RNASeq to globally analyse changes in the host cell translatome and transcriptome upon infection with mouse hepatitis virus, strain A59 (MHV-A59), a model murine coronavirus in the same genus as SARS-CoV and MERS-CoV. We observed translational upregulation of ATF4, ATF5 and Ddit3 and activation of the unfolded protein response (UPR). Phosphorylation of eIF2α led to the global inhibition of translation and a substantial increase in empty 80S ribosomes. A drug that inhibits the UPR attenuates virus growth suggesting that MHV may have evolved to subvert the UPR to its own advantage. We also investigated an artefact of cycloheximide pretreatment in ribosome profiling whereby ribosomes accumulate at the 5’ end of coding sequences in stressed cells but not in unstressed or untreated cells, thus extending earlier studies in yeast to mammalian cells. The study sheds light on the mechanisms of CoV translational shutoff and reveals a potential new therapeutic strategy.Coronaviruses (CoVs) are enveloped, positive-sense RNA viruses with an unusually large RNA genome and a unique replication strategy. They cause important diseases in mammals and birds ranging from enteritis in cows and pigs and upper respiratory disease in chickens, to potentially lethal human respiratory infections. Here, we apply ribosome profiling and parallel RNA sequencing to analyse global changes in host cell transcriptome and translatome upon infection with mouse hepatitis virus strain A59 (MHV-A59), a model murine coronavirus in the same genus as the human pathogens severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). Amongst differentially-regulated cellular genes, we observed up-regulation of all arms of the unfolded protein response (UPR), including translational activation of transcription factors ATF4, ATF5 and Chop. Polysome profiling of infected-cells revealed an accumulation of empty 80S ribosomes, consistent with increased phosphorylation of eIF2α leading to translational shut-off via inhibited initiation. Ribosomal footprints on phosphorylated-eIF2α-resistant mRNAs revealed unambiguous upstream open reading frame (uORF) occupancy consistent with host maintenance of the UPR. Unexpectedly, an inhibitor of PERK that blocks the UPR and relieves translation inhibition was found to attenuate virus growth suggesting that MHV may subvert the UPR to its own advantage. This study sheds new light on the complex interactions between MHV and host during infection and provides new potential targets for antiviral intervention.

The translational landscape of Zika virus during infection of mammalian and insect cells

Zika virus is a single-stranded, positive-sense RNA virus of the family Flaviviridae, which has recently undergone a rapid expansion among humans in the Western Hemisphere. Here, we report a high-resolution map of ribosomal occupancy of the Zika virus genome during infection of mammalian and insect cells, obtained by ribosome profiling. In contrast to some other flaviviruses such as West Nile, we find no evidence for substantial frameshift-induced ribosomal drop-off during translation of the viral polyprotein, indicating that Zika virus must use alternative mechanisms to downregulate levels of catalytically active viral polymerase. We also show that high levels of ribosome-protected fragments map in-frame to two previously overlooked upstream open reading frames (uORFs) initiating at CUG and UUG codons, with likely consequences for the efficiency of polyprotein expression. Curiously, in African isolates of Zika virus, the two uORFs are fused in-frame into a single uORF. A parallel RNA-Seq analysis reveals the 5′ end position of the subgenomic flavivirus RNA in mammalian and insect cells. Together, these provide the first analysis of flavivirus gene expression by ribosome profiling. Author Summary Recent Zika virus outbreaks have been associated with congenital diseases and neurological complications. An enhanced understanding of the molecular biology of this pathogen may contribute towards the development of improved treatment and control methods. We present a single-codon resolution analysis of Zika virus translation in mammalian and mosquito cells using ribosome profiling. The analysis revealed two hitherto uncharacterized uORFs in the 5′ leader of Zika virus Brazilian isolate PE243, both of which are occupied by ribosomes during infection. In contrast, these two uORFs are fused into a single uORF in African isolates. This observation provides a new avenue for further investigations into potential factors involved in the emergence of Zika virus from a rarely detected pathogen into a major epidemic.