Betty Y.-W. Chung

An overlapping essential gene in the Potyviridae

The family Potyviridae includes >30% of known plant virus species, many of which are of great agricultural significance. These viruses have a positive sense RNA genome that is ≈10 kb long and contains a single long ORF. The ORF is translated into a large polyprotein, which is cleaved into ≈10 mature proteins. We report the discovery of a short ORF embedded within the P3 cistron of the polyprotein but translated in the +2 reading-frame. The ORF, termed pipo, is conserved and has a strong bioinformatic coding signature throughout the large and diverse Potyviridae family. Mutations that knock out expression of the PIPO protein in Turnip mosaic potyvirus but leave the polyprotein amino acid sequence unaltered are lethal to the virus. Immunoblotting with antisera raised against two nonoverlapping 14-aa antigens, derived from the PIPO amino acid sequence, reveals the expression of an ≈25-kDa PIPO fusion product in planta. This is consistent with expression of PIPO as a P3-PIPO fusion product via ribosomal frameshifting or transcriptional slippage at a highly conserved G1-2A6-7 motif at the 5′ end of pipo. This discovery suggests that other short overlapping genes may remain hidden even in well studied virus genomes (as well as cellular organisms) and demonstrates the utility of the software package MLOGD as a tool for identifying such genes.

Novel virus discovery and genome reconstruction from field RNA samples reveals highly divergent viruses in dipteran hosts.

We investigated whether small RNA (sRNA) sequenced from field-collected mosquitoes and chironomids (Diptera) can be used as a proxy signature of viral prevalence within a range of species and viral groups, using sRNAs sequenced from wild-caught specimens, to inform total RNA deep sequencing of samples of particular interest. Using this strategy, we sequenced from adult Anopheles maculipennis s.l. mosquitoes the apparently nearly complete genome of one previously undescribed virus related to chronic bee paralysis virus, and, from a pool of Ochlerotatus caspius and Oc. detritus mosquitoes, a nearly complete entomobirnavirus genome. We also reconstructed long sequences (1503-6557 nt) related to at least nine other viruses. Crucially, several of the sequences detected were reconstructed from host organisms highly divergent from those in which related viruses have been previously isolated or discovered. It is clear that viral transmission and maintenance cycles in nature are likely to be significantly more complex and taxonomically diverse than previously expected.

Transcriptional slippage in the positive-sense RNA virus family Potyviridae

The family Potyviridae encompasses ~30% of plant viruses and is responsible for significant economic losses worldwide. Recently, a small overlapping coding sequence, termed pipo, was found to be conserved in the genomes of all potyvirids. PIPO is expressed as part of a frameshift protein, P3N‐PIPO, which is essential for virus cell‐to‐cell movement. However, the frameshift expression mechanism has hitherto remained unknown. Here, we demonstrate that transcriptional slippage, specific to the viral RNA polymerase, results in a population of transcripts with an additional “A” inserted within a highly conserved GAAAAAA sequence, thus enabling expression of P3N‐PIPO. The slippage efficiency is ~2% in Turnip mosaic virus and slippage is inhibited by mutations in the GAAAAAA sequence. While utilization of transcriptional slippage is well known in negative‐sense RNA viruses such as Ebola, mumps and measles, to our knowledge this is the first report of its widespread utilization for gene expression in positive‐sense RNA viruses.

Discovery of a Small Non-AUG-Initiated ORF in Poleroviruses and Luteoviruses That Is Required for Long-Distance Movement.

Viruses in the family Luteoviridae have positive-sense RNA genomes of around 5.2 to 6.3 kb, and they are limited to the phloem in infected plants. The Luteovirus and Polerovirus genera include all but one virus in the Luteoviridae. They share a common gene block, which encodes the coat protein (ORF3), a movement protein (ORF4), and a carboxy-terminal extension to the coat protein (ORF5). These three proteins all have been reported to participate in the phloem-specific movement of the virus in plants. All three are translated from one subgenomic RNA, sgRNA1. Here, we report the discovery of a novel short ORF, termed ORF3a, encoded near the 5’ end of sgRNA1. Initially, this ORF was predicted by statistical analysis of sequence variation in large sets of aligned viral sequences. ORF3a is positioned upstream of ORF3 and its translation initiates at a non-AUG codon. Functional analysis of the ORF3a protein, P3a, was conducted with Turnip yellows virus (TuYV), a polerovirus, for which translation of ORF3a begins at an ACG codon. ORF3a was translated from a transcript corresponding to sgRNA1 in vitro, and immunodetection assays confirmed expression of P3a in infected protoplasts and in agroinoculated plants. Mutations that prevent expression of P3a, or which overexpress P3a, did not affect TuYV replication in protoplasts or inoculated Arabidopsis thaliana leaves, but prevented virus systemic infection (long-distance movement) in plants. Expression of P3a from a separate viral or plasmid vector complemented movement of a TuYV mutant lacking ORF3a. Subcellular localization studies with fluorescent protein fusions revealed that P3a is targeted to the Golgi apparatus and plasmodesmata, supporting an essential role for P3a in viral movement.

Powerful sequence similarity search methods and in-depth manual analyses can identify remote homologs in many apparently "orphan" viral proteins.

