Discontinuous RNA Research Articles

Experimental and analytical pipeline for sub-genomic RNA landscape of coronavirus by Nanopore sequencer.

Coronaviruses (CoVs), including severe acute respiratory syndrome coronavirus 2, can infect a variety of mammalian and avian hosts with significant medical and economic consequences. During the life cycle of CoV, a coordinated series of subgenomic RNAs, including canonical subgenomic messenger RNA and non-canonical defective viral genomes (DVGs), are generated with different biological implications. Studies that adopted the Nanopore sequencer (ONT) to investigate the landscape and dynamics of viral RNA subgenomic transcriptomes applied arbitrary bioinformatics parameters without justification or experimental validation. The current study used bovine coronavirus (BCoV), which can be performed under biosafety level 2 for library construction and experimental validation using traditional colony polymerase chain reaction and Sanger sequencing. Four different ONT protocols, including RNA direct and cDNA direct sequencing with or without exonuclease treatment, were used to generate RNA transcriptomic libraries from BCoV-infected cell lysates. Through rigorously examining the k-mer, gap size, segment size, and bin size, the optimal cutoffs for the bioinformatic pipeline were determined to remove the sequence noise while keeping the informative DVG reads. The sensitivity and specificity of identifying DVG reads using the proposed pipeline can reach 82.6% and 99.6% under the k-mer size cutoff of 15. Exonuclease treatment reduced the abundance of RNA transcripts; however, it was not necessary for future library preparation. Additional recovery of clipped BCoV nucleotide sequences with experimental validation expands the landscape of the CoV discontinuous RNA transcriptome, whose biological function requires future investigation. The results of this study provide the benchmarks for library construction and bioinformatic parameters for studying the discontinuous CoV RNA transcriptome.IMPORTANCEFunctional defective viral genomic RNA, containing all the cis-acting elements required for translation or replication, may play different roles in triggering cell innate immune signaling, interfering with the canonical subgenomic messenger RNA transcription/translation or assisting in establishing persistence infection. This study does not only provide benchmarks for library construction and bioinformatic parameters for studying the discontinuous coronavirus RNA transcriptome but also reveals the complexity of the bovine coronavirus transcriptome, whose functional assays will be critical in future studies.

Expression dynamics of the aplysia abyssovirus

Two recent studies documented the genome of a novel, extremely large (35.9 kb), nidovirus in RNA sequence databases from the marine neural model Aplysia californica. The goal of the present study was to document the distribution and transcriptional dynamics of this virus, Aplysia abyssovirus 1 (AAbV), in maricultured and wild animals. We confirmed previous findings that AAbV RNA is widespread and reaches extraordinary levels in apparently healthy animals. Transmission electron microscopy identified viral replication factories in ciliated gill epithelial cells but not in neurons where viral RNA is most highly expressed. Viral transcripts do not exhibit evidence of discontinuous RNA synthesis as in coronaviruses but are consistent with production of a single leaderless subgenomic RNA, as in the Gill-associated virus of Penaeus monodon. Splicing patterns in chronically infected adults suggested high levels of defective genomes, possibly explaining the lack of obvious disease signs in high viral load animals.

SARS-CoV-2, SARS-CoV, and MERS-CoV encode circular RNAs of spliceosome-independent origin.

Circular RNAs (circRNAs) are a newly recognized component of the transcriptome with critical roles in autoimmune diseases and viral pathogenesis. To address the importance of circRNA in RNA viral transcriptome, we systematically identified and characterized circRNAs encoded by the RNA genomes of betacoronaviruses using both bioinformatical and experimental approaches. We predicted 351, 224, and 2764 circRNAs derived from severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), SARS‐CoV, and Middle East respiratory syndrome coronavirus, respectively. We experimentally identified 75 potential SARS‐CoV‐2 circRNAs from RNA samples extracted from SARS‐CoV‐2‐infected Vero E6 cells. A systematic comparison of viral and host circRNA features, including abundance, strand preference, length distribution, circular exon numbers, and breakpoint sequences, demonstrated that coronavirus‐derived circRNAs had a spliceosome‐independent origin. We further showed that back‐splice junctions (BSJs) captured by inverse reverse‐transcription polymerase chain reaction have different level of resistance to RNase R. Through northern blotting with a BSJ‐spanning probe targeting N gene, we identified three RNase R‐resistant bands that represent SARS‐CoV‐2 circRNAs that are detected cytoplasmic by single‐molecule and amplified fluorescence in situ hybridization assays. Lastly, analyses of 169 sequenced BSJs showed that both back‐splice and forward‐splice junctions were flanked by homologous and reverse complementary sequences, including but not limited to the canonical transcriptional regulatory sequences. Our findings highlight circRNAs as an important component of the coronavirus transcriptome, offer important evaluation of bioinformatic tools in the analysis of circRNAs from an RNA genome, and shed light on the mechanism of discontinuous RNA synthesis.

Insights into the specificity for the interaction of the promiscuous SARS-CoV-2 nucleocapsid protein N-terminal domain with deoxyribonucleic acids

The SARS-CoV-2 nucleocapsid protein (N) is a multifunctional promiscuous nucleic acid-binding protein, which plays a major role in nucleocapsid assembly and discontinuous RNA transcription, facilitating the template switch of transcriptional regulatory sequences (TRS). Here, we dissect the structural features of the N protein N-terminal domain (N-NTD) and N-NTD plus the SR-rich motif (N-NTD-SR) upon binding to single and double-stranded TRS DNA, as well as their activities for dsTRS melting and TRS-induced liquid-liquid phase separation (LLPS). Our study gives insights on the specificity for N-NTD(-SR) interaction with TRS. We observed an approximation of the triple-thymidine (TTT) motif of the TRS to β-sheet II, giving rise to an orientation difference of ~25° between dsTRS and non-specific sequence (dsNS). It led to a local unfavorable energetic contribution that might trigger the melting activity. The thermodynamic parameters of binding of ssTRSs and dsTRS suggested that the duplex dissociation of the dsTRS in the binding cleft is entropically favorable. We showed a preference for TRS in the formation of liquid condensates when compared to NS. Moreover, our results on DNA binding may serve as a starting point for the design of inhibitors, including aptamers, against N, a possible therapeutic target essential for the virus infectivity.

Duplex formation between the template and the nascent strand in the transcription-regulating sequences is associated with the site of template switching in SARS – CoV-2

ABSTRACT Recently published transcriptomic data of the SARS-CoV-2 coronavirus show that there is a large variation in the frequency and steady state levels of subgenomic mRNA sequences. This variation is derived from discontinuous subgenomic RNA synthesis, where the polymerase switches template from a 3ʹ proximal genome body sequence to a 5ʹ untranslated leader sequence. This leads to a fusion between the common 5ʹ leader sequence and a 3ʹ proximal body sequence in the RNA product. This process revolves around a common core sequence (CS) that is present at both the template sites that make up the fusion junction. Base-pairing between the leader CS and the nascent complementary minus strand body CS, and flanking regions (together called the transcription regulating sequence, TRS) is vital for this template switching event. However, various factors can influence the site of template switching within the same TRS duplex. Here, we model the duplexes formed between the leader and complementary body TRS regions, hypothesizing the role of the stability of the TRS duplex in determining the major sites of template switching for the most abundant mRNAs. We indicate that the stability of secondary structures and the speed of transcription play key roles in determining the probability of template switching in the production of subgenomic RNAs. We speculate on the effect of reported variant nucleotide substitutions on our models.

Les enzymes de la réplication/transcription chez les coronavirus.

The discovery of a new coronavirus (CoV) as the causative agent of the severe acute respiratory syndrome (SARS) pandemic outbreak in 2003 has stimulated a number of studies on the molecular biology of SARS-CoV and related viruses. This research has provided significant new insight into functions and activities of the CoV replication-transcription complex, a multi-protein complex that directs coordinated processes of both continuous and discontinuous RNA synthesis to replicate and transcribe the large CoV genome, a single-stranded, positive-sense RNA of ∼30kilobases. In this review, we summarize current understanding of the expression and functions of key replicative enzymes, such as RNA polymerases, ribonucleases, methyltransferases and other replicase gene encoded proteins involved in genome expression, virus-host interactions and other processes. Collectively, these recent studies reveal fascinating details of a huge enzymatic machinery unique in the RNA virus world.

Transcriptional and Translational Landscape of Equine Torovirus.

The genus Torovirus (subfamily Torovirinae, family Coronaviridae, order Nidovirales) encompasses a range of species that infect domestic ungulates, including cattle, sheep, goats, pigs, and horses, causing an acute self-limiting gastroenteritis. Using the prototype species equine torovirus (EToV), we performed parallel RNA sequencing (RNA-seq) and ribosome profiling (Ribo-seq) to analyze the relative expression levels of the known torovirus proteins and transcripts, chimeric sequences produced via discontinuous RNA synthesis (a characteristic of the nidovirus replication cycle), and changes in host transcription and translation as a result of EToV infection. RNA sequencing confirmed that EToV utilizes a unique combination of discontinuous and nondiscontinuous RNA synthesis to produce its subgenomic RNAs (sgRNAs); indeed, we identified transcripts arising from both mechanisms that would result in sgRNAs encoding the nucleocapsid. Our ribosome profiling analysis revealed that ribosomes efficiently translate two novel CUG-initiated open reading frames (ORFs), located within the so-called 5' untranslated region. We have termed the resulting proteins U1 and U2. Comparative genomic analysis confirmed that these ORFs are conserved across all available torovirus sequences, and the inferred amino acid sequences are subject to purifying selection, indicating that U1 and U2 are functionally relevant. This study provides the first high-resolution analysis of transcription and translation in this neglected group of livestock pathogens.IMPORTANCE Toroviruses infect cattle, goats, pigs, and horses worldwide and can cause gastrointestinal disease. There is no treatment or vaccine, and their ability to spill over into humans has not been assessed. These viruses are related to important human pathogens, including severe acute respiratory syndrome (SARS) coronavirus, and they share some common features; however, the mechanism that they use to produce sgRNA molecules differs. Here, we performed deep sequencing to determine how equine torovirus produces sgRNAs. In doing so, we also identified two previously unknown open reading frames "hidden" within the genome. Together these results highlight the similarities and differences between this domestic animal virus and related pathogens of humans and livestock.

New insights about the regulation of Nidovirus subgenomic mRNA synthesis

The members of the Order Nidovirales share a similar genome organization with two overlapping nonstructural polyproteins encoded in the 5′ two-thirds and the structural proteins encoded in the 3′ third. They also express their 3′ region proteins from a nested set of 3′ co-terminal subgenomic messenger RNAs (sg mRNAs). Some but not all of the Nidovirus sg mRNAs also have a common 5′ leader sequence that is acquired by a discontinuous RNA synthesis mechanism regulated by multiple 3′ body transcription regulating sequences (TRSs) and the 5′ leader TRS. Initial studies detected a single major body TRS for each 3′ sg mRNA with a few alternative functional TRSs reported. The recent application of advanced techniques, such as next generation sequencing and ribosomal profiling, in studies of arteriviruses and coronaviruses has revealed an expanded sg mRNA transcriptome and coding capacity.

Continuous and Discontinuous RNA Synthesis in Coronaviruses.

Replication of the coronavirus genome requires continuous RNA synthesis, whereas transcription is a discontinuous process unique among RNA viruses. Transcription includes a template switch during the synthesis of subgenomic negative-strand RNAs to add a copy of the leader sequence. Coronavirus transcription is regulated by multiple factors, including the extent of base-pairing between transcription-regulating sequences of positive and negative polarity, viral and cell protein-RNA binding, and high-order RNA-RNA interactions. Coronavirus RNA synthesis is performed by a replication-transcription complex that includes viral and cell proteins that recognize cis-acting RNA elements mainly located in the highly structured 5' and 3' untranslated regions. In addition to many viral nonstructural proteins, the presence of cell nuclear proteins and the viral nucleocapsid protein increases virus amplification efficacy. Coronavirus RNA synthesis is connected with the formation of double-membrane vesicles and convoluted membranes. Coronaviruses encode proofreading machinery, unique in the RNA virus world, to ensure the maintenance of their large genome size.

Crystallization and preliminary X-ray crystallographic analysis of a nonstructural protein 15 mutant from Human coronavirus 229E.

Nonstructural protein 15 (nsp15), also called endoribonuclease, is a gene product of open reading frame 1b (ORF 1b) in coronaviruses. It is an important enzyme in the transcription/replication process involved in discontinuous negative-strand RNA synthesis. In this work, mutants of nsp15 from Human coronavirus 229E (HCoV-229E) were made based on structural analysis of the homologous nsp15s in Severe acute respiratory syndrome coronavirus (SARS-CoV) and Mouse hepatitis virus (MHV). The I26A/N52A mutant of nsp15 was overexpressed, purified and crystallized, and this mutant led to a trimeric form rather than hexamers or monomers. Crystals of trimeric nsp15 were obtained by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant and diffracted to 2.5 Å resolution. The crystals belonged to space group C2221, with unit-cell parameters a = 85.9, b = 137.5, c = 423.1 Å, α = β = γ = 90°.

Enhancers compete with a long non-coding RNA for regulation of the Kcnq1 domain.

The imprinted Kcnq1 domain contains a differentially methylated region (KvDMR) in intron 11 of Kcnq1. The Kcnq1ot1 non-coding RNA emerges from the unmethylated paternal KvDMR in antisense direction, resulting in cis-repression of neighboring genes. The KvDMR encompasses the Kcnq1ot1 promoter, CTCF sites and other DNA elements, whose individual contribution to regulation of the endogenous domain is unknown. We find that paternal inheritance of a deletion of the minimal Kcnq1ot1 promoter derepresses the upstream Cdkn1c gene. Surprisingly, Kcnq1ot1 transcripts continue to emerge from alternative sites, evidence that silencing depends, not on the ncRNA, but on the promoter sequence. Detailed analyses of Kcnq1ot during cardiogenesis show substantial chromatin reorganization coinciding with discontinuous RNA production in both wild-type and mutant mice, with loss of imprinting. We show that CTCF binds to both methylated and unmethylated alleles of the KvDMR. Furthermore, we report a multitude of enhancers within the Kcnq1ot1 region, and present conformational dynamics of a novel heart enhancer engaged in Kcnq1 expression. Our results have important implications on tissue-specific imprinting patterns and how transcriptional mechanisms compete to maximize the expression of vital genes, in addition to shifting our perception on the role of the long ncRNA in regulating this imprinted domain.

Insights into RNA synthesis, capping, and proofreading mechanisms of SARS-coronavirus

The successive emergence of highly pathogenic coronaviruses (CoVs) such as the Severe Acute Respiratory Syndrome (SARS-CoV) in 2003 and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in 2012 has stimulated a number of studies on the molecular biology. This research has provided significant new insight into functions and activities of the replication/transcription multi-protein complex. The latter directs both continuous and discontinuous RNA synthesis to replicate and transcribe the large coronavirus genome made of a single-stranded, positive-sense RNA of ∼30kb.In this review, we summarize our current understanding of SARS-CoV enzymes involved in RNA biochemistry, such as the in vitro characterization of a highly active and processive RNA polymerase complex which can associate with methyltransferase and 3′–5′ exoribonuclease activities involved in RNA capping, and RNA proofreading, respectively. The recent discoveries reveal fascinating RNA-synthesizing machinery, highlighting the unique position of coronaviruses in the RNA virus world.

Long-distance RNA-RNA interactions in the coronavirus genome form high-order structures promoting discontinuous RNA synthesis during transcription.

Coronavirus (CoV) transcription requires a high-frequency recombination process that links newly synthesized minus-strand subgenomic RNA copies to the leader region, which is present only once, at the 5' end of the genome. This discontinuous RNA synthesis step is based on the complementarity between the transcription-regulating sequences (TRSs) at the leader region and those preceding each gene in the nascent minus-strand RNA. Furthermore, the template switch requires the physical proximity of RNA genome domains located between 20,000 and 30,000 nucleotides apart. In this report, it is shown that the efficacy of this recombination step is promoted by novel additional long-distance RNA-RNA interactions between RNA motifs located close to the TRSs controlling the expression of each gene and their complementary sequences mapping close to the 5' end of the genome. These interactions would bring together the motifs involved in the recombination process. This finding indicates that the formation of high-order RNA structures in the CoV genome is necessary to control the expression of at least the viral N gene. The requirement of these long-distance interactions for transcription was shown by the engineering of CoV replicons in which the complementarity between the newly identified sequences was disrupted. Furthermore, disruption of complementarity in mutant viruses led to mutations that restored complementarity, wild-type transcription levels, and viral titers by passage in cell cultures. The relevance of these high-order structures for virus transcription is reinforced by the phylogenetic conservation of the involved RNA motifs in CoVs.

Gene N Proximal and Distal RNA Motifs Regulate Coronavirus Nucleocapsid mRNA Transcription

Coronavirus subgenomic mRNA (sgmRNA) transcription requires a discontinuous RNA synthesis mechanism driven by the transcription-regulating sequences (TRSs), located at the 3' end of the genomic leader (TRS-L) and also preceding each gene (TRS-B). In transmissible gastroenteritis virus (TGEV), the free energy of TRS-L and cTRS-B (complement of TRS-B) duplex formation is one of the factors regulating the transcription of sgmRNAs. In addition, N gene sgmRNA transcription is controlled by a transcription-regulating motif, including a long-distance RNA-RNA interaction between complementary proximal and distal elements. The extension of complementarity between these two sequences increased N gene transcription. An active domain, a novel essential component of the transcription-regulating motif, has been identified. The active domain primary sequence was necessary for its activity. Relocation of the active domain upstream of the N gene TRS core sequence in the absence of the proximal and distal elements also enhanced sgmRNA N transcription. According to the proposed working model for N gene transcriptional activation, the long-distance RNA-RNA interaction relocates the distant active domain in close proximity with the N gene TRS, which probably increases the frequency of template switching during the synthesis of negative RNA. The transcription-regulating motif has been optimized to a minimal sequence showing a 4-fold activity increase in relation to the native RNA motif. Full-length TGEV infectious viruses were generated with the optimized transcription-regulating motif, which enhanced by 5-fold the transcription of the 3a gene and can be used in expression vectors based in coronavirus genomes.

Structure and functional relevance of a transcription-regulating sequence involved in coronavirus discontinuous RNA synthesis.

Transmissible gastroenteritis coronavirus (TGEV) genomic RNA transcription generates 5'- and 3'-coterminal subgenomic mRNAs. This process involves a discontinuous step during the synthesis of minus-sense RNA that is modulated by transcription-regulating sequences located at the 3' end of the leader (TRS-L) and also preceding each viral gene (TRS-Bs). TRSs include a highly conserved core sequence (CS) (5'-CUAAAC-3') and variable flanking sequences. It has been previously proposed that TRS-Bs act as attenuation or stop signals during the synthesis of minus-sense RNAs. The nascent minus-stranded RNA would then be transferred by a template switch process to the TRS-L, which acts as the acceptor RNA. To study whether the TRS-L is structured and to determine whether this structure has a functional impact on genomic and subgenomic viral RNA synthesis, we have used a combination of nuclear magnetic resonance (NMR) spectroscopy and UV thermal denaturation approaches together with site-directed mutagenesis and in vivo transcriptional analyses. The results indicated that a 36-nucleotide oligomer encompassing the wild-type TRS-L forms a structured hairpin closed by an apical AACUAAA heptaloop. This loop contains most of the CS and is isolated from a nearby internal loop by a short Watson-Crick base-paired stem. TRS-L mutations altering the structure and the stability of the TRS-L hairpin affected replication and transcription, indicating the requirement of a functional RNA hairpin structure in these processes.

Subgenomic messenger RNAs: Mastering regulation of (+)-strand RNA virus life cycle

Many (+)-strand RNA viruses use subgenomic (SG) RNAs as messengers for protein expression, or to regulate their viral life cycle. Three different mechanisms have been described for the synthesis of SG RNAs. The first mechanism involves internal initiation on a (−)-strand RNA template and requires an internal SGP promoter. The second mechanism makes a prematurely terminated (−)-strand RNA which is used as template to make the SG RNA. The third mechanism uses discontinuous RNA synthesis while making the (−)-strand RNA templates. Most SG RNAs are translated into structural proteins or proteins related to pathogenesis: however other SG RNAs regulate the transition between translation and replication, function as riboregulators of replication or translation, or support RNA–RNA recombination. In this review we discuss these functions of SG RNAs and how they influence viral replication, translation and recombination.

Arterivirus Nsp1 Modulates the Accumulation of Minus-Strand Templates to Control the Relative Abundance of Viral mRNAs

The gene expression of plus-strand RNA viruses with a polycistronic genome depends on translation and replication of the genomic mRNA, as well as synthesis of subgenomic (sg) mRNAs. Arteriviruses and coronaviruses, distantly related members of the nidovirus order, employ a unique mechanism of discontinuous minus-strand RNA synthesis to generate subgenome-length templates for the synthesis of a nested set of sg mRNAs. Non-structural protein 1 (nsp1) of the arterivirus equine arteritis virus (EAV), a multifunctional regulator of viral RNA synthesis and virion biogenesis, was previously implicated in controlling the balance between genome replication and sg mRNA synthesis. Here, we employed reverse and forward genetics to gain insight into the multiple regulatory roles of nsp1. Our analysis revealed that the relative abundance of viral mRNAs is tightly controlled by an intricate network of interactions involving all nsp1 subdomains. Distinct nsp1 mutations affected the quantitative balance among viral mRNA species, and our data implicate nsp1 in controlling the accumulation of full-length and subgenome-length minus-strand templates for viral mRNA synthesis. The moderate differential changes in viral mRNA abundance of nsp1 mutants resulted in similarly altered viral protein levels, but progeny virus yields were greatly reduced. Pseudorevertant analysis provided compelling genetic evidence that balanced EAV mRNA accumulation is critical for efficient virus production. This first report on protein-mediated, mRNA-specific control of nidovirus RNA synthesis reveals the existence of an integral control mechanism to fine-tune replication, sg mRNA synthesis, and virus production, and establishes a major role for nsp1 in coordinating the arterivirus replicative cycle.

A Discontinuous RNA Platform Mediates RNA Virus Replication: Building an Integrated Model for RNA–based Regulation of Viral Processes

Plus-strand RNA viruses contain RNA elements within their genomes that mediate a variety of fundamental viral processes. The traditional view of these elements is that of local RNA structures. This perspective, however, is changing due to increasing discoveries of functional viral RNA elements that are formed by long-range RNA–RNA interactions, often spanning thousands of nucleotides. The plus-strand RNA genomes of tombusviruses exemplify this concept by possessing different long-range RNA–RNA interactions that regulate both viral translation and transcription. Here we report that a third fundamental tombusvirus process, viral genome replication, requires a long-range RNA–based interaction spanning ∼3000 nts. In vivo and in vitro analyses suggest that the discontinuous RNA platform formed by the interaction facilitates efficient assembly of the viral RNA replicase. This finding has allowed us to build an integrated model for the role of global RNA structure in regulating the reproduction of a eukaryotic RNA virus, and the insights gained have extended our understanding of the multifunctional nature of viral RNA genomes.

Role of RNA chaperones in virus replication

RNA molecules are functionally diverse in part due to their extreme structural flexibility that allows rapid regulation by refolding. RNA folding could be a difficult process as often molecules adopt a spatial conformation that is very stable but not biologically functional, named a kinetic trap. RNA chaperones are non-specific RNA binding proteins that help RNA folding by resolving misfolded structures or preventing their formation. There is a large number of viruses whose genome is RNA that allows some evolutionary advantages, such as rapid genome mutation. On the other hand, regions of the viral RNA genomes can adopt different structural conformations, some of them lacking functional relevance and acting as misfolded intermediates. In fact, for an efficient replication, they often require RNA chaperone activities. There is a growing list of RNA chaperones encoded by viruses involved in different steps of the viral cycle. Also, cellular RNA chaperones have been involved in replication of RNA viruses. This review briefly describes RNA chaperone activities and is focused in the roles that viral or cellular nucleic acid chaperones have in RNA virus replication, particularly in those viruses that require discontinuous RNA synthesis.

Arterivirus subgenomic mRNA synthesis and virion biogenesis depend on the multifunctional nsp1 autoprotease.

Many groups of plus-stranded RNA viruses produce additional, subgenomic mRNAs to regulate the expression of part of their genome. Arteriviruses and coronaviruses (order Nidovirales) are unique among plus-stranded RNA viruses for using a mechanism of discontinuous RNA synthesis to produce a nested set of 5'- and 3'-coterminal subgenomic mRNAs, which serve to express the viral structural protein genes. The discontinuous step presumably occurs during minus-strand synthesis and joins noncontiguous sequences copied from the 3'- and 5'-proximal domains of the genomic template. Nidovirus genome amplification ("replication") and subgenomic mRNA synthesis ("transcription") are driven by 13 to 16 nonstructural proteins (nsp's), generated by autocatalytic processing of two large "replicase" polyproteins. Previously, using a replicon system, the N-terminal nsp1 replicase subunit of the arterivirus equine arteritis virus (EAV) was found to be dispensable for replication but crucial for transcription. Using reverse genetics, we have now addressed the role of nsp1 against the background of the complete EAV life cycle. Mutagenesis revealed that nsp1 is in fact a multifunctional regulatory protein. Its papain-like autoprotease domain releases nsp1 from the replicase polyproteins, a cleavage essential for viral RNA synthesis. Several mutations in the putative N-terminal zinc finger domain of nsp1 selectively abolished transcription, while replication was either not affected or even increased. Other nsp1 mutations did not significantly affect either replication or transcription but still dramatically reduced the production of infectious progeny. Thus, nsp1 is involved in at least three consecutive key processes in the EAV life cycle: replicase polyprotein processing, transcription, and virion biogenesis.