Cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer.

The tremendous pandemic potential of coronaviruses was demonstrated twice in the past few decades by two global outbreaks of deadly pneumonia. Entry of coronaviruses into cells is mediated by the transmembrane spike glycoprotein S, which forms a trimer carrying receptor-binding and membrane fusion functions. S also contains the principal antigenic determinants and is the target of neutralizing antibodies. Here we present the structure of a mouse coronavirus S trimer ectodomain determined at 4.0 Å resolution by single particle cryo-electron microscopy. It reveals the metastable pre-fusion architecture of S and highlights key interactions stabilizing it. The structure shares a common core with paramyxovirus F proteins, implicating mechanistic similarities and an evolutionary connection between these viral fusion proteins. The accessibility of the highly conserved fusion peptide at the periphery of the trimer indicates potential vaccinology strategies to elicit broadly neutralizing antibodies against coronaviruses. Finally, comparison with crystal structures of human coronavirus S domains allows rationalization of the molecular basis for species specificity based on the use of spatially contiguous but distinct domains.

Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC.

Most human coronaviruses cause mild upper respiratory tract disease but may be associated with more severe pulmonary disease in immunocompromised individuals. However, SARS coronavirus caused severe lower respiratory disease with nearly 10% mortality and evidence of systemic spread. Recently, another coronavirus (human coronavirus-Erasmus Medical Center (hCoV-EMC)) was identified in patients with severe and sometimes lethal lower respiratory tract infection. Viral genome analysis revealed close relatedness to coronaviruses found in bats. Here we identify dipeptidyl peptidase 4 (DPP4; also known as CD26) as a functional receptor for hCoV-EMC. DPP4 specifically co-purified with the receptor-binding S1 domain of the hCoV-EMC spike protein from lysates of susceptible Huh-7 cells. Antibodies directed against DPP4 inhibited hCoV-EMC infection of primary human bronchial epithelial cells and Huh-7 cells. Expression of human and bat (Pipistrellus pipistrellus) DPP4 in non-susceptible COS-7 cells enabled infection by hCoV-EMC. The use of the evolutionarily conserved DPP4 protein from different species as a functional receptor provides clues about the host range potential of hCoV-EMC. In addition, it will contribute critically to our understanding of the pathogenesis and epidemiology of this emerging human coronavirus, and may facilitate the development of intervention strategies.

Deletion of accessory genes 3a, 3b, 5a or 5b from avian coronavirus infectious bronchitis virus induces an attenuated phenotype both in vitro and in vivo.

Avian coronavirus infectious bronchitis virus (IBV) infects domestic fowl, resulting in respiratory disease and causing serious losses in unprotected birds. Its control is mainly achieved by using live attenuated vaccines. Here we explored the possibilities for rationally attenuating IBV to improve our knowledge regarding the function of IBV accessory proteins and for the development of next-generation vaccines with the recently established reverse genetic system for IBV H52 based on targeted RNA recombination and selection of recombinant viruses in embryonated eggs. To this aim, we selectively removed accessory genes 3a, 3b, 5a and 5b individually, and rescued the resulting recombinant (r) rIBV-Δ3a, rIBV-Δ3b, rIBV-Δ5a and rIBV-Δ5b. In vitro inoculation of chicken embryo kidney cells with recombinant and wild-type viruses demonstrated that the accessory protein 5b is involved in the delayed activation of the interferon response of the host after IBV infection. Embryo mortality after the inoculation of 8-day-old embryonated chicken eggs with recombinant and wild-type viruses showed that rIBV-Δ3b, rIBV-Δ5a and rIBV-Δ5b had an attenuated phenotype in ovo, with reduced titres at 6 h p.i. and 12 h p.i. for all viruses, while growing to the same titre as wild-type rIBV at 48 h p.i. When administered to 1-day-old chickens, rIBV-Δ3a, rIBV-Δ3b, rIBV-Δ5a and rIBV-Δ5b showed reduced ciliostasis in comparison to the wild-type viruses. In conclusion, individual deletion of accessory genes in IBV H52 resulted in mutant viruses with an attenuated phenotype.

Cell Attachment Domains of the Porcine Epidemic Diarrhea Virus Spike Protein Are Key Targets of Neutralizing Antibodies.

Porcine epidemic diarrhea virus (PEDV) causes enteric disease in pigs, resulting in significant economic losses to the swine industry worldwide. Current vaccination approaches against this emerging coronavirus are only partially effective, though natural infection protects pigs against reinfection and provides lactogenic immunity to suckling piglets. The viral spike (S) glycoprotein, responsible for receptor binding and cell entry, is the major target for neutralizing antibodies. However, knowledge of antibody epitopes, their nature and location in the spike structure, and the mechanisms by which the antibodies interfere with infection is scarce. Here we describe the generation and characterization of 10 neutralizing and nonneutralizing mouse monoclonal antibodies raised against the S1 receptor binding subunit of the S protein. By expression of different S1 protein fragments, six antibody epitope classes distributed over the five structural domains of the S1 subunit were identified. Characterization of antibodies for cross-reactivity and cross-neutralization revealed antigenic differences among PEDV strains. The epitopes of potent neutralizing antibodies segregated into two epitope classes and mapped within the N-terminal sialic acid binding domain and in the more C-terminal receptor binding domain. Antibody neutralization escape mutants displayed single amino acid substitutions that impaired antibody binding and neutralization and defined the locations of the epitopes. Our observations picture the antibody epitope landscape of the PEDV S1 subunit and reveal that its cell attachment domains are key targets of neutralizing antibodies.IMPORTANCE Porcine epidemic diarrhea virus (PEDV), an emerging porcine coronavirus, causes an economically important enteric disease in pigs. Effective PEDV vaccines for disease control are currently lacking. The spike (S) glycoprotein on the virion surface is the key player in virus cell entry and, therefore, the main target of neutralizing antibodies. To understand the antigenic landscape of the PEDV spike protein, we developed monoclonal antibodies against the spike protein's S1 receptor binding region and characterized their epitopes, neutralizing activity, and cross-reactivity toward multiple PEDV strains. Epitopes of antibodies segregated into six epitope classes dispersed over the multidomain S1 structure. Monoclonal antibodies revealed antigenic variability in B-cell epitopes between PEDV strains. The epitopes of neutralizing antibodies mapped to two distinct domains in S1 that are involved in binding to carbohydrate and proteinaceous cell surface molecules, respectively, indicating the importance of these cell attachment sites on the PEDV spike protein in eliciting a protective humoral immune response.

Characterization of Epitope-Specific Anti-Respiratory Syncytial Virus (Anti-RSV) Antibody Responses after Natural Infection and after Vaccination with Formalin-Inactivated RSV.

UNLABELLED: Antibodies against the fusion (F) protein of respiratory syncytial virus (RSV) play an important role in the protective immune response to this important respiratory virus. Little is known, however, about antibody levels against multiple F-specific epitopes induced by infection or after vaccination against RSV, while this is important to guide the evaluation of (novel) vaccines. In this study, we analyzed antibody levels against RSV proteins and F-specific epitopes in human sera and in sera of vaccinated and experimentally infected cotton rats and the correlation thereof with virus neutralization. Analysis of human sera revealed substantial diversity in antibody levels against F-, G (attachment)-, and F-specific epitopes between individuals. The highest correlation with virus neutralization was observed for antibodies recognizing prefusion-specific antigenic site Ø. Nevertheless, our results indicate that high levels of antibodies targeting other parts of the F protein can also mediate a potent antiviral antibody response. In agreement, sera of experimentally infected cotton rats contained high neutralizing activity despite lacking antigenic site Ø-specific antibodies. Strikingly, vaccination with formalin-inactivated RSV (FI-RSV) exclusively resulted in the induction of poorly neutralizing antibodies against postfusion-specific antigenic site I, although antigenic sites I, II, and IV were efficiently displayed in FI-RSV. The apparent immunodominance of antigenic site I in FI-RSV likely explains the low levels of neutralizing antibodies upon vaccination and challenge and may play a role in the vaccination-induced enhancement of disease observed with such preparations. IMPORTANCE: RSV is an importance cause of hospitalization of infants. The development of a vaccine against RSV has been hampered by the disastrous results obtained with FI-RSV vaccine preparations in the 1960s that resulted in vaccination-induced enhancement of disease. To get a better understanding of the antibody repertoire induced after infection or after vaccination against RSV, we investigated antibody levels against fusion (F) protein, attachment (G) protein, and F-specific epitopes in human and animal sera. The results indicate the importance of prefusion-specific antigenic site Ø antibodies as well as of antibodies targeting other epitopes in virus neutralization. However, vaccination of cotton rats with FI-RSV specifically resulted in the induction of weakly neutralizing, antigenic site I-specific antibodies, which may play a role in the enhancement of disease observed after vaccination with such preparations.

A reverse genetics system for avian coronavirus infectious bronchitis virus based on targeted RNA recombination.

BACKGROUND: Avian coronavirus infectious bronchitis virus (IBV) is a respiratory pathogen of chickens that causes severe economic losses in the poultry industry worldwide. Major advances in the study of the molecular biology of IBV have resulted from the development of reverse genetics systems for the highly attenuated, cell culture-adapted, IBV strain Beaudette. However, most IBV strains, amongst them virulent field isolates, can only be propagated in embryonated chicken eggs, and not in continuous cell lines. METHODS: We established a reverse genetics system for the IBV strain H52, based on targeted RNA recombination in a two-step process. First, a genomic and a chimeric synthetic, modified IBV RNA were co-transfected into non-susceptible cells to generate a recombinant chimeric murinized (m) IBV intermediate (mIBV). Herein, the genomic part coding for the spike glycoprotein ectodomain was replaced by that of the coronavirus mouse hepatitis virus (MHV), allowing for the selection and propagation of recombinant mIBV in murine cells. In the second step, mIBV was used as the recipient. To this end a recombination with synthetic RNA comprising the 3'-end of the IBV genome was performed by introducing the complete IBV spike gene, allowing for the rescue and selection of candidate recombinants in embryonated chicken eggs. RESULTS: Targeted RNA recombination allowed for the modification of the 3'-end of the IBV genome, encoding all structural and accessory genes. A wild-type recombinant IBV was constructed, containing several synonymous marker mutations. The in ovo growth kinetics and in vivo characteristics of the recombinant virus were similar to those of the parental IBV strain H52. CONCLUSIONS: Targeted RNA recombination allows for the generation of recombinant IBV strains that are not able to infect and propagate in continuous cell lines. The ability to introduce specific mutations holds promise for the development of rationally designed live-attenuated IBV vaccines and for studies into the biology of IBV in general.

A Single Point Mutation Creating a Furin Cleavage Site in the Spike Protein Renders Porcine Epidemic Diarrhea Coronavirus Trypsin Independent for Cell Entry and Fusion.

The emerging porcine epidemic diarrhea virus (PEDV) requires trypsin supplementation to activate its S protein for membrane fusion and virus propagation in cell culture. By substitution of a single amino acid in the S protein, we created a recombinant PEDV with an artificial furin protease cleavage site N terminal of the putative fusion peptide (PEDV-SFCS). PEDV-SFCS exhibited trypsin-independent cell-cell fusion and was able to replicate in culture cells independently of trypsin, though to low titer.

ATP1A1-mediated Src signaling inhibits coronavirus entry into host cells.

UNLABELLED: In addition to transporting ions, the multisubunit Na(+),K(+)-ATPase also functions by relaying cardiotonic steroid (CTS)-binding-induced signals into cells. In this study, we analyzed the role of Na(+),K(+)-ATPase and, in particular, of its ATP1A1 α subunit during coronavirus (CoV) infection. As controls, the vesicular stomatitis virus (VSV) and influenza A virus (IAV) were included. Using gene silencing, the ATP1A1 protein was shown to be critical for infection of cells with murine hepatitis virus (MHV), feline infectious peritonitis virus (FIPV), and VSV but not with IAV. Lack of ATP1A1 did not affect virus binding to host cells but resulted in inhibited entry of MHV and VSV. Consistently, nanomolar concentrations of the cardiotonic steroids ouabain and bufalin, which are known not to affect the transport function of Na(+),K(+)-ATPase, inhibited infection of cells with MHV, FIPV, Middle East respiratory syndrome (MERS)-CoV, and VSV, but not IAV, when the compounds were present during virus inoculation. Cardiotonic steroids were shown to inhibit entry of MHV at an early stage, resulting in accumulation of virions close to the cell surface and, as a consequence, in reduced fusion. In agreement with an early block in infection, the inhibition of VSV by CTSs could be bypassed by low-pH shock. Viral RNA replication was not affected when these compounds were added after virus entry. The antiviral effect of ouabain could be relieved by the addition of different Src kinase inhibitors, indicating that Src signaling mediated via ATP1A1 plays a crucial role in the inhibition of CoV and VSV infections. IMPORTANCE: Coronaviruses (CoVs) are important pathogens of animals and humans, as demonstrated by the recent emergence of new human CoVs of zoonotic origin. Antiviral drugs targeting CoV infections are lacking. In the present study, we show that the ATP1A1 subunit of Na(+),K(+)-ATPase, an ion transporter and signaling transducer, supports CoV infection. Targeting ATP1A1 either by gene silencing or by low concentrations of the ATP1A1-binding cardiotonic steroids ouabain and bufalin resulted in inhibition of infection with murine, feline, and MERS-CoVs at an early entry stage. Infection with the control virus VSV was also inhibited. Src signaling mediated by ATP1A1 was shown to play a crucial role in the inhibition of virus entry by ouabain and bufalin. These results suggest that targeting the Na(+),K(+)-ATPase using cardiotonic steroids, several of which are FDA-approved compounds, may be an attractive therapeutic approach against CoV and VSV infections.

Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner.

Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fusion proteins leading to viral and host membrane fusion. In the present study we investigated the entry of coronaviruses (CoVs). Using siRNA gene silencing, we found that proteins known to be important for late endosomal maturation and endosome-lysosome fusion profoundly promote infection of cells with mouse hepatitis coronavirus (MHV). Using recombinant MHVs expressing reporter genes as well as a novel, replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV to lysosomes is required for fusion and productive entry to occur. Nevertheless, MHV was shown to be less sensitive to perturbation of endosomal pH than vesicular stomatitis virus and influenza A virus, which fuse in early and late endosomes, respectively. Our results indicate that entry of MHV depends on proteolytic processing of its fusion protein S by lysosomal proteases. Fusion of MHV was severely inhibited by a pan-lysosomal protease inhibitor, while trafficking of MHV to lysosomes and processing by lysosomal proteases was no longer required when a furin cleavage site was introduced in the S protein immediately upstream of the fusion peptide. Also entry of feline CoV was shown to depend on trafficking to lysosomes and processing by lysosomal proteases. In contrast, MERS-CoV, which contains a minimal furin cleavage site just upstream of the fusion peptide, was negatively affected by inhibition of furin, but not of lysosomal proteases. We conclude that a proteolytic cleavage site in the CoV S protein directly upstream of the fusion peptide is an essential determinant of the intracellular site of fusion.

Rapid Emergence of Highly Pathogenic Avian Influenza Subtypes from a Subtype H5N1 Hemagglutinin Variant.

In 2014, novel highly pathogenic avian influenza A H5N2, H5N5, H5N6, and H5N8 viruses caused outbreaks in Asia, Europe, and North America. The H5 genes of these viruses form a monophyletic group that evolved from a clade 2.3.4 H5N1 variant. This rapid emergence of new H5Nx combinations is unprecedented in the H5N1 evolutionary history.

Proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture.

Isolation of porcine epidemic diarrhea coronavirus (PEDV) from clinical material in cell culture requires supplementation of trypsin. This may relate to the confinement of PEDV natural infection to the protease-rich small intestine of pigs. Our study focused on the role of protease activity on infection by investigating the spike protein of a PEDV isolate (wtPEDV) using a reverse genetics system based on the trypsin-independent cell culture-adapted strain DR13 (caPEDV). We demonstrate that trypsin acts on the wtPEDV spike protein after receptor binding. We mapped the genetic determinant for trypsin-dependent cell entry to the N-terminal region of the fusion subunit of this class I fusion protein, revealing a conserved arginine just upstream of the putative fusion peptide as the potential cleavage site. Whereas coronaviruses are typically processed by endogenous proteases of the producer or target cell, PEDV S protein activation strictly required supplementation of a protease, enabling us to study mechanistic details of proteolytic processing. Importance: Recurring PEDV epidemics constitute a serious animal health threat and an economic burden, particularly in Asia but, as of recently, also on the North-American subcontinent. Understanding the biology of PEDV is critical for combatting the infection. Here, we provide new insight into the protease-dependent cell entry of PEDV.

Identification and characterization of a proteolytically primed form of the murine coronavirus spike proteins after fusion with the target cell.

UNLABELLED: Enveloped viruses carry highly specialized glycoproteins that catalyze membrane fusion under strict spatial and temporal control. To prevent premature activation after biosynthesis, viral class I fusion proteins adopt a locked conformation and require proteolytic cleavage to render them fusion-ready. This priming step may occur during virus exit from the infected cell, in the extracellular milieu or during entry at or in the next target cell. Proteolytic processing of coronavirus spike (S) fusion proteins during virus entry has been suggested but not yet formally demonstrated, while the nature and functionality of the resulting subunit is still unclear. We used a prototype coronavirus--mouse hepatitis virus (MHV)--to develop a conditional biotinylation assay that enables the specific identification and biochemical characterization of viral S proteins on virions that mediated membrane fusion with the target cell. We demonstrate that MHV S proteins are indeed cleaved upon virus endocytosis, and we identify a novel processing product S2* with characteristics of a fusion-active subunit. The precise cleavage site and the enzymes involved remain to be elucidated. IMPORTANCE: Virus entry determines the tropism and is a crucial step in the virus life cycle. We developed an approach to characterize structural components of virus particles after entering new target cells. A prototype coronavirus was used to illustrate how the virus fusion machinery can be controlled.

Adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase 4-mediated entry of the Middle East respiratory syndrome coronavirus.

Middle East respiratory syndrome coronavirus (MERS-CoV) replicates in cells of different species using dipeptidyl peptidase 4 (DPP4) as a functional receptor. Here we show the resistance of ferrets to MERS-CoV infection and inability of ferret DDP4 to bind MERS-CoV. Site-directed mutagenesis of amino acids variable in ferret DPP4 thus revealed the functional human DPP4 virus binding site. Adenosine deaminase (ADA), a DPP4 binding protein, competed for virus binding, acting as a natural antagonist for MERS-CoV infection.

Influenza A virus entry into cells lacking sialylated N-glycans.

Influenza A virus (IAV) enters host cells after attachment of its hemagglutinin (HA) to surface-exposed sialic acid. Sialylated N-linked glycans have been reported to be essential for IAV entry [Chu VC, Whittaker GR (2004) Proc Natl Acad Sci USA 102:18153-18158], thereby implicating the requirement for proteinaceous receptors in IAV entry. Here we show, using different N-acetylglucosaminyl transferase 1 (GnT1)-deficient cells, that N-linked sialosides can mediate, but are not required for, entry of IAV. Entry into GnT1-deficient cells was fully dependent on sialic acid. Although macropinocytic entry appeared to be affected by the absence of sialylated N-glycans, dynamin-dependent entry was not affected at all. However, binding of HA to GnT1-deficient cells and subsequent entry of IAV were reduced by the presence of serum, which could be reversed by back-transfection of a GnT1-encoding plasmid. The inhibitory effect of serum was significantly increased by inhibition of the viral receptor-destroying enzyme neuraminidase (NA). Our results indicate that decoy receptors on soluble serum factors compete with cell surface receptors for binding to HA in the absence of sialylated N-glycans at the cell surface. This competition is particularly disturbed by the additional presence of NA inhibitors, resulting in strongly reduced IAV entry. Our results indicate that the balance between HA and NA is important not only for virion release, but also for entry into cells.

The receptor binding domain of the new Middle East respiratory syndrome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies.

The spike (S) protein of the recently emerged human Middle East respiratory syndrome coronavirus (MERS-CoV) mediates infection by binding to the cellular receptor dipeptidyl peptidase 4 (DPP4). Here we mapped the receptor binding domain in the S protein to a 231-amino-acid fragment (residues 358 to 588) by evaluating the interaction of spike truncation variants with receptor-expressing cells and soluble DPP4. Antibodies to this domain--much less so those to the preceding N-terminal region--efficiently neutralize MERS-CoV infection.

Evolution of the hemagglutinin protein of the new pandemic H1N1 influenza virus: maintaining optimal receptor binding by compensatory substitutions.

Pandemic influenza A H1N1 (pH1N1) virus emerged in 2009. In the subsequent 4 years, it acquired several genetic changes in its hemagglutinin (HA). Mutations may be expected while virus is adapting to the human host or upon evasion from adaptive immune responses. However, pH1N1 has not displayed any major antigenic changes so far. We examined the effect of the amino acid substitutions found to be most frequently occurring in the pH1N1 HA protein before 1 April 2012 on the receptor-binding properties of the virus by using recombinant soluble HA trimers. Two changes (S186P and S188T) were shown to increase the receptor-binding avidity of HA, whereas two others (A137T and A200T) decreased binding avidity. Construction of an HA protein tree revealed the worldwide emergence of several HA variants during the past few influenza seasons. Strikingly, two major variants harbor combinations of substitutions (S186P/A137T and S188T/A200T, respectively) with opposite individual effects on binding. Stepwise reconstruction of the HA proteins of these variants demonstrated that the mutations that increase receptor-binding avidity are compensated for by the acquisition of subsequent mutations. The combination of these substitutions restored the receptor-binding properties (avidity and specificity) of these HA variants to those of the parental virus. The results strongly suggest that the HA of pH1N1 was already optimally adapted to the human host upon its emergence in April 2009. Moreover, these results are in agreement with a recent model for antigenic drift, in which influenza A virus mutants with high and low receptor-binding avidity alternate.

CD200 receptor controls sex-specific TLR7 responses to viral infection.

Immunological checkpoints, such as the inhibitory CD200 receptor (CD200R), play a dual role in balancing the immune system during microbial infection. On the one hand these inhibitory signals prevent excessive immune mediated pathology but on the other hand they may impair clearance of the pathogen. We studied the influence of the inhibitory CD200-CD200R axis on clearance and pathology in two different virus infection models. We find that lack of CD200R signaling strongly enhances type I interferon (IFN) production and viral clearance and improves the outcome of mouse hepatitis corona virus (MHV) infection, particularly in female mice. MHV clearance is known to be dependent on Toll like receptor 7 (TLR7)-mediated type I IFN production and sex differences in TLR7 responses previously have been reported for humans. We therefore hypothesize that CD200R ligation suppresses TLR7 responses and that release of this inhibition enlarges sex differences in TLR7 signaling. This hypothesis is supported by our findings that in vivo administration of synthetic TLR7 ligand leads to enhanced type I IFN production, particularly in female Cd200(-/-) mice and that CD200R ligation inhibits TLR7 signaling in vitro. In influenza A virus infection we show that viral clearance is determined by sex but not by CD200R signaling. However, absence of CD200R in influenza A virus infection results in enhanced lung neutrophil influx and pathology in females. Thus, CD200-CD200R and sex are host factors that together determine the outcome of viral infection. Our data predict a sex bias in both beneficial and pathological immune responses to virus infection upon therapeutic targeting of CD200-CD200R.

Genome-wide gene expression analysis of anguillid herpesvirus 1.

BACKGROUND: Whereas temporal gene expression in mammalian herpesviruses has been studied extensively, little is known about gene expression in fish herpesviruses. Here we report a genome-wide transcription analysis of a fish herpesvirus, anguillid herpesvirus 1, in cell culture, studied during the first 6 hours of infection using reverse transcription quantitative PCR. RESULTS: Four immediate-early genes - open reading frames 1, 6A, 127 and 131 - were identified on the basis of expression in the presence of a protein synthesis inhibitor and unique expression profiles during infection in the absence of inhibitor. All of these genes are located within or near the terminal direct repeats. The remaining 122 open reading frames were clustered into groups on the basis of transcription profiles during infection. Expression of these genes was also studied in the presence of a viral DNA polymerase inhibitor, enabling classification into early, early-late and late genes. In general, clustering by expression profile and classification by inhibitor studies corresponded well. Most early genes encode enzymes and proteins involved in DNA replication, most late genes encode structural proteins, and early-late genes encode non-structural as well as structural proteins. CONCLUSIONS: Overall, anguillid herpesvirus 1 gene expression was shown to be regulated in a temporal fashion, comparable to that of mammalian herpesviruses.

Visualizing coronavirus RNA synthesis in time by using click chemistry.

Coronaviruses induce in infected cells the formation of replicative structures, consisting of double-membrane vesicles (DMVs) and convoluted membranes, where viral RNA synthesis supposedly takes place and to which the nonstructural proteins (nsp's) localize. Double-stranded RNA (dsRNA), the presumed intermediate in RNA synthesis, is localized to the DMV interior. However, as pores connecting the DMV interior with the cytoplasm have not been detected, it is unclear whether RNA synthesis occurs at these same sites. Here, we studied coronavirus RNA synthesis by feeding cells with a uridine analogue, after which nascent RNAs were detected using click chemistry. Early in infection, nascent viral RNA and nsp's colocalized with or occurred adjacent to dsRNA foci. Late in infection, the correlation between dsRNA dots, then found dispersed throughout the cytoplasm, and nsp's and nascent RNAs was less obvious. However, foci of nascent RNAs were always found to colocalize with the nsp12-encoded RNA-dependent RNA polymerase. These results demonstrate the feasibility of detecting viral RNA synthesis by using click chemistry and indicate that dsRNA dots do not necessarily correspond with sites of active viral RNA synthesis. Rather, late in infection many DMVs may harbor dsRNA molecules that are no longer functioning as intermediates in RNA synthesis.

Anguillid herpesvirus 1 transcriptome.

We used deep sequencing of poly(A) RNA to characterize the transcriptome of an economically important eel virus, anguillid herpesvirus 1 (AngHV1), at a stage during the lytic life cycle when infectious virus was being produced. In contrast to the transcription of mammalian herpesviruses, the overall level of antisense transcription from the 248,526-bp genome was low, amounting to only 1.5% of transcription in predicted protein-coding regions, and no abundant, nonoverlapping, noncoding RNAs were identified. RNA splicing was found to be more common than had been anticipated previously. Counting the 10,634-bp terminal direct repeat once, 100 splice junctions were identified, of which 58 were considered likely to be involved in the expression of functional proteins because they represent splicing between protein-coding exons or between 5' untranslated regions and protein-coding exons. Each of the 30 most highly represented of these 58 splice junctions was confirmed by RT-PCR. We also used deep sequencing to identify numerous putative 5' and 3' ends of AngHV1 transcripts, confirming some and adding others by rapid amplification of cDNA ends (RACE). The findings prompted a revision of the AngHV1 genome map to include a total of 129 protein-coding genes, 5 of which are duplicated in the terminal direct repeat. Not counting duplicates, 11 genes contain integral, spliced protein-coding exons, and 9 contain 5' untranslated exons or, because of alternative splicing, 5' untranslated and 5' translated exons. The results of this study sharpen our understanding of AngHV1 genomics and provide the first detailed view of a fish herpesvirus transcriptome.