ABSTRACT The genome sequences of new viruses often contain many “orphan” or “taxon-specific” proteins apparently lacking homologs. However, because viral proteins evolve very fast, commonly used sequence similarity detection methods such as BLAST may overlook homologs. We analyzed a data set of proteins from RNA viruses characterized as “genus specific” by BLAST. More powerful methods developed recently, such as HHblits or HHpred (available through web-based, user-friendly interfaces), could detect distant homologs of a quarter of these proteins, suggesting that these methods should be used to annotate viral genomes. In-depth manual analyses of a subset of the remaining sequences, guided by contextual information such as taxonomy, gene order, or domain cooccurrence, identified distant homologs of another third. Thus, a combination of powerful automated methods and manual analyses can uncover distant homologs of many proteins thought to be orphans. We expect these methodological results to be also applicable to cellular organisms, since they generally evolve much more slowly than RNA viruses. As an application, we reanalyzed the genome of a bee pathogen, Chronic bee paralysis virus (CBPV). We could identify homologs of most of its proteins thought to be orphans; in each case, identifying homologs provided functional clues. We discovered that CBPV encodes a domain homologous to the Alphavirus methyltransferase-guanylyltransferase; a putative membrane protein, SP24, with homologs in unrelated insect viruses and insect-transmitted plant viruses having different morphologies (cileviruses, higreviruses, blunerviruses, negeviruses); and a putative virion glycoprotein, ORF2, also found in negeviruses. SP24 and ORF2 are probably major structural components of the virions.

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.

Most microRNAs in the single-cell alga Chlamydomonas reinhardtii are produced by Dicer-like 3-mediated cleavage of introns and untranslated regions of coding RNAs

We describe here a forward genetic screen to investigate the biogenesis, mode of action, and biological function of miRNA-mediated RNA silencing in the model algal species,Chlamydomonas reinhardtii Among the mutants from this screen, there were three at Dicer-like 3 that failed to produce both miRNAs and siRNAs and others affecting diverse post-biogenesis stages of miRNA-mediated silencing. The DCL3-dependent siRNAs fell into several classes including transposon- and repeat-derived siRNAs as in higher plants. The DCL3-dependent miRNAs differ from those of higher plants, however, in that many of them are derived from mRNAs or from the introns of pre-mRNAs. Transcriptome analysis of the wild-type and dcl3 mutant strains revealed a further difference from higher plants in that the sRNAs are rarely negative switches of mRNA accumulation. The few transcripts that were more abundant in dcl3 mutant strains than in wild-type cells were not due to sRNA-targeted RNA degradation but to direct DCL3 cleavage of miRNA and siRNA precursor structures embedded in the untranslated (and translated) regions of the mRNAs. Our analysis reveals that the miRNA-mediated RNA silencing in C. reinhardtii differs from that of higher plants and informs about the evolution and function of this pathway in eukaryotes.

mRNA turnover through CDS-targeting is the primary role of miRNA in the green alga Chlamydomonas.

MicroRNAs regulate gene expression as part of the RNA-induced silencing complex, where the sequence identity of the miRNA provides the specificity to the target messenger RNA, and the result is target repression. The mode of repression can be through target cleavage, RNA destabilization and/or decreased translational efficiency. Here, we provide a comprehensive global analysis of the evolutionarily distant unicellular green alga Chlamydomonas reinhardtii to quantify the effects of miRNA on protein synthesis and RNA abundance. We show that, similar to metazoan systems, miRNAs in Chlamydomonas regulate gene-expression primarily by destabilizing mRNAs. However, unlike metazoan miRNA where target site utilization localizes mainly to 3’UTRs, in Chlamydomonas utilized target sites lie predominantly within coding regions. These results demonstrate that destabilization of mRNA is the main evolutionarily conserved mode of action for miRNAs, but details of the mechanism diverge between plant and metazoan kingdoms.

Endogenous miRNA in the green alga Chlamydomonas regulates gene expression through CDS-targeting

MicroRNAs (miRNAs) are 21–24-nucleotide RNAs present in many eukaryotes that regulate gene expression as part of the RNA-induced silencing complex. The sequence identity of the miRNA provides the specificity to guide the silencing effector Argonaute (AGO) protein to target mRNAs via a base-pairing process1. The AGO complex promotes translation repression and/or accelerated decay of this target mRNA2. There is overwhelming evidence both in vivo and in vitro that translation repression plays a major role3–7. However, there has been controversy about which of these three mechanisms is more significant in vivo, especially when effects of miRNA on endogenous genes cannot be faithfully represented by reporter systems in which, at least in metazoans, the observed repression vastly exceeds that typically observed for endogenous mRNAs8,9. Here, we provide a comprehensive global analysis of the evolutionarily distant unicellular green alga Chlamydomonas reinhardtii to quantify the effects of miRNA on protein synthesis and RNA abundance. We show that, similar to metazoan steady-state systems, endogenous miRNAs in Chlamydomonas can regulate gene expression both by destabilization of the mRNA and by translational repression. However, unlike metazoan miRNA where target site utilization localizes mainly to 3′ UTRs, in Chlamydomonas utilized target sites lie predominantly within coding regions. These results demonstrate the evolutionarily conserved mode of action for miRNAs, but details of the mechanism diverge between the plant and metazoan kingdoms.MicroRNAs regulate gene expression as part of the RNA-induced silencing complex. An analysis of miRNA in Chlamydomonas reinhardtiishows that algal miRNAs target sites predominantly within coding regions to destabilize mRNA and repress translation